Environmental Biotechnology: Proceedings of the International Symposium on Biotechnology, Bratislava, Czecho-Slovakia, June 27-29, 1990 (Studies in Environmental Science)

ENVI R 0 N M ENTA L B IOTEC H N0LOGY

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Studies in Environmental Science 42

E NVI R 0 N M E NTA L BIOTECHNOLOGY Proceedings of the International Symposium on Biotechnology, Bratislava, Czecho-Slovakia, June 27-29, 1990 edited by

A. Blaiej and V. Privarova Institute of Biotechnology, Slovak Technical University, Bratislava, Czech0-Slovakia

ELSEVIER Amsterdam-Oxford-New

York-Tokyo,

1991

Distributors for the United States and Canada: ELSEVIER SCI ENCE PUB LIS H IN G CO M PANY I NC. 655 Avenue of the Americas New York, NY 10010, U S A . for the East European Countries, Democratic Republic of Vietnam, Mongolian People's Republic, People's Republic of Korea, People's Republic of China, Republic of Cuba: ALFA, Hurbanovo nhm. 3, 81 5 8 9 Bratislava, Czechoslovakia for all remaining areas: ELSEVIER SCIENCE PUBLISHERS B. V. Sara Burgerhartstraat 25 P. 0. Box 21 1, 1000 AE Amsterdam, The Netherlands

Library of Congress Cataloging-in-Publication Data International Symposium on Biotechnology (1 990: Bratislava, Czecho-Slovakia) Environmental biotechnology: proceedings of the International Symposium on Biotechnology, Bratislava, Czecho-Slovakia, June 27-29,1990 / edited by A. Blaiej and V. Privarovh. 446 p. (Studies in environmental sciences; 42) ISBN 0-444-98720-7 1. Environmental biotechnology - Congresses. 2. Microbial biotechnology - Congresses. 3. Biotechnology - Environmental aspects - Congresses. 4. Biomass energy - Environmental aspects - Congresses. I . Blaiej, Anton. II. Privarovh, V. Ill. Title. IV. Series. TD192.5.157 1990 628-dc20 90-29865 CIP ISBN 0-444-98720-7 (Vol. 42) ISBN 0-444-41696-X (Series)

0A.

Blaiej and V. Privarova, Bratislava 1991

All rights reserved. No part of this publication may be reproduced, stored in a retrieval system or transmitted in any form or by any means, electronic, mechanical, photocopying, recording or otherwise, without the prior written permission of the copyright owners. Printed in Czecho-Slovakia

PREFACE

F o r t h e l a s t f o u r y e a r s we h a v e r e g u l a r l y o r g a n i z e d t h e i n t e r n a t i o n a l symposium INTERBIOTECH, technology.

devoted t o selected t o p i c s o f b i o -

The symposium i s a c o n c o m i t a n t s c i e n t i f i c e v e n t sccom-

p a n y i n g t h e I n t e r n a t i o n a l C h e m i c a l F a i r INCHEBA i n B r a t i s l a v a . The firqt I n t e r b i o t e c h ' 8 7 (Progress i n Biotechnology,

was d e v o t e d t o Enzyme T e c h n o l o g i e s

Volume 4,

E l s e v i e r Amsterdam 1 9 0 8 ) .

The s e c o n d I n t e r b i o t e c h '88 was d e v o t e d t o B i o c h e m i c a l s . The t h i r d I n t e r b i o t e c h ' 8 9 was d e v o t e d t o M a t h e m a t i c a l M o d e l l i n g i n Biotechnology

(Progress i n Biotechnology,

volume

6, ~

1

v i e r Amsterdam 1 9 9 0 ) . The f o u r t h I n t e r b i o t e c h ' 9 0 was d e v o t e d t o B i o t e c h n o l o g y a n d t h e Environment.

The symposium l e c t u r e s a r e d i v i d e d i n t o t h r e e s u b -

j e c t areas: M i c r o b i a l waste t r e a t m e n t and p r o c e s s i n g P o s s i b l e p o s i t i v e a n d n e g a t i v e i m p a c t s o f b i o t e c h n o l o g y on the environment B i o e n e r g y and t h e e n v i r o n m e n t The g r o w i n g a w a r e n e s s o f e n v i r o n m e n t a l p r o b l e m s h a s s t i m u l a t e d o u r a t t e n t i o n t o devote t h i s I n t e r b i o t e c h '90 t o t h e r e l a t i o n between b i o t e c h n o l o g y and t h e e n v i r o n m e n t .

We a r e e n t e r i n g a new age,

a g e o f a c i e n c e and s c i e n c e b a s e d i n d u s t r y . a g e o f ENVIRONMENTALISM.

the

We a r e a l s o e n t e r i n g t h e

The p r o s p e r i t y i n t h e n e a r f u t u r e p s r t i c u -

l a r l y i n t h e d e v e l o p e d i n d u s t r i a l c o u n t r i e s d e p e n d s on how we s h a l l b e a b l e t o c r e a t e a new l i v i n g s t y l e a n d a new " c o n s u m e r c u l t u r e " b a a e d on r a t i o n a l c o n s u m p t i o n o f g o o d s ,

environmental q u a l i t y o f

p r o d u c t i o n and e n v i r o n m e n t a l q u a l i t y o f p r o d u c t s .

Advances i n s c i e n -

c e a n d t e c h n o l o g y a n d t h e wisdom o f man c a n s h a p e an o r d e r l y h e a l t h y q u a l i t y o f l i f e o f s o c i e t y and i n d i v i d u a l s . B i o t e c h n o l o g y has a chance t o accept t h e c h a l l e n g e o f t h e f u t u r e and p o s i t i v e l y i n f l u e n c e and i m p r o v e t h e q u a l i t y o f t h e human e n v i r o n m e n t s i n c e b i o t e c h n o l o g y w i l l b e a new n a t i o n a l s e c t o r b a s e d on :

~

~

- 6 -

-

renewable raw m a t e r i a l s

- more e n v i r o n m e n t a l l y sound t e c h n o l o g y and p r o c e s s e s

-

very h i g h environmental q u a l i t y o f bioproducts

- b i o p r o d u c t s as biodegradable n o n - t o x i c m a t t e r s

-

r e c y c l a b l e wastes from t h e m a n u f a c t u r e r and consumer Many e s s e n t i a l human g o a l s ,

through processes,

n e e d s and demands c a n o n l y b e met

goods and s e r v i c e s p r o v i d e d b y b i o t e c h n o l o g y

.

This i n d u s t r i a l s e c t o r i s a t y p i c a l sample f o r s u s t a i n a b l e developm e n t a n d w i l l b e p o w e r e d b y a c o n t i n u i n g f l o w o f human w e a l t h f r o m bioindustry.

Biotechnology o f f e r s p o t e n t i a l p o s s i b i l i t i e s t o con-

t r i b u t e t o t h e i m p r o v e m e n t o f t h e human e n v i r o n m e n t .

We b e l i e v e

t h a t t h e s y m p o s i u m I n t e r b i o t e c h ' 9 0 h a s s h o w n how b i o t e c h n o l o g y c a n h e l p t o s o l v e some e n v i r o n m e n t a l p r o b l e m s .

CONTENTS

Preface

.......................................................

R e l a t i o n b e t w e e n t h e economy, t h e environment A. Blafej

5

b i o t e c h n o l o g y and

................................................

11

I. M i c r o b i o l o g i c a l w a s t e t r e a t m e n t a n d p r o c e s s i n g M i c r o b i a l waste water and waste t r e a t m e n t J . H o l l b , P. M i h d l t z , L . Czakb a n d L .

Morvai

.............

A n a e r o b i c t r e a t m e n t o f e f f l u e n t f r o m epoxy r e s i n s p r o d u c t i o n units P . Senna, E . D ’ A d d a r i o and A . R o b e r t i e l l o

................

Anaerobic treatment o f e f f l u e n t s : process monitoring C. S o r l i n i

use o f b i o i n d i c a t o r s

............................................... ...................... -

47

for

Simultaneous n i t r i f i c a t i o n - d e n i t r i f i c a t i o n i n a c t i v a t e d s l u d g e p r o c e s s combined w i t h b i o f i l m c u l t i v a t i o n M. D r t i l , J. T G l g y e s s y a n d I. B o d f k Hexamethylenetetraamine removal i n s i n g l e system I . B o d f k , J . D e r c o a n d M. D r t i l

27

55

65

sludge a c t i v a t i o n

..........................

71

E x p e r i m e n t a l and m a t h e m a t i c a l m o d e l l i n g o f a c t i v a t e d s l u d g e process P . F a r k a S o v A , J. D e r c o a n d M . K r A l i k

77

Aerobic thermophilic M. B o m i o

85

..................... ................................................. sludge treatment

E l i m i n a t i o n o f s p e c i a l b a c t e r i a from treatment e f f l u e n t by ciliates M. Macek a n d P. H a r t m a n

113

M i c r o b i o l o g i c a l t r e a t m e n t o f m u n i c i p a l sewage s l u d g e a n d r e f u s e as means o f d i s i n f e c t i o n p r i o r t o r e c y c l i n g i n agriculture D. Strauch

121

Modeling o f organic matter d e s t r u c t i o n by microorganisms community V.A. Vavilin

137

E f f e c t o f p r o t o z o a on b a c t e r i a l d e g r a d a t i o n o f a r o m a t i c hydrocarbons Yu.L. G u r e v i c h a n d V.P. L a d y g i n a

147

P h e n o l and n a p h t h a l e n e d e g r a d a t i o n b y m i x e d c u l t u r e o f microorganisms N . S . M a n u k o v s k i , M. I . Teremova,. Yu.L. G u r e v i c h a n d I.M. Pan’kovs

155

S p e c i f i c a d s o r p t i o n o f m e t a l c a t i o n s on t h e s u r f a c e o f l i p i d membrane s y s t e m s A.M. Omel’chenko

.........................................

165

The i n v e r s e f l u i d i z a t i o n a new a p p r o a c h t o b i o f i l m r e a c t o r d e s i a~. n, t o a e r o b i c wastewater t r e a t m e n t L . N i k o l o v a n d D. Karamanev

177

..................................

............................................... .............................................

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- 8 -

P r o d u c t i o n o f sugars from l i g n o c e l l u l o s i c wastes b a s i c r e s e a r c h and p i l o t s t u d i e s H . E s t e r b a u e r , M . Hayn, W . S a t t l e r , W . S t e i n e r , H . S t e i n m h l l e r , Th. S t e i n e r a n d M . S i n n e r The

............... f u t u r e o f t h e l i q n o c e l l u l o s i c wastes b i o c o n v e r s i o n I. S p i l d a , A . B l a f e j a n d M. K o 3 i k .......................

183 20 1

D e t o x i f i c a t i o n o f p h e n o l p o l l u t e d s o i l b y some N o c a r d i a a n d Basidiomycetes E . M a l a r c z y k , 2 . L e w i c k a - K r b l , J. Kochmahska-Rdest, R . A p a l o v i c , M. S t a s z c z a k a n d A . L e o n o w i c z

209

The d e t o x i f y i n g r o l e o f f e r r o - p h e n o l i c c o m p l e x e s p r o d u c e d by Nocardia E . M a l a r c z y k , J. Kochmahska-Rdest, M . W o j t a s - W a s i l e w s k a and A . Leonowicz

215

M i c r o b i a l t r e a t m e n t o f i n d u s t r i a l wastes L.I. V o r o b j e v a , L . V . Modyanova, P . B . F.M. Chasaeva a n d E . V . D o v g i l e v i c h

221

..............

........................................ Terentijev,

......................

H i g h e r f u n g i as a p o t e n t i a l f e e d a n d f o o d s o u r c e f r o m l i g n o c e l l u l o s i c wastes A . L e o n o w i c z , M. W o j t a s - W a s i l e w s k a , J. R o g a l s k i a n d J. L u t e r e k

.............................................

2 29

11. B i o e n e r g y a n d e n v i r o n m e n t I m m o b i l i z e d p h o t o s y n t h e t i c systems f o r t h e p r o d u c t i o n o f f u e l s and c h e m i c s l s D.O. H a l l , K . K . Rad a n d I . H . P a r k

......................

Anaerobic t r e a t m e n t o f excrements from l a r g e - s c a l e animal farms J . K e r e k r b t y , B . P e t r o v i E o v B , K . Boaa a n d 0. Adamec

259

.....

277

I n t e n s i f i c a t i o n and e c o l o g i c a l a s p e c t s o f m e t h a n e f e r m e n t a t i o n o f a g r i c u l t u r a l wastes M.J. B e k e r , A.P. G r i n b e r g s , V.E. D a v i d s , L . J . L a b a n e , J.E. Blumbergs and M . K . Marauska

287

........................

Economic and b i o e n e r g e t i c a s p e c t s o f methanogenesis quantitative investigations S . V a s s i l i e v a , M. Robeva a n d 5. M u t a f o v

-

.................

297

Recovery o f energy from m u n i c i p a l s o l i d waste i n f a b r i c a t e d digesters J . Coombs a n d Y . R . Coombs

305

L a n d f i l l gas f u e l and e c o l o g i c a l p r o b l e m s 2. P i e t r z y k

317

............................... ............................................. Biomass and t h e p r o b l e m s o f e c o l o g y , a g r o c h e m i s t r y and e n e r g y E.S. P a n t s k h a v a .........................................

329

111. P o s s i b l e p o s i t i v e a n d n e g a t i v e i m p a c t s o f b i o t e c h n o l o g y on e n v i r o n m e n t P o s i t i v e a n d n e g a t i v e i m p a c t s o f b i o t e c h n o l o g y on t h e e n v i r o n m e n t Dr. L. H u b e r 341

............................................

Release o f g e n e t i c a l l y - engineered microorganisms i n t h e environment: r i s k of h o r i z o n t a l g e n e t i c - t r a n s f e r W.P.M. Hoekstra

.........................................

351

- 9 -

The r o l e o f c u l t u r e c o l l e c t i o n s t o s a f e g u a r d n a t u r e ’ s m i c r o b i o l o g i c a l resources K.A. Malik

359

I s biotechnology a blessing i n disguise? R. K o k k e

369

.............................................. ................................................ Improvements o f a g r i c u l t u r a l c r o p s by g e n e t i c e n g i n e e r i n g J. B o t t e r m a n ............................................

379

The c y c l o d e x t r i n s a n d t h e i r a p p l i c a t i o n i n e n v i r o n m e n t a l biotechnology J. Szejtli

387

B i o t e c h n o l o g y o f m e t a l s and t h e e n v i r o n m e n t F. S p a l d o n , M. K u b n i e r o v B , D. Kupka, E . J. E e c h o v s k B a n d V . S e p e l d k

399

..............................................

A small A.

PastirikovB,

............................. s i m u l a t i o n system and e c o l o g i c a l f o r e c a s t i n g G. Degermendzky ......................................

413

A s t e r i l i z a b l e c e n t r i f u g a l s e p a r a t i o n system f o r a s e p t i c and c o n t a i n e d c e l l h a r v e s t and r e c y c l e G. K r o o k , H. A x e l s s o n a n d C. T h o r s s o n

...................

427

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RELATION BETWEEN THE E C O N O M Y ,

11

-

BIOTECHNOLOGY AND

THE ENVIRONMENT A.

BLATEJ

Slovak Technical U n i v e r s i t y ,

Bratislava

The g r o w i n g a w a r e n e s s o f e n v i r o n m e n t a l p r o b l e m s h a s s t i m u l a t e d a t t e n t i o n t o t h e r e l a t i o n s h i p between economic development and i t s i m p a c t on t h e human e n v i r o n m e n t .

I n the p a s t 50 years world-wide

t e n d e n c i e s have been d i r e c t e d towards:

-

speeding-up economic and s o c i a l development s t i m u l a t i n g consumption a c t i v i t i e s forming

m a t e r i a l and f i n a n c i a l r e s o u r c e s t h a t m a i n l y

ensure t h e increase o f the m a t e r i a l wealth o f populations. S t r e s s has been and i s s t i l l p l a c e d on economic growth.

The c u l t o f

e c o n o m i c g r o w t h i s deep i n t h e s o c i a l c o n s c i o u s n e s s a s t h e m a i n s o u r ce o f m a t e r i a l w e a l t h o f an

i n d i v i d u a l a s w e l l as s o c i e t y .

t i o n , economic t h i n k i n g and t h e l o n g - t e r m ture

y e a r s i n many c o u n t r i e s i s b a s e d on t h e t h e o r y o f m a t e r i a l

production growth.

T h i s a l s o r e s u l t s i n an i n c r e a s e o f i n n o v a t i v e

a c t i v i t y end b o t h s c i e n t i f i c - t e c h n o l o g i c a l subordinated t o t h i s fact. ggle

I n addi-

economic s t r a t e g y f o r f u -

f o r spheres o f i n f l u e n c e ,

markets are p r i o r i t i e s .

and t r a d e p o l i c i e s a r e

A t present, global competition, raw m a t e r i a l ,

the stru-

e n e r g y s o u r c e s and

The g r o w t h o f t h e g r o s s n a t i o n a l p r o d u c t i s

s t i l l t h e m a i n i n d i c a t o r o f t h e s u c c e s s o f t h e n a t i o n a l economy. higher the inter-annual

growth o f the gross product,

The

t h e more r e a l

a r e t h e p o s s i b i l i t i e s f o r s a t i s f y i n g t h e n e e d s o f an i n d i v i d u a l a n d society.

This development i s a p p r o p r i a t e ,

the natural ecological equilibrium.

p r o v i d e d i t does n o t a f f e c t

I n the past,

n a t u r e was a b l e t o

c o u n t e r b a l a n c e man’s n e g a t i v e i n f l u e n c e s b y s e l f - r e g u l a t i o n . highly-developed i n d u s t r i a l c o u n t r i e s today,

man’a

In

i n f l u e n c e on t h e

environment has reached such a l e v e l t h a t n a t u r e l o s e s i t s s e l f - r e gulating ability.

T h i s i s why t h e w h o l e s o c i e t y i s a n d w i l l b e

negatively affected.

12

-

Anthropogenic s t r e s s o f n a t u r a l systems r e s u l t s

i n r e d u c i n g t h e p o t e n t i a l s o u r c e s o f economic development, v i t y o f b i o t i c resources i s lowered, vironment accelerates, ned, that,

c o n d i t i o n s f o r s o c i a l development a r e worse-

m o r b i d i t y and m o r t a l i t y a r e i n c r e a s i n g l y h i g h e r ,

evidence of

t h e decreased q u a l i t y o f t h e environment.

a t present, however,

and t h e r e i s We c a n s a y

t h e economic g r o w t h and t h e e n v i r o n m e n t a r e two

phenomena s t a n d i n g a t c o n t r a d i c t o r y p o s i t i o n s . ment,

producti-

d e s t r u c t i o n o f t h e manmade en-

An e x p e c t e d d e v e l o p -

i s very c l o s e l y connected with t h e problem o f t h e

r e a l e x i s t e n c e o f man a n d h i s s u r v i v a l . The e x i s t i n g d e v e l o p m e n t o f e c o n o m i c g r o w t h i s a c c o m p a n i e d b y a n immense s q u a n d e r i n g o f r a w m a t e r i a l a n d e n e r g y s o u r c e s a n d s i m u l t a n e o u s l y by h i g h e c o l o g i c a l s t r e s s i n n a t u r e caused by p r o d u c t i o n a n d consumer w a s t e . cological,

Man's

v a l u e o r i e n t a t i o n s were i n essence a n t i e -

connected w i t h m a t e r i a l w e l f a r e t h a t g r e a t l y exceeds r a -

t i o n a l consumption i n t h e h i g h l y developed c o u n t r i e s .

P r a c t i c a l wis-

dom ( l i f e p h i l o s o p h y ) i s b a s e d on t h e k n o w l e d g e t h a t h e h o l d s much

i n possession,

w a n t s e v e n more a n d s i m u l t a n e o u s l y h a s a d e q u a t e means

and energy t o o b t a i n more.

T h a t i s why t h e d i s t r i b u t i o n o f w e a l t h

p r o c e e d s e v e n more i l n e v e n l y ;

w e a l t h y men g r o w w e a l t h i e r a n d p o o r

men become p o o r e r . The p r o g r e s s o f s o c i e t y i s i d e n t i f i e d o n l y w i t h m a t e r i a l w e l f a r e a n d n o t w i t h an i n c r e a s e o f l i f e q u a l i t y . m u s t b e d i r e c t e d a t a new q u a l i t y ,

Development o f s o c i e t y

where e n v i r o n m e n t a l q u a l i t y i s

a p r i m a r y f a c t o r t o which economic g r o w t h i s s u b o r d i n a t e d .

T h i s me-

ans t h a t r e g e n e r a t i o n o f t h e environment i s t h e b a s i s and economic p r o d u c t i o n muat b e d e r i v e d f r o m i t . I n t h e economy, luate factor

coats,

i.e.

we m a i n l y e v a -

r a w m a t e r i a l and e n e r g y c o s t s a n d t h e i r

conversion t o the f i n a l product.

We a r e n o t v e r y i n t e r e s t e d i n t h e

n a t u r a l resources renewal costs,

n o r i n t h e l o s s e s caused by e n v i -

ronmental contamination,

air,

water,

f o r e s t s and s o i l p o l l u t i o n .

Ig-

norance o f t h e s e l o s s e s and n o n - a c q u a i n t a n c e w i t h t h e m a r g i n o f " a d m i s s i b l e l o a d i n g " o f n a t u r e h a v e i r r e v o c a b l y d e s t r o y e d some e c o systems. ciency,

Enterpriae i s not i d e n t i f i e d with social production e f f i and l a c k o f e c o l o g i c a l knowledge g i v e s no p o s s i b i l i t y t o

objectively quantify

consequences,

r e s u l t o f t h e i n f l u e n c e o f man.

damage a n d l o s s e s a r i s i n g a s a

T e c h n o l o g i c a l p r o g r e s s has i t s r o o t s

i n t h e i n d u s t r i a l r e v o l u t i o n and,

l a t e r on,

technical o r i e n t a t i o n o f production,

i t developed i n t o t h e

a n d t o t h e maximum u t i l i z a t i o n

and c o n t r o l o v e r n a t u r a l r e s o u r c e s and f o r c e s .

Problems connected

-

-

13

w i t h t h e b i o s p h e r e , e c o l o g y a n d human e n v i r o n m e n t w e r e a l w a y s s e condary.

An a p p r o a c h t o t h e phenomena a n d new p r o b l e m s , introduced by scientific-technological ture,

w h i c h have been

p r o g r e s s i n r e l a t i o n t o na-

r e q u i r e s a new s t r a t e g y o f s o c i o e c o n o m i c d e v e l o p m e n t . T h e r e f o r e i t i s n e c e s s a r y t o t a k e measures,

de a n d i n t h e f u t u r e

beyond t h e year

l e r a t e environmental problem s o l v i n g ,

2000.

f o r t h e 90 s deca-

I t i s i m p o r t a n t t o acce-

from t h e v i e w p o i n t o f

investment

i n t h e s o - c a l l e d e c o l o g i c a l s t r u c t u r e s a n d e c o l o g i c a l m e a s u r e s as w e l l as t h e p r o d u c t i o n o f needs.

technological

devices f o r environmental

A c c e l e r a t i o n o f t h e s o c i a l - e c o n o m i c a l development i s condi-

t i o n e d by t h e d i s c o n t i n u a t i o n o f d e t e r i o r a t i o n and by g r a d u a l ,

plan-

ned r e c o v e r y o f t h e environment. We h a v e t o a t t e m p t ' a n e l a b o r a t i o n o f t h e s c i e n t i f i c a l l y

deter-

m i n e d l o a d s t a n d a r d s o f - t h e i n d i v i d u a l b r a n c h e s o f n a t i o n a l economy on t h e environment.

The" sum o f t h e damage p l u s t h e c o s t s c o v e r i n g

environmental protection represents the basis f o r ascertaining the o b j e c t i v e e c o l o g i c a l consequences o f economic development o f t h e i n d i v i d u a l branches.

We m u s t r e a l i z e t h e n e c e s s i t y o f c o s t s f o r t h e

p r o t e c t i o n and f o r m a t i o n o f t h e environment n o t as t h e s o c i e t y ' s loss,

b u t v i c e versa,

nomic development.

a s an a l l - s o c i e t y

contribution t o social-eco-

I t w i l l become o b v i o u s s i m u l t a n e o u s l y w h i c h b r a n -

ches cause c o n t a m i n a t i o n ,

w h i c h a r e t o o e x p e n s i v e f o r s o c i e t y and

which p r o d u c t i o n a c t i v i t i e s a r e uneconomical from such a complex viewpoint. The r e a l c h a l l e n g e i n t h i s f i e l d i s t o make i n d u s t r i a l t e c h nologies

-

more e n v i r o n m e n t a l l y sound

-

more e n v i r o n m e n t a l l y

-

l e s s d i s r u p t i v e t o the environment

friendly

I n d u s t r y i s c e n t r a l t o t h e economies o f modern s o c i e t i e s a n d dispensable engine o f growth.

an i n -

Many e s s e n t i a l human n e e d s c a n b e m e t

o n l y t h r o u g h g o o d s a n d s e r v i c e s p r o v i d e d by t h e i n d u s t r y .

There has

been remarkable t e c h n o l o g i c a l p r o g r e s s s i n c e t h e i n d u s t r i a l r e v o lution.

I n many f i e l d s ,

extraordinary technological breakthroughs

have been a c h i e v e d and o t h e r s w i l l

follow.

In fact,

one o f t h e g r e a t

paradoxes o f technology i s t h a t p r i n c i p a l l y i t has c o n t r i b u t e d t o human p r o g r e s s ,

m o d e r n c i v i l i z a t i o n e n d t o human w e a l t h b u t may c a u -

s e t h e d e g r a d a t i o n o f t h e human e n v i r o n m e n t a s w e l l aa a c t i n g aga-

i n s t man.

Take, ture.

f o r instance,

14

-

the application o f pesticides i n agricul-

U s i n g more p e s t i c i d e s and s i m i l a r c h e m i c a l s has caused u n p r e -

cedented growth o f crop production.

They h a v e u n q u e s t i o n a b l y i m p r o -

ved t h e w e l f a r e o f m i l l i o n s o f p e o p l e .

However,

s c i e n t i s t s and t h e

p u b l i c a r e concerned about t h e r e s i d u e s o f p e s t i c i d e s i n food,

owing

t o t h e i r n e g a t i v e i m p a c t on human h e a l t h . Take a n o t h e r p r o b l e m ,

that o f chlorofluorcarbons.

S o many p e o p -

l e have b e n e f i t e d f r o m t h e i r a p p l i c a t i o n i n r e f r i g e r a t i o n , d i t i o n i n g and i n s u l a t i o n .

a i r con-

B u t t h i s t e c h n o l o g i c a l m a r v e l has a l s o

c r e a t e d a s e r i o u s e n v i r o n m e n t a l t h r e a t t o t h e ozone l a y e r , t h i n n i n g and c r e a t i n g t h e s o - c a l l e d ozone h o u l s

which i s

/l/.

A good d e a l o f r e s e a r c h h a s a l r e a d y b e e n done t o d e s i g n t e c h nologies which p r o t e c t t h e environment.

However,

e x p e r i e n c e shows

X o r more o f caX i n running costs.

t h a t e n v i r o n m e n t a l s a f e g u a r d s c a n a d d up t o 1 5 - 1 8 p i t a l i n v e s t m e n t c o s t a and up t o 10

A t p r e s e n t we a r e f a c i n g some g l o b a l p r o b l e m s ( T a b l e 1). The m o s t i m p o r t a n t i s t h e e n v i r o n m e n t .

Similarly,

a t present i n

t h e environment there a r e g l o b a l problems as w e l l (Table 2 ) .

TABLE 1 Global problems

Peace a n d s e c u r i t y Environment Food s e c u r i t y Raw m a t e r i e l s Population growth Energy r e a o u r c e s Economic and s o c i a l development

TABLE 2 Global environmental problems Pollution

-

wastes

G l o b a l warming D e p l e t i o n o f ozone l a y e r Acid r a i n Deforestation Disappearing o f b i o l o g i c a l species

-

15

-

HUMAN E N V I R O N M E N T

The human e n v i r o n m e n t i s a c o m p l e x m u l t i c o m p o n e n t

system crea-

t e d b y some p a r t s / 2 / : a) biosphere

-

i t s phyaical,

c h e m i c a l and b i o l o g i c a l c o m p o s i t i o n ,

where man i s i n i n t e r a c t i o n w i t h h i s s u r r o u n d i n g a b ) socioeconomic environment, material,

w h e r e man r e a l i z e s h i s b i o l o g i c a l ,

a o c i s l and c u l t u r a l needs

c ) man-made e n v i r o n m e n t - c o n s t r u c t e d b y human a c t i v i t i e a

d ) w o r k i n g e n v i r o n m e n t - man-made o r c o m b i n e d n a t u r a l s y s t e m a n d man-made m a t e r i a l c o m p o n e n t s a n d p r o c e s s e s w i t h human s o c i o e c o nomic a c t i v i t i e a T h e i r i n t e r a c t i o n i a shown i n F i g .

1. The human e n v i r o n m e n t becomes

t h e m a i n component o f q u a l i t y o f l i f e a n d t h e r e f o r e m u s t h a v e p r i o r i t y o v e r a l l o t h e r human p r i o r i t i e s ( F i g .

2).

NATURAL ENVIRONMENT

t

SOCIOECONOMIC ENVIRONMENT

ENVIRONMENT

Fig.

ENVIRONMENT

1. Human E n v i r o n m e n t .

L I F E STYLE

\ MATERIAL WEALTH

SOCIAL S E C U R I T Y

/ QUALITY OF L I F E

ENVIRONMENT

F i g . 2.

Quality o f Life.

-

16

-

INDUSTRIAL BIOTECHNOLOGY T h e r e a r e many d i f f e r e n t d e f i n i t i o n s o f b i o t e c h n o l o g y .

What

concerns i n d u s t i a l b i o t e c h n o l o g y can be d e f i n e d as t e c h n o l o g y which uses b i o l o g i c a l organisms,

i n t h e man-made

t h e i r components and b i o l o g i c a l p r o c e s s e s

environment f o r i n d u s t r i a l manufacture.

I n d u s t r i a l b i o t e c h n o l o g y i s i n t e g r a t e d t e c h n o l o g y which u s e s s c i e n t i f i c and t e c h n o l o g i c a l knowledge from b i o l o g i c a l , and t e c h n o l o g i c a l s c i e n c e s

chemical

3).

(Fig.

CHEMICAL

BIOLOGICAL

SCIENCES

SCIENCES

INDUSTRIAL BIOTECHNOLOGY

I TECHNOLOGICAL SCIENCES

Fig.

3 . I n d u s t r i a l Biotechnology.

The m o s t i m p o r t a n t s c i e n c e s a r e :

-

from biology-

genetics,

mol

e t i

ula

enzymology,

protein engineering

-

from chemistry-

biochemistry,

organic chemistry,

bioorganic

c h e m i s t r y and p h y s i c a l c h e m i s t r y

-

from technology-

bioengineering,

computer sciences, engineering,

chemical engineering,

microelectronics,

automation,

system

c y b e r n e t i c s and o p t i m a l i s a t i o n .

B i o t e c h n o l o g y i s very i m p o r t a n t management o f t h e g l o b a l e c o s y s t e m .

f o r human p r o g r e s s a n d f o r t h e B i o t e c h n o l o g y w i l l b e one o f t h e

m o s t i n t e r e s t i n g i n d u a t r i a l a e c t o r s a n d w i l l g r a d u a l l y c r e a t e a new i n d u s t r i a l c o m p l e x o f t h e n a t i o n a l economy, (Fig.

the so-called bioindustry

4).

BIOTECHNOLOGY AND THE E N V I R O N M E N T I n d u s t r i a l b i o t e c h n o l o g y has p o t e n t i a l t o c o n t r i b u t e t o s o l v i n g many e n v i r o n m e n t a l p r o b l e m s :

Biometallurgy

a Renewable r a w m a t e r i a l s a P l a n t s and A n i m a l s

a Bioenergy a Biofood

a New k i n d s o f m i c r o o r g a n i s m s

a Biotechnological treatment

a Biofodder

a Agriculture

o f wastes

I

0

Biotechnological mechanical engineering

0

Forest products industry

I-

4

a Food i n d u s t r y

a Bioinformatics 0

Biocybernetics a Bioelectronics a Biochemical industry

F i g . 4. B i o l o g i z a t i o n o f n a t i o n a l economy ( b i o i n d u s t r y ) .

a Pharmaceutical i n d u s t r y a Microbiological industry

I

-

18

-

c o m p l e x u t i l i z a t i o n o f r a w m a t e r i a l s on t h e b a s e o f c a r b o n r e sources

-

u t i l i z a t i o n o f organic solids,

l i q u i d s and gas wastes f r o m t r a d i -

t i o n a l t e c h n o l o g i e s and c o n v e r t i n g them t o u s e f u l p r o d u c t s

-

s u b s t i t u t i o n o f aome t r a d i t i o n a l c h e m i c a l p r o c e s s e s ,

c h e m i c a l s and

c h e m i c a l man-made m a t e r i a l s

-

p r o d u c t i o n o f many b i o c h e m i c a l s a n d b i o m a t e r i a l s w h i c h c a n b e ma-

-

p r o d u c t i o n o f new s u b s t a n c e s t h a t h a v e n e v e r b e e n m a n u f a c t u r e d

de i n e x p e n s i v e l y w i t h h i g h e r v a l u e a d d e d p r o d u c t s before

-

-

a l l b i o c h e m i c a l s a n d b i o m a t e r i a l s aa b i o d e g r a d a b l e p r o d u c t s p r o v i d i n g low wastes o r wasteless processes enabling manufacture o f higher e c o l o g i c a l q u a l i t y o f products b e t t e r e c o l o g i c a l p r o p e r t i e s o f goods high index o f r e c y c l i z a t i o n

I n t h e c a s e o f t h e c h e m i c a l i n d u s t r y we c a n show a g o o d e x a m p l e o f how many p r o d u c t s a n d p r o c e s s e s c a n be s u b s t i t u t e d t h r o u g h b i o t e c h nology.

The c h e m i c a l i n d u s t r y h a s a s p e c i a l p o s i t i o n i n t h i s a r e a .

The e n v i r o n m e n t i s o v e r l o a d e d b y c h e m i c a l s a n d c h e m i c a l m a t e r i a l s . The number o f i n d i v i d u a l c h e m i c a l s i s a b o u t 4.5 t u r a l e n d s y n t h e t i c compounds).

m i l l i o n ( b o t h na-

A t t h e p r e s e n t t i m e commercial che-

m i c a l p r o d u c t s r e p r e s e n t s about 100.000

c h e m i c a l s . Each y e a r about

1 0 0 0 new p r o d u c t s s r e a d d e d t o t h e m a r k e t . I n d u s t r i a l c h e m i c a l processes a r e based on i n c o m p l e t e c h e m i c a l r e a c t i o n s w h i c h depend on t h e s t a t e o f c h e m i c a l e q u i 1 i b r i u m . A duct,

pro-

r e s c t i o n s u b s t a n c e s and i m p u r i t i e s need s e p a r a t i o n and p u r i -

f i c a t i o n during or a f t e r reaction.

A l l t h e s e o p e r a t i o n s make was-

tes. The s e c o n d s o u r c e o f c h e m i c a l p o l l u t i o n a r i s e s f r o m t h e a p p l i c a t i o n o f chemicals,

c h e m i c a l m a t e r i a l s and c h e m i c a l p r o c e s s e s i n

o t h e r s e c t o r s o f t h e n a t i o n a l economy.

P o l l u t i o n problems o f t h i s

k i n d a r e v e r y o f t e n caused by a m i s a p p l i c a t i o n o f t h e c h e m i c a l s o r chemical m a t e r i e l s by users. S p e c i f i c problems o f chemical p o l l u t i o n :

-

chemical substances are u s u a l l y t o x i c ,

mutagenic and even can-

c e r o g e n i c compounds

-

many p r o d u c t s o f s y n t h e t i c o r g a n i c c h e m i s t r y a r e x e n o b i o t i c s ,

-

p o l l u t i o n problems o f t e n c o m p l i c a t e h i g h odor and s m e l t even i n

t h e i r b i o d e g r a d a b i l i t y i s very low very low concentration

HOW

-

can b i o t e c h n o l o g y c o n t r i b u t e t o i m p r o v i n g c h e m i c a l po-

Ilutj.on? I n the future

t h e s y n t h e t i c o r g a n i c c h e m i s t s h a v e t o use

mnre b i o l o g i c a l p a t h w a y s f o r

-

19

b i o s y n t h e s i s o f new compounds t o a v o i d :

high temperatures high pressures the p r o d u c t i o n o f t o x i c wastes

The c h e m i s t s have t o l e a r n t o u s e s u b s t r a t e - s p e c i f i c ,

high turnover

number c a t a l y s t s s u c h as enzymes a n d h i g h l y r e a c t i v e ,

but specific

s u b s t r a t e s s u c h as coenzymes.

T a b l e 3 shows some c o m p a r i s o n s b e t -

ween c h e m i c a l i n d u s t r y a n d i n d u s t r i a l b i o t e c h n o l o g y .

T a b l e 4 shows

p o t e n t i a l p o s s i b i l i t i e s o f s u b s t i t u t i o n o f t r a d i t i o n a l p r o d u c t s by biotechnological products i n agriculture, food industry.

Fig.

energy,

t r a n s p o r t and t h e

5 shows t h e t e n d e n c y o f how t o r e d u c e w a s t e s .

R e d u c t i o n o f w a s t e s has t o b e m a i n s t r e a m l i n e i n i n d u s t r y .

Wasteless

a n d l o w w a s t e t e c h n o l o g y t h r o u g h w a s t e r e c y c l i n g a n d r e u s e have g r a d u a l l y become a c c e p t e d p r a c t i c e s i n mnny i n d u s t r i a l s e c t o r s .

i t i s n o t p o s s i b l e t o reuse o r g a n i c waste,

I f

i t can be p r o c e s s e d b i o -

technologically f o r biogas production ( i n anaerobic reactors or land-

fills y s t e m ) o r b i o c o m p o s t f o r a g r i c u l t u r e . New p o t e n t i a l f o r b i o t e c h n o l o g y h a s b e e n opened b y r e c o m b i n a n t

DNA t e c h n i q u e s and c e l l f u s i o n .

These gene m a n i p u l a t i o n t e c h n i q u e s

h o l d t h e p r o m i s e f o r d e v e l o p m e n t o f new b i o p r o c e s s i n g s y s t e m s t h r o ugh t h e b i o c h e m i c a l m o d i f i c a t i o n o f e x i s t i n g organisms,

f o r the i n -

creased production o f u s e f u l metabolites o f "the manufacture"

of

new s u b s t a n c e s . T h e r e i s no s p e c i f i c r e a s o n f o r s p e c i a l s a f e t y i n b i o t e c h n o l o g y

/4/

a n d no b a s i s f o r d e v e l o p i n q s p e c i f i c l e g i s l a t i o n f o r r e c o m b i n a n t

DNA o r g a n i s m s . Dn t h e one h a n d , of

the e x i s t i n g l e g a l p r o v i s i o n s i n d i f f e r e n t areas

t h e economic and s o c i a l a c t i v i t i e s can be a p p l i e d t o work w i t h

these organisms.

On t h e o t h e r h a n d ,

t o prevent p o t e n t i a l r i s k ,

guidelines elaborated purposely

a r e i n use i n most c o u n t r i e s .

The h a z a r d s a r i s i n g f r o m r D N A o r g a n i s m s a r e i n g e n e r a l o f t h e same n a t u r e a s t h o s e o f c o n v e n t i o n a l o r g a n i s m s . Any r i s k s a s s o c i a t e d w i t h t h e a p p l i c a t i o n o f r D N A o r g a n i s m s may be a s s e s s e d i n g e n e r a l l y t h e same way a s t h o s e a s s o c i a t e d w i t h non-recombinant

DNA organisms.

I n d u s t r y h a s a l o n g e x p e r i e n c e a n d a good s a f e t y r e c o r d i n hand l i n g l i v i n g organisms,

i n c l u d i n g pathogens.

The i n t r i n s i c a l l y l o w

r i s k organisms used i n most cases i n i n d u s t r i a l p r o d u c t i o n r e q u i r e o n l y a minimum l e v e l o f c o n t a i n m e n t .

TABLE 3 Comparison o f p r o d u c t s m a n u f a c t u r e d by c h e m i c a l

i n d u s t r y and i n d u s t r i a l ~

PRODUCT OR PROCESS

Raw m a t e r i a l s

biotechnology ~

CHEMICAL INDUSTRY

INDUSTRIAL BIOTECHNOLOGY

TREND

Crude o i l

Organic carbon-sources

S u b s t i t u t e f o s s i l raw

N a t u r a l gas

(starch,

m a t e r i a l s by renewable

Coal

lulose,

Synthetic organic

M i c r o b i a l organic chemicals

I n c r e a s i n g o f biodegra-

c e l l u l o s e , hemicelsaccharides)

resources

chemicals

Fine biochemicals

d a b i l i t y and d e c r e a s i n g

Fine chemicals

Biochemical s p e c i a l i t i e s

o f t o x i c i t y , mutagenity

Specialities

B u l k b i o c h e m i c a l commodities

and c a n c e r o g e n i t y I

Bulk commodities

N

D

Chemical

S y n t h e t i c polymers

Biopolymers

Increasing o f

materials

Synthetic f i b r e s

Biofibres

biodegradability

Synthetic rubbers

Biorubbers

Chemical d r u g s

Microbial products

Natural products

Chemical c a t a l y s e

Biocatalyse

Low t e m p e r a t u r e

Processes

Low p r e s s u r e Transformation

Chemical pathways

B i o c h e m i c a l pathways

I n many cases much more e f f e c t i v e

I

TABLE 4 Some I n s t a n c e s o f S u b s t i t u t i o n o f T r a d i t i o n a l P r o d u c t s b y B i o t e c h n o l o q i c a l ones

SECTOR

BIOTECHNOLOGICAL

TRADITIONAL PRODUCTS

TREND

PRODUCTS ~

Agriculture

Energy

Reduction o f chemicals

Pesticides

Biopesticides

Industrial pesticides

Biofertilizers

i n agriculture

Synthetic plastics

Bioplastics

S o i l improvement

Naphtha

Bio-oil

Reduction of a i r

Heavy o i l

Biogas

P o l l u t i o n c a u s e d by

N a t u r a l gas

Hydrogen (biopho-

pollutants (acid rain)

t o l y s i s o f water)

I N I-

I

Transport

Petrol Naphtha

Alcohols

Reduction o f a i r

Natural o i l s

p o l l u t i o n c a u s e d by

Hydrogen (biopho-

pollutants (acid rain)

t o l y s i s o f water) Foodstuff

Meat o f a n i m a l s ( e n e r -

Single c e l l proteins

Higher n u t r i t i o n value

g e t i c a l l y very inten-

Decreasing o f n a t u r a l

sive)

fat Energy s a v i n g processes

Lo

a, Lo 10

0 .-i

t

-c

x f-l

m

Lo 4 '4

E

m

a,

r4-

'CI

U

m

0 0 P)

0

22 -

I

w

r 3

z

0

v)

u

P) 10

m

Lo

U

3

?!

v ) 4 02= z

I

a, 3

m

n

-

23

-

The s a f e t y o f o r g a n i s m s i n t r o d u c e d i n t o t h e e n v i r o n m e n t m u s t be e v a l u a t e d o n a c a s e - b y - c a s e

basis,

r e a i s n o t y e t w e l l developed.

The c a s e - b y - c a s e

as r i s k assessment

i n t h i s a-

a p p r o a c h may n e v e r -

t h e l e s s n o t n e c e s s a r i l y be a p p l i e d t o a l l a p p l i c a t i o n s .

CONCLUSIONS A l l around us,old ment,

forms and methods o f socioeconomic d e v e l o p -

organizational structures,

o f human w e a l t h ,

s y s t e m s o f management,

philosophy

and t h e o l d c o n v e n t i o n s o f t h i n k i n g a b o u t n a t u r e

a r e u n d e r g o i n g r a d i c a l changes.

People a r e awakening t o t h e u r g e n t

n e e d f o r a f u n d s m e n t a l r e v i s i o n i n t h e r e l a t i o n s h i p b e t w e e n man s n d nature.

The g l o b a l e n v i r o n m e n t a l

planet Earth.

c r i s i s now f a c e s m a n k i n d a n d t h e

The t i m e h a s now come t o r e s o l v e t h o s e c r i s e s i n t h e

1 9 9 0 s on t h e b a s i s o f s u r v i v a l o f a l l l i v e s on t h e E a r t h . succeed i n e n s u r i n g e c o l o g i c a l s e c u r i t y . ternational,

We c a n

A series o f action i n in-

r e g i o n a l and n a t i o n a l frameworks c o u l d be t a k e n :

i t m u s t be r e c o g n i z e d t h a t i n t h e d e v e l o p e d w o r l d ,

First,

the

o v e r c o n s u m p t i o n o f g o o d s a n d s e r v i c e s m u s t d e c l i n e a n d a new c o n s u mer c u l t u r e i s n e e d e d .

On t h e o t h e r h a n d ,

poverty i n the developing

w o r l d i s one o f t h e g r e a t e s t t h r e a t s t o t h e g l o b s 1 e n v i r o n m e n t a s w e l l /l/. The c e n t r a l p r o b l e m i s i n r e d u c i n g o v e r c o n s u m p t i o n o n t h e one h a n d a n d r e m o v i n g p o v e r t y on t h e o t h e r hand. Second,

we m u s t r e s p e c t t h e d i f f e r i n g e n v i r o n m e n t a l p r i o r i t i e s

o f i n d u s t r i a l countries.

One may h a v e a s h o r t a g e o f d r i n k i n g w a t e r ,

w h i l e t h e o t h e r i s more c o n c e r n e d w i t h w a t e r p o l l u t i o n a n d s t i l l o t h e r c o u n t r i e s concerned w i t h r a p i d s o i l d e g r a d a t i o n or a i r p o l l u t i o n etc.

A u n i f y i n g c o n c e r n f o r t h e e n v i r o n m e n t does n o t mandate a

search f o r uniform environmental p r i o r i t i e s or uniform environmental standards.

B u t t h e g l o b a l e n v i r o n m e n t a l c r i s i s must seek t o i n t e g -

r a t e d i f f e r i n g national perceptions,

s e n s i t i v i e s and p r i o r i t i e s w i t -

h i n a new f r a m e w o r k o f c o o p e r s t i o n i n e n v i r o n m e n t a l l y s o u n d d e v e l o p ment. Third,

t h e v i s i o n a n d p e r c e p t i o n o f o u r common f u t u r e e x i s t s

i n a wide-ranging

agreement t h a t s u s t a i n a b l e development s t r a t e g i e s

a h o u l d meet t h e n e e d s o f t h e p r e s e n t g e n e r a t i o n w i t h o u t c o m p r o m i s i n g t h e a b i l i t y o f t h e f u t u r e g e n e r a t i o n t o meet t h e i r own n e e d s / 3 / .

The c o n c e p t o f s u s t a i n a b l e d e v e l o p m e n t h a s n o t y e t b e e n r i g o r o u s l y and s c c e p t a b l y d e f i n e d ,

I t must i n c l u d e t h e p r o t e c t i o n o f f u t u r e eco-

n o m i c g r o w t h r a t e s a s w e l l a s f u t u r e human

development l e v e l s .

Fourth, re-thinking

24

-

t h e s e r e a l i t i e s i n t h e human e n v i r o n m e n t r e q u i r e s t h e o f people.

They m u s t a d j u s t t h e i r b e h a v i o r t o t h e e n v i -

ronment and f o r m u l a t e a s o c i o - e c o n o m i c a l

s y s t e m t h a t f o c u s e s a s much

a t t e n t i o n on t h e p e o p l e a s i t d o e s o n t e c h n o l o g y .

I t m u s t f o c u s on:

-

n a t u r a l r e s o u r c e s as much a s o n p r o d u c t i o n

-

t h e l o n g t e r m d e v e l o p m e n t a s much a s on t h e s h o r t - t e r m lopment / 3 / .

deve-

O n l y s u c h a s y s t e m c a n meet t h e c h a l l e n g e o f t h e

f u t u r e development o f mankind. age o f s c i e n c e ,

We a r e e n t e r i n g a new age,

the

h i g h t e c h n o l o g y and s c i e n c e based i n d u s t r y .

We a r e a l s o e n t e r i n g t h e age o f e n v i r o n m e n t a l i s m a n d t h e age o f b i o l o g i s a t i o n o f t h e n a t i o n a l economy. We c o n c l u d e t h a t i m m e d i a t e l y t a r g e t e d r e d u c t i o n s o f a l l t y p e s o f p o l l u t a n t s and wastes a r e r e q u i r e d .

I n d u s t r i a l b i o t e c h n o l o g y can

c o n t r i b u t e t o s o l v i n g many g l o b a l e n v i r o n m e n t a l p r o b l e m s o v e r t h e c o m i n g decade.

REFERENCES

1 2

3 4

W.H. D r a p e r , T e c h n o l o g y , I n d u s t r y a n d t h e E n v i r o n m e n t , P a p e r o n t h e G l o b a l Forum on E n v i r o n m e n t a n d D e v e l o p m e n t f o r S u r v i v a l , Moscow 1 9 9 0 . A . B l a f e j a n d c o - w o r k e r s : The C h e m i c a l A s p e c t s o f Human E n v r i r o n ment, A l f a B r a t i s l a v a 1981 ( i n S l o v a k ) . G.H. B r u n d t l a n d , Our common f u t u r e , O x f o r d U n i v e r s i t y P r e s s , O x f o r d 1987. R e c o m b i n a n t -DNA S a f e t y C o n s i d e r a t i o n , OECD P a r i s , 1 9 8 6 .

1. Microbiological Waste Treatment and Processing

This Page Intentionally Left Blank

MICROBIAL WASTE W A T E R AND W A S T E T R E A T M E N T

J. HOLLb, P. MIHALTZ,

L.

C Z A K b and L.

MORVAI

U n i v e r s i t y o f T e c h n i c a l Sciences, I n s t i t u t e o f A g r i c u l t u r a l C h e m i c a l T e c h n o l o g y , H-1521 B u d a p e s t , G e l l g r t t 6 r 4 , H u n g a r y

A common f e a t u r e o f t h e m a j o r i t y o f t h e s e t r e a t m e n t p r o c e s s e s i s that the substrate i s a non-specified dingly,

organic matter.

Correspon-

t h e a i m o f t h e t e c h n i q u e s t o be e v a l u a t e d i s b i o d e g r a d a t i o n

f o r e n v i r o n m e n t a l c o n t r o l p u r p o s e s and sometimes f o r g a i n i n g marketable products. T h i s p a p e r b r i e f l y summarizes a c t u a l t r e n d s o f e n v i r o n m e n t a l biotechnologies.

More s p e c i f i c a l l y ,

r e s u l t s from three main areas

a r e d i s c u s s e d w i t h a r e s e a r c h and development c o n t r i b u t i o n f r o m t h e authors’

institute:

-

communal sewage t r e a t m e n t w i t h s p e c i a l r e g a r d t o n u t r i e n t removal

-

h i g h l y i n t e n s i v e d e n i t r i f i c a t i o n and n i t r i f i c a t i o n o f d r i n k i n g - and waste w a t e r s

-

a n a e r o b i c t r e a t m e n t o f c o n c e n t r a t e d waste w a t e r s and s o l i d wastes.

Communal sewaqe t r e a t m e n t w i t h n u t r i e n t r e m o v a l The a c t i v a t e d s l u d g e p r o c e s s i s , me d e c a d e s ,

and w i l l s u r e l y r e m a i n f o r so-

t h e m o s t i m p o r t a n t means o f communal sewage t r e a t m e n t .

C o n s i d e r i n g t h e huge volumes t r e a t e d ,

t h e most i m p o r t a n t p r o b -

lems o f a p p l i c a t i o n are:

-

-

e f f i c i e n t oxygenation, together with r e l i a b l e modelling and p r o c e s s c o n t r o l f o r d e s i g n and o p e r a t i o n enhanced i n t e g r a t i o n o f n u t r i e n t r e m o v a l s t e p s i n t o t h e process.

The r e v i e w ( 1 )

i n c l u d i n g the f i r s t statement,

underlines this

b y t h e 1 3 0 0 MW a e r a t i o n power c o n a u m p t i o n a t US a c t i v a t e d s l u d g e plants,

r e s p o n s i b l e f o r 40 % o f t o t a l o p e r a t i n g c o a t s .

Aerstion effi-

c i e n c i e s under process c o n d i t i o n s i n t h e UK proved t o be as low as 1,l-2,2 systems,

kg02/kWh a n d 0,5-1,7 respectively.

kg02/kWh a t d i f f u s e d a n d m e c h a n i c a l

-

-

28

With f i n e b u b b l e p o r o u s s p a r g e r s , tained.

much b e t t e r r e s u l t s w e r e ob-

-

P e r h a p s t h e r e c e n t l y d e v e l o p e d Messner p a n e l s

n i q u e hydrodynamic f e a t u r e a v o i d i n g l i q u i d c i r c u l a t i o n best o f t h i s type.

An a e r a t i o n e f f i c i e n c y o f 5 , 6

t e d under i n d u s t r i a l c o n d i t i o n s ( 2 ) ,

are the

kg02/kWh was r e p o r -

a s s u r i n g some a d d i t i o n a l a d v a n -

tages,

as i n s i t u s l u d g e t h i c k e n i n g a n d s e p a r a t i o n .

depths

-

A t high aeration

as i n t h e s u c c e s s f u l Hoechst and Bayer systems

g r a d i e n t s o f oxygen s o l u b i l i t y ,

w i t h t h e u-

-

-

important

t o g e t h e r w i t h l i q u i d d i s p e r s i o n may

l e a d t o s u p e r f i c i a l O2 desorption,

w h i c h may a f f e c t e n e r g e t i c c o n -

d i t i o n s as p o i n t e d o u t i n ( 3 ) . Oxygenation economics i s perhaps t h e most i m p o r t a n t p r a c t i c a l g o a l o f t h e Water Research C e n t e r (UK)

a c t i v a t e d s l u d g e model,

con-

s i d e r i n g a l s o t a n k b a c k m i x i n g b e h s v i o u r and t h e d i f f e r e n t i a t i o n b e t ween v i a b l e a n d n o n - v i a b l e m i c r o b e s i n s l u d g e .

From p l a n t dimensio-

n i n g a s p e c t s K r o i s s e v a l u a t e s 3 methods p r e f e r r i n g t h e I A W P R C dynam i c s i m u l a t i o n model t o g e t h e r w i t h i t s f u r t h e r N e c e s s i t y f o n u t r i e n t ( N a n d P compounds) a major goal f o r f u r t h e r From t h e e a r l y 7 0 s on, s e r t i n g a mixed,

r e s e a r c h needs ( 4 ) . r e m o v a l h a s become

development o f t h e a c t i v a t e d s l u d g e system.

the so-called

non-aerated

load n i t r i f i c a t i o n plant,

e n s u r e d an ammonia-and

ent with r e l a t i v e l y l i t t l e d i f f i c u l t y .

nitrate-free

p h o r o u s r e m o v a l b y a b i o l o g i c a l one

c h e m i c a l phos-

a t a s k s o l v e d b y t h e 3 r d ge-

From t h e f i r s t ,

solution without n i t r i f i c a t i o n ,

of

-

efflu-

The m o r e d i f f i c u l t p r o b l e m

was t o r e p l a c e c o s t l y a n d e x c e s s i v e s l u d g e - p r o d u c i n g n e r a t i o n systems i n t h e 80s.

in-

2nd g e n e r a t i o n p r o c e s s e s ,

anoxic step i n t o t h e t r a d i t i o n a l low

s t i l l "chemical

l i k e Phostrip,

hybride"

an impressive t r e n d

development can be observed. Environmental f a c t o r s favoring the overgrowth o f P sccumulating

Acinetobacters,

together with those o f n i t r i f i c a t i o n ,

and o r g a n i c removal a r e o f t e n c o n t r a d i c t o r y .

denitrification

The p r o b l e m i s s o l v e d

by a s o p h i s t i c a t e d s e r i e s o f a n a e r o b i c a n o x i c and a e r o b i c t r e a t m e n t s t e p s i n t e r l i n k e d by m u l t i p l e r e c i r c u l a t i o n ( 7 ) .

Our m e t h o d c o m b i n e d a n a e r o b i c a n d a n o x i c s p a c e s i n a s i n g l e v e r t i c a l s l u d g e bed r e a c t o r ,

as can be seen i n F i g .

t a k e s p l a c e i n t h e a e r o b i c (AER),

(ANOX)

reactor sections

(5).

1. N i t r i f i c a t i o n

d e n i t r i f i c a t i o n i n the anoxic

The e f f i c i e n c y

T N o f t h e l a t t e r de-

p e n d s m o s t l y o n t h e r e c i r c u l a t i o n r a t i o R w h i c h i s t h e r a t i o o f recirculated/influent

flow rates,

a s shown i n t h e f i g u r e .

E x p e r i m e n t a l r e s u l t s a r e g i v e n i n F i g . 2. With t h e i n f l u e n t N 3 t h e e f f i c i e n c i e s were f o u n d t o be s u p e r i o r c o n t e n t l e s s t h a n 80 g/m

,

t o theoretical

29

values (dotted l i n e ) ,

s u g g e s t i n g as an e x p l i c a t i o n

a f t e r - d e n i t r i f i c s t i o n under a n o x i c c o n d i t i o n s i n t h e s e t t l i n g tank.

IR+llO

lR111

a

RECRCULATION RATE R 1-1

F i g . 1. F l o w s h e e t o f b i o l o g i c a l organics-N-P removal.

F i g . 2. D e n i t r i f i c a t i o n e f f i c i e n cy i n f u n c t i o n o f t h e r e c i r c u l a t i o n rate.

Perhaps t h e most c o m p l i c a t e d p r o b l e m i s t o t h o r o u g h l y undera t a n d t h e mechanism o f b a c t e r i a l P r e l e a s e a n d u p t a k e . p o t e n t i a l and r e a d i l y degradable o r g a n i c s u b s t r a t e a c i d ) a n d i n h i b i t i n g NO;

c o n c e n t r a t i o n i n t h e a n a e r o b i c t a n k seemed

t o be t h e most i m p o r t a n t e n v i r o n m e n t a l

factors.

enough i n f o r m a t i o n f o r c o n t r o l l i n g t h e p r o c e s s , batch sludge t e s t .

The r e d o x

(volatile fatty

I n order t o gain we i n t r o d u c e d a

F o r t h e s u b s t r a t e we u s e d a c e t i c p r o p i o n i c - o r

b u t y r i c a c i d as p r e c u r s o r s o f b e t a - h y d r o x y - b u t y - r a t e

t y p e compound

f o r m a t i o n ( 6 ) . F i g u r e s 3 , 4 a n d 5 show t h e t i m e d e p e n d e n c e o f aubstrste,

phosphorous c o n c e n t r a t i o n and r e d o x p o t e n t i a l ,

under a n a e r o b i c and a e r o b i c c o n d i t i o n s .

a n d p h o s p h o r o u s r e l e s a e were s t r i c t l y p r o p o r t i o n a l . w i t h the redox p o t e n t i a l ,

respectively,

Fluxes o f substrate uptake Both,

together

followed f i r s t order kinetics.

From t h e a p p l i e d m a t h e m a t i c a l m o d e l

we o b t a i n e d k r e a c t i o n r a t e c o n s t a n t s a n d C tions,

depicted i n Fig.

6.,as

oo

terminal P concentra-

a function o f the s p e c i f i c organic

a c i d dose.

P r e l e a s e and d e n i t r i f i c a t i o n a r e c o m p e t i n g f o r o r g a n i c substrates.

The l a t t e r b e i n g an e n e r g y p r o d u c i n g p r o c e s s ,

a t l o w COD/N

r a t i o s may i n h i b i t P r e l e a s e w h i c h l e a d s t o a r e d u c e d u p t a k e u n d e r

- 30 -

a e r o b i c c o n d i t i o n s ( 6 ) . T h i s e f f e c t i s shown i n F i g . s u l t s gained w i t h laboratory-scale

equipment,

7 . w i t h re-

accordinq t o Fig.

1.

F i g . 3. Batch anaerobic s u b s t r a t e depletion i n time.

TIME [ min 1

F i g . 5. Batch redox p o t e n t i a l decrease i n time.

F i g . 4. B a t c h a n a e r o b i c p h o s p h o r o u s r e l e a s e and a e r o b i c u p t a k e i n time.

I

a 1

7

F i g . 6. K i n e t i c parameters o f P release i n function o f specific s u b s t r a t e dose.

I

n

17 CODlN RATIO

[-I

F i g . 7 . Dependence o f P r e m o v a l e f f i c i e n c y on o r g a n i c s / n i t r o g e n ratio.

-

31

-

The i n d u s t r i a l p r o c e s s d e v e l o p e d a p p l i e s s u p p l e m e n t a r y s e p a r a t i o n o f a e r o b i c end s n o x i c - a n a e r o b i c

p r o c e s s s t e p s by a p e r i o d i -

t h u s e c o n o m i z i n g more o r g a n i c

cal aeration shut-off

t i m i n g program,

matter f o r P release.

That helped us t o maintain a h i g h P removal

e f f i c i e n c y down t o a COD/N Fig.

8.

r a t i o a s low a s 5 : l .

shows t h e s k e t c h o f a p r e f a b r i c a t e d p a c k a g e p l a n t .

A u n i q u e f e a t u r e o f t h e common a n o x i c a n d a n a e r o b i c s p a c e i s t h a t t h e r e l a t i v e s e t t l i n g v e l o c i t y o f t h e sludge i n t h e upflow stream p e r f o r m s a t h i c k e n i n g e f f e c t 2-3

t i m e s as h i g h s t s t r i c t l y e q u a l

i n - and o u t l e t s u s p e n d e d s o l i d c o n c e n t r a t i o n s .

T

u EXCESS SLUDGE

Fig.

8.Disgrammatic view o f t h e p r e f a b r i c a t e d equipment.

The s o l u t i o n r e q u i r e s ,

i n addition t o the i n t e n s i f i c a t i o n effect,

no m e c h a n i c a l s t i r r i n g a s i n any o t h e r known p r o c e s s e s ( 7 ) .

Fig.

9.

shows e q u i p m e n t l i k e t h i s made o f p l a s t i c . The p r o c e s s was r e a l i z e d o n l a r g e s c a l e b y t r a n s f o r m a t i o n o f a conventional activated sludge p l a n t

3

c i t y o f 600 m 3 t r e a t s 1000 m / d

(Fig.

10).

The t o t a l t a n k c a p a -

communal w a s t e w a t e r w i t h c o m p l e t e

n i t r i f i c a t i o n a t s l u d g e r e s i d e n c e t i m e a s l o w a s 6 d, f i c a t i o n e f f i c i e n c y and approx.

90

X

denitri-

0,3 g/m3 e f f l u e n t P c o n c e n t r a t i o n .

H i q h l y i n t e n s i v e d e n i t r i f i c a t i o n and n i t r i f i c a t i o n Although the t e c h n i c a l l y simple modifications o f the a c t i v a t e d . sludge process y i e l d h i g h l y valuable supplementary performances, s e a r c h h a s b e e n c o n d u c t e d f o r b a s i c a l l y new s o l u t i o n s .

re-

Mainly i n the

-

32

-

Fig. 10. A modified l a r g e scale a c t i v a t e d sludge plant. f i e l d o f d e n i t r i f i c a t i o n a n d n i t r i f i c a t i o n t w o t r e n d s seem t o a t t r a c t most i n t e r e s t :

n o v e l p r o c e s s p r i n c i p l e s i n m i c r o b i o l o g y and b i o c h e -

m i s t r y , a n d t h e u s e o f new b i o r e a c t o r c o n f i g u r a t i o n s . D e n i t r i f i e r s are mostly p r o c a r i o t e t a x o n s ; mainly h e t e r o t r o p h s . Among c h e m o l i t o t r o p h s t h e r e a r e s few d e n i t r i f i c a n t s ,

s u c h as T h i o -

b a c i l l u s , which o x i d i z e s u l p h u r , o r t h e p r o c e s s r e q u i r e s t h e u s e o f m o l e c u l a r H 2 a s e l e c t r o n d o n o r w i t h A l c a l i g e n e s e u t r o p h a ( 8 , 9 ) Mo d est p r o d u c t i v i t i e s ( 2 - t o 8 kg N03/m 3 d ) may b e a t t r i b u t e d t o h i g h e x i g e n c i e s t o w a r d s u b s t r a t e mass t r a n s f e r s .

The p r o c e s s e s ,

33

-

c o m b i n i n g t h e two c o n s e c u t i v e N r e m o v a l s t e p s ,

m i g h t o f f e r c o n s i d e r a b l e s d v a n t s g e s i f s p e c i a l c o n d i t i o n s were filled tion,

(i.e.

simultaneous methanogenic o r g a n i c s removal,

ful-

reduc-

SO4

w i t h T h i o s p h a e r a p a n t o t r o p h a ( 1 0 ) o r an a n o x i c ammonium

etc.)

oxidation process with s t i l l u n i d e n t i f i e d c u l t u r e (11).

As f o r w a s t e w a t e r n i t r i f i c a t i o n , a f t e r o v e r l o a d e d m u n i c i p a l plants,

c l a s s i c t r i c k l i n g f i l t e r s a r e r u n n i n g i n t h e US, a l t h o u g h 3 kgNH3/m d (14). R e a l l y

t h e i r p r o d u c t i v i t y h a r d l y exceeds 0,3-0,4

i n t e n s i v e f l u i d bed systems w i t h a h i g h e r c a p a c i t y e x h i b i t s e v e r a l disadvantages,

l i k e t h e h i g h p r i c e and s h o r t l i f e t i m e o f a c t i v a t e d

carbon support, abrasion,

or the excessive costs o f a pure O2 supply, b i o f i l m

p r o c e s s i n s t a b i l i t y w i t h 3-phase f l u i d i z a t i o n ,

etc.

On t h e b a s i s o f f l u i d i z e d b i o r e a c t o r c o n c e p t i o n and o p e r a t i o n e x p e r t i s e g a i n e d d u r i n g a decsde o f d e n i t r i f i c a t i o n r e s e a r c h ,

we

r e a l i z e d a h i g h l y s a f e and e c o n o m i c a l p o s t - n i t r i f i c a t i o n system. 3 W i t h a r o u g h s a n d s u p p o r t we r e a c h e d 2 kgNH3/m d a t a maximum o f

1 5 g/kg

s u p p o r t biomsss c o n c e n t r a t i o n .

n a t i o n system assures, mass t r a n s f e r

The s p e c i a l f r e e a i r o x y g e -

on t h e p i l o t s c a l e ,

coefficients,

about 3 times as h i g h

as a t t a i n e d i n b u b b l e columns (31,

duced r e c i r c u l a t i o n r a t e s and v e r y s i m p l e c o n s t r u c t i o n . method, o v e r l o s d e d n o n - n i t r i f y i n g

p l a n t s r e q u i r e o n l y 10-20

m e n t a r y r e a c t o r volume compared t o 200-400

re-

With t h i s

X supple-

% necessary f o r t r a d i t i o -

n a l methods.

I n s p i t e o f numerous r e s e a r c h r e p o r t s on b i o l o q i c a l f l u i d bed denitrification,

p r a c t i c a l l y no i n d u s t r i a l r e a l i z a t i o n has been

a t t s i n e d so f a r .

I n d u s t r i a l scale d e n i t r i f i c a t i o n plants are b u i l t

on t h e b a s i s o f a f i x e d b e d r e a c t o r d e s i g n ( 1 2 1 , 3 mum p r o d u c t i v i t y t o a b o u t 3 k g N O 3 - N / m d ( 1 3 ) . C o n s i d e r i n g t h e above,

l i m i t i n g t h e maxi-

we t h i n k s p e c i a l d i f f i c u l t i e s may b e

associated with:

-

-

s a f e a n d u n i f o r m m i c r o b i a l a d h e s i o n on s u p p o r t f o r m a t i o n o f a dense b i o f i l m s t r u c t u r e , non-denitrifying contsminetion,

t e c h n i c a l l y f e a s i b l e bed r e g e n e r a t i o n p r o c e d u r e , f o r m removal o f excess biomass.

Therefore,

As f o r effects

surface,

p o s s i b l y f r e e from

our research e f f o r t s the physico-chemical

uni-

h a v e b e e n f o c u s e d on t h e s e p r o b l e m s . aspect,

we t h o r o u g h l y s t u d i e d t h e

o f support surface characteristics

t o h y d r a u l i c shear,

i.e.

and m i c r o b i a l r e s i s t a n c e

and e l a b o r a t e d a n a p p r o p r i a t e p r e t r e a t m e n t met-

hod f o r t h e sand c a r r i e r p a r t i c l e s ( p s t e n t e d ) .

I n principle,

-

34

v a r i o u s o r g a n i c compounds c a n be a p p l i e d a s e l e c -

t r o n donors f o r d e n i t r i f i c a t i o n .

Accordingly,

s e l e c t i o n seems t o b e

governed b y economic and p u b l i c h e a l t h c o n s i d e r a t i o n s . out,

however,

a t an e a r l y s t a g e o f t h e e x p e r i m e n t s ,

I t turned

that organic

s u b s t a n c e s h a v e a s i g n i f i c a n t e f f e c t on b i o f i l m s t r u c t u r e , quently,

conse-

a l s o on f l u i d b e d e x p a n s i o n we f i n a l l y c h o s e Pseudomonas

d e n i t r i f i c a n s as an i n o c u l a t i n g c u l t u r e .

11. P r e s u m a b l y ,

the wider

This i s presented i n Fig.

t h e r a n g e o f b a c t e r i a c a p a b l e o f metabo-

l i z i n g t h e o r g a n i c compound,

the looser i s the structure o f the bio-

film. Fig.

12.

O

shows an e l e c t r o n m i c r o g r a p h o f a b i o c o a t e d p a r t i c l e .

lo

30

Lo

5o

~xhrrn95slgl

F i g . 11. Bed e x p a n s i o n a t d i f f e r e n t C sources, i n f u n c t i o n o f biomass c o n c e n t r a t i o n .

F i g . 12. E l e c t r o n m i c r o g r a p h o f a biocoated particle.

F o r k i n e t i c s t u d i e s we f o l l o w e d tion profile.

Fig.

13.

the a x i a l n i t r a t e concentra-

m o s t l y shows l i n e a r d i s t r i b u t i o n s ,

which

means t h a t t h e r e i s n o d i f f u s i o n l i m i t a t i o n o f t h e i n t r i n s i c z e r o o r d e r r e a c t i o n , as a l s o s t a t e d by Harremoes ( 1 5 ) . r a m e t e r s and c a l c u l a t e d v o l u m e t r i c

E n v i r o n m e n t a l pa-

( r N ) a n d s p e c i f i c (pN) d e n i t r i -

f i c e t i o n r a t e s are also given.

A b a s i c requirement f o r d r i n k i n g water d e n i t r i f i c a t i o n i s t o minimize e f f l u e n t organic matter flux, e x a c t dosage o f t h e C source.

C and N b a l a n c e i n t h e p r o c e s s ,

t h e C/N

w h i c h c a n b e p r o v i d e d b y an

For t h i s purpose,

we e s t a b l i s h e d

full

i n o r d e r t o s t u d y t h e dependence o f

r a t i o on p r o c e s s p a r a m e t e r s .

Fig.

14.

shows t h e e f f e c t o f X

biomass c o n c e n t r a t i o n s ,

i.e.

35

age.

-

As e x c e s s C 0 2 i s g e n e r a t e d f r o m

endogeneous o x i d a t i o n o f t h e b i o m a s s ,

t h e C s o u r c e c a n be " s a v e d "

a t higher X levels.

-E

P)

"

'h 120 -

am

uf = 28 mlh t = 14 ' C CSOUKES:PROP~ONC ACID

ImgKs/gl Icml 1 2Q +

3.4 4 5 6m

*

60

-

PN

V '

25,6 26.2 325 37.7 46.2 66.6

131 141 147 158 176 198

0.021 0,022 0,023 0,026 0,026 0,017

16,O 17.2 18,6 20.4 228 31.3

40.6 41,2 458

525 63.6 581

Lcl-

20

0 10 Fig.

13.

-

'[

0 1.3

40

55

77

85

100 115 I30 145 160

OF BED

hf

1.1' 10

N i t r a t e c o n c e n t r a t i o n p r o f i l e s i n t h e f l u i d i z e d bed.

20

I

I

30

40

14. Carbon/nitrogen

After satisfactory

50 xh[mg/gl r a t i o a t d i f f e r e n t biomass c o n c e n t r a t i o n s .

laboratory experience,

a f u l l y automated p i -

l o t p l a n t f o r d r i n k i n g w a t e r was b u i l t and t e s t e d .

3

p h o t o o f t h e 50 m /d

Fig.

1 5 shows t h e

c a p a c i t y equipment.

S i n c e d r i n k i n g w a t e r q u a l i t y had t o be a t t a i n e d ,

the reactor

was c o m p l e t e d w i t h e f f l u e n t a f t e r t r e a t m e n t f a c i l i t i e s , Fig.

[cml

REKTRON DONOR: PROPONIC Q + ETANOL OINF. Sw,o = CONSTANT

a

Fig.

25

as shown i n

16. S p e c i f i c n i t r a t e r e m o v a l d a t a were measured

-

i n order o f higher

-

36

-

magnitude.

The r e s u l t s a r e g i v e n i n F i g .

17.

e t two d i f f e r e n t tem-

peratures,

a s a f u n c t i o n o f h f b e d h e i g h t ( r e a c t o r d i a m e t e r was

300 m m ) .

F i g 15. F u l l y automated p i l o t s c a l e d r i n k i n g w a t e r d e n i t r i f i c a t i o n . plant.

1. FLUID-BED DENlTRlFlCATlON COLUMN 2.FILTER CARTRIDGE 3.AIRING AND N2 STRIPPING COLUMN 4 . SAND FILTER 5.ACTlVE -CARBON FILTER

6. SAND REGENERATOR 7. CHEMICAL FEEDER Fig.

(C AND P I

16. Flow sheet o f f l u i d i z e d bed d e n i t r i f i c e t i o n p l a n t .

-

37

-

A t a p p r o p r i a t e a d j u s t m e n t o f t h e C/N i n c r e a s e a o n l y by 1-1,2

g/m

3

.A

ratio,

t h e e f f l u e n t TOC

v e r y l o w l e v e l o f suspended s o l i d s

d i s c h a r g e c a n b e f u l l y r e t a i n e d on t h e s a n d f i l t e r . e n t l i v i n g c e l l c o u n t o f 5.103-5.10

4

Typical efflu-

can be e l i m i n a t e d by a d d i t i o n

o f 3 mg c h l o r i n e . T o t a l o p e r a t i n g c o s t s o f t h e d r i n k i n g w a t e r p l a n t were c a l c u l a t e d t o be r o u g h l y mose p r o c e s s e s ,

1/10

mental p o l l u t i o n aspects. about 3-6

o f c o n c u r r i n g i o n e x c h a n g e a n d r e v e r s e os-

n o t t o mention c r u c i a l advantages r e g a r d i n g e n v i r o n The r e g e n e r a t e d e x c e s s b i o m a s s r e p r e s e n t s

X o f t h e t r e a t e d w a t e r ( w i t h max. 30 kgVSS/m3 b e i n g abso-

l u t e l y h a r m l e s s and r e a d i l y d i s p o s a b l e m a t e r i a l ) .

-

110. U m E I r n 100 -

-

0

z

9080 -

/'

60

HIGH OF BED hf[cml F i g . 17. height.

V o l u m e t r i c d e n i t r i f i c a t i o n r a t e s i n f u n c t i o n o f t h e bed

The p i l o t p l a n t r e a c t o r t o g e t h e r w i t h a n o v e l r e g e n e r a t i o n s y s t e m was a l s o t e s t e d f o r w a s t e w a t e r .

A n i t r i f y i n g activated slud-

ge m u n i c i p a l sewage t r e a t m e n t p l a n t s u p p l i e d i t s e f f l u e n t as r e a c t o r feed (Fig.

18).

After a f a i r l y short start-up period,

3

the r e a c t o r reached a

8 5 m / d c a p a c i t y w h i l e t h e i n i t i a l 8 5 - 1 0 0 g/m3 was r e d u c e d p r a c t i c a l l y t o z e r o . very low r e s i d u a l organic matter,

n i t r a t e concentration

As t h e t r e a t e d sewage c o n t a i n e d

a 0,57

g methanol/gNO;

e l e c t r o n d o n o r d o s e was f o u n d t o b e an o p t i m a l s o l u t i o n , m i c a l aspects,

specific f r o m econo-

aa w e l l .

Anaerobic waste t r e a t m e n t

I n highly concentrated ( i n d u s t r i a l )

waste water,

severe l i m i -

t a t i o n s impede t h e a p p l i c a t i o n o f a e r o b i c p r o c e s s e s f o r o r g a n i c s

- 38 -

Fig.

18.

F l u i d i z e d b e d sewage d e n i t r i f i c a t i o n p l a n t .

removal (excessive oxygenation costs, sludge production,

diffusion limitations,

high

etc.).

P r o d u c t s o f t h e a n a e r o b i c d e g r a d a t i o n a r e e n e r g y r i c h compounds (biogas)

-

c o m p a r e d t o t h o s e o f t h e a e r o b i c p r o c e s s where o x y g e n i s

the terminal e l e c t r o n acceptor

i n s t e a d o f COz.

Such b a s i c b i o e n e r g e -

t i c a l differences manifest i n the o v e r a l l C balance,

i.e.

i n the

d i s t r i b u t i o n o f i n p u t C f l u x between c e l l s y n t h e s e s and gaseous p r o ducts:

50-50

pectively

X i n a e r o b i c and 5-95 % i n a n a e r o b i c p r o c e s s e s , r e s -

(16).

On t h e o t h e r h a n d ,

technical feasibility

o f the process requi-

r e s c o s t l y and s o p h i s t i c a t e d s o l u t i o n s t o e n s u r e h i g h enough r e a c t i o n rates,

-

e.g.:

h e a t e d and t h e r m a l l y

insulated reactors,

important increase o f X c e l l concentration, i . e . t o a v o i d wash-out from c o n t i n u o u s r e a c t o r s .

The l a t t e r c a n b e p r a c t i c a l l y e n s u r e d b y c e l l i m m o b i l i z a t i o n . p r o c e s s can o n l y be promoted o r c o n t r o l l e d i n d i r e c t l y , s t e r i l e mixed c u l t u r e c o n d i t i o n s ; factors.

i.e.

by i n f l u e n c i n g e n v i r o n m e n t a l

As t h i s j o b h a s b e e n c a r r i e d o u t o n l y e m p i r i c a l l y ,

a systematic study o f physico-chemical,

The

under nonwe made

p h y s i o l o g i c a l and e c o l o g i c a l

-

39

-

f a c t o r s a f f e c t i n g m i c r o b i a l a d h e s i o n and a g g r e g a t i o n .

The g r e a t e s t

e f f o r t s h a v e b e e n d e v o t e d t o d e v e l o p i n g o b j e c t i v e e x p e r i m e n t a l meth o d s . The s l u d g e g r a n u l a t i o n p r o c e s s w a s f o l l o w e d i n 5 p a r a l l e l UASB b i o r e a c t o r s o f e q u a l h y d r a u l i c s b u t ,

f r o m No.1.

t o 5.,

reduced

o r g a n i c a c i d l o a d s ( F i g . 19.).

1 9 . 5 p a r a l l e l l a b o r a t o r y UASB r e a c t o r s .

C a p i l ary s u c t i o n t i m e ( C S T )

is a rapid f i l t e r a b i l i t y test

i f y i n g a g g r e g a t i o n a g a i n s t time ( F i g . 20.). c

100

? ; E U A !

61,

80

LO

/;A00

20

I

0

I

I

20

LO

60

80

I

100 120

140

TIME [ d 1

F i g . 20. C a p i l l a r y s u c t i o n time e v o l u t i o n a t d i f f e r e n t o r g a n i c loads of the 5 reactors.

-

40

With dynamic f r a c t i o n i n g sedimen-

- 100 E

s

+

tation,

particle size distribu-

t i o n can be d e s c r i b e d w i t h m i c -

80 60

roscopic calibration,

LO

The l o w e s t

g r a p h shows t h i s i n t h e c a s e o f

20

a non-granulated

0

3

0 -

inoculum.

The u p p e r a n d m i d d l e o n e s a r e made o f s m a l l and h i g h l o a d g r a -

I

60

3 40

nulated sludges (Fig.

g

5 m/h u p f l o w v e l o c i t y ,

20

n

7

3

A t

21).

8 8 and

5 % o f t h e t o t a l s l u d g e mass we-

50 100 80 60

r e washed o u t ,

a t raw and g r a n u -

l a t e d sludges,

respectively (17).

On t h e s c a n n i n g e l e c t r o m i c -

40 20

rographs (Fig.

22. a n d 2 3 . ) t h e

7

0

granule forming r o l e o f filamen-

10 20 30 LO 50 UPFLOW VELOCITY [mlhl

t o u s o r g a n i s m s ( p r o b a b l y Methanot r i x sp.)

F i g . 21. S e t t l i n g v e l o c i t y d i s t r i b u t i o n o f raw ( l o w e r ) and g r a n u l a t e d ( u p p e r and m i d d l e ) sludges.

c a n b e seen.

As g r a n u l a t i o n i s m a i n l y a

m i c r o b i a l s e l e c t i o n process, F420 c o f a c t o r

sity)

level

-

the

(fluorescence inten-

w h i c h i s n o t o n l y c h a r a c t e r i s t i c o f methanogens b u t

a l s o s e r v e s a s a means f o r d i f f e r e n t i a t i n g M e t h a n o t r i x and M e t h a n o -

-

s a r c i n a type organisms system,

as seen i n F i g .

d e s c r i b e s q u i t e w e l l t h e change i n t h e eco24.

As t h e o r g a n i s m s a l s o e x h i b i t d i f f e r e n t

(Ks h a l f - r a t e ,

k i n e t i c parameters

i n h i b i t i o n c o n s t a n t s and n e x p o n e n t ) ,

Ki

a disconti-

nuous methanogenic a c t i v i t y t e s t h e l p e d t o f u r t h e r q u a n t i f y t h e d i s tinctions,

as s e e n i n F i g .

25.

With t h e m o d i f i e d Haldane k i n e t i c s (18) evolution)

r a t e s determined: q

--

1

1 + Ks/S

qmax

+ (S/KiIn

gave m a r k e d l y d i f f e r e n t c o n s t a n t s ( K s a n d 4,38 resp. )

s p e c i f i c growth (gas

kg/m3;

n

=

4,08

and 1,0

=

0,125

and 0,036;

Ki

=

2,Ol

f o r raw and g r a n u l a t e d s l u d g e s ,

.

I n a d d i t i o n t o proving the reduced i n h i b i t i o n s e n s i t i v i t y o f the granulated sludge,

the f i n d i n g s helped us t o conceive a novel

start-up strategy (19).

41

-

I t s most important f e a t u r e i s t h e substan-

t i a l l y higher i n i t i a l organic load, a s reported before.

F i g . 23. Filamentous structure o f a granule.

-

42

TIME [dl F i g . 24. F 4 2 0 c o f a c t o r c o n t e n t evolution a t d i f f e r e n t organic loads o f the 5 reactors.

-

SUBSTRATE CONCENTRATlONIgCm31 F i g . 25. D i f f e r e n t s u b s t r a t e i n h i b i t i o n e f f e c t on r a w a n d granulated sludges.

A t a UASB-fixed bed h y b r i d e r e a c t o r c o n f i g u r a t i o n ( F i g .

26.)

b i o f i l m e v a l u a t i o n waa a l s o q u a l i t a t i v e l y f o l l o w e d b y t h e p r o p o r t i o n o f a c i d o g e n e a n d m e t h a n o g e n e o r g a n i s m s e x p r e s s e d by t h e e f f l u ent/influent (Fig.

27.).

6 kg/m3

organic a c i d r a t i o evolution,

as a f u n c t i o n o f t i m e

The s t e r i c c o n c e n t r a t i o n o f t h e f i x e d b i o f i l m g r e w t o

(see F i g .

28.1,

b u t f l o a t i n g sludge r e t e n t i o n o f the c a r r i e r

seemed t o b e a t l e a s t a s i m p o r t a n t .

TIME [ d l F i g . 26. U A S B de r e a c t o r

-

f i x e d bed h y b r i -

F i g . 27. E v o l u t i o n o f t h e m e t h a n o g e n / a c i d i f y i n g a c t i v i t y i n t h e fixed b e d p a r t o f t h e h y b r i d e r e a c t o r

As a p r a c t i c a l a p p l i c a t i o n ,

43

-

we s t u d i e d m a i n l y t h e t r e a t m e n t

o f p o l y s a c c h a r i d e c o n t a i n i n g i n d u s t r i a l waste w a t e r s

tillery,

pharmaceutical industries).

(canning,

dis-

With t h e e x c e p t i o n o f c a n n i n g ,

h i g h s u l p h a t e c a u s e s s e r i o u s p r o b l e m s by t h e i n h i b i t i o r o f m e t h a n o g e n e s i s . We f o u n d t h a t t h e r a t e o f s u l p h a t e r e d u c t i o n is much mo-

r e f a v o u r a b l e under c o n d i t i o n s o f t h e a c i d i c phase. T o x i c and c o r r o s i v e e f f e c t s o f t h e r e s u l t i n g H 2 S c a n b e m i n i m i z e d by i n e r t g a s s t r i p p i n g and c h e m i c a l a b s o r p t i o n ( 2 0 ) .

Fig.

28. B i o f i l m coated p l a s t i c c a r r i e r .

F u r f u r a l p r o d u c t i o n from p l a n t pentosanes i n v o l v e s t h e format i o n o f waste water

c o n t a i n i n g up t o 3 0 kg/m3

acetic acid.

The t o -

x i c e f f e c t s o f 0,6-1,4

kg/m3

t i o n o f l o w c o s t waste

( a c i d i f i e d p i g manure) a s s u r i n g a l s o enough

N,

P and g r o w t h f a c t o r l e v e l .

f u r f u r a l were e l i m i n a t e d b y t h e a d d i -

A h i g h l y economical i n s i t u n e u t r a l i -

z a t i o n p r o c e d u r e was e l a b o r a t e d , lation

-

NaOH a d d i t i o n

-

applying a combination o f recircu-

and Cog s t r i p p i n g ( Z l . ,

22.

As a r e s u l t o f r e s e a r c h c o n d u c t e d o v e r a d e c a d e ,

23). we e l a b o r a t e d

a n d t e s t e d on p i l o t p l a n t s c a l e (24,251 a c o m p l e t e p i g manure t r e a t 3 ment s y s t e m ( 1 5 m , F i g . 2 9 , 3 0 1 . T h i s c o n s i s t s o f a n a n a e r o b i c t r e atment o f 6 - 9 d r e s i d e n c e t i m e ,

mechanical s o l i d - l i q u i d separation

h e l p e d by t h e r m a l a n d c h e m i c a l e f f e c t s .

NH3 d i s t i l l a t i o n a n d t e r t i a -

r y b i o l o g i c a l t r e a t m e n t f o r r e s i d u a l o r g a n i c s a n d N,

P r e m o v a l was

-

Fig.

29-30.

44

-

P i l o t p l a n t p i g manure t r e a t m e n t p l a n t .

c o m p l e t e d i n o r d e r t o meet s u r f a c e w a t e r q u a l i t y r e q u i r e m e n t s .

In-

d i v i d u a l l y o p t i m i z e d process s t e p s a r e o r g a n i z e d so as t o m u t u a l l y support e f f i c i e n c y . tion,

S p e c i a l a t t e n t i o n i s devoted t o avoid a i r p o l l u -

t o a t t a i n v a l u a b l e p r o d u c t s (NH3

tilizer)

solution,

concentrated fer-

and t o combine p r o c e s s e s b y a most e c o n o m i c a l e n e r g y s a v i n g

method ( 2 6 ) . An application o f increasing interest

senohold wastes,

i s extended t o s o l i d hou-

m a i n l y i n EC c o u n t r i e s (27).

Classic low i n t e n s i t y

c o v e r e d d e p o s i t s a r e s u b s t i t u t e d by m o d e r n h e a t e d r e a c t o r s e n s u r i n g c o n s i d e r a b l e volume r e d u c t i o n and gas y i e l d ,

with reduced residence

times (28).

REFERENCES Healey: Improvements i n t h e a c t i v a t e d sludge process i n t h e U.K. a n d U.S., J o u r n a l WPCF., V.61, No.4, 4 4 7 - 4 5 1 ( 1 9 8 9 ) H. B u r k h a l t e r ; E r g e b n i s a e v o n S a u e r s t o f f z u f u h r v e r s u c h e n i n A l t e h e i n . I n : W i e n e r M i t t e i l u n g e n . Wasser Abwasser-Gewasser B d 6 4 . ,

M.J.

69-84, Wien, 1 9 8 6 . J. H o l l b , P. M i h d l t z , L . Czak6 a n d J . T b t h : C o r r e l a t i o n o f liq i u d d i s p e r s i o n and oxygen t r a n a f e r i n b u b b l e column, s p p l . M i c r o b i a l B i o t e c h n o l . 27:260-264 (1987). H. K r o i a s , Neue B e m e s s u n g s v o r s c h r i f t e n b e i B e l e b u n g s a n l a g e n h i n s i c h t l i c h S t i c k s t o f f - und P h o s p h o r e n t f e r n u n g i n Proc. UTEC ' 8 9 , 3 2 8 - 3 3 4 , T r e n d Commerz GmbH, L i n r , 1 9 8 9 . L . Czakb, P. M i h d l t z a n d J. Tbth, P r o c e s s a n d e q u i p m e n t f o r b i o l . t r e a t m e n t o f o r g a n i c c o n t a i n i n g , s p e c i f i c a l l y f o r communal wast e w a t e r . Hung. P a t e n t , 1 8 8 5 0 2 , 1 9 8 5 . Y . Comeau e t a l . , B i o c h e m i c a l m o d e l f o r e n h a n c e d b i o l o g i c a l p h o s p h o r u s r e m o v a l w a t e r r e s . 20, 1 2 , 1 5 1 1 - 1 5 2 1 , 1 9 8 6 .

7 8 9 10

11 12 13 14 15 16

17

18 19 20

21

22

23 24

25 26 27 28

45

-

J. P i n n e k a m p , G r u n d l a g e n , V e r f a h r e n u. L e i s t u n g s f a h i g k e i t d e r Erhbhten biologischen Phosphorelimination, Abwassertechnik 39, 4 , 2 1 - 2 6 , 1 9 8 8 . G. B l e c o n e t e l . , P r o c B d 6 de d e n i t r i f i c a t i o n b i o l o g i q u e a u t o t r o p h e p a r T h i o b a c i l l u s d e n i t r i f i c a n s s u r s o u f r e - m a e r l , Revue f r a n c a i s e d e s S c i e n c e s de l ' e a u , 2, 2 6 7 - 2 7 9 , 1 9 8 3 . C. Reisinger; Die b i o l o g i s c h e Trinkwasseraufbereitung i n F l i e s s b e t t r e a k t o r e n - k y b e r n e t i s c h e und t e c h n i s c h e Aspekte Proc. U T E C ' 8 9 , 1 9 3 - 1 9 7 , T r e n d Commerz GmbH, L i n z , 1989. C . M . H o o i j m a n s e t a l . , N i t r i f i c a t i o n - d e n i t r i f i c a t i o n by i m m o b i l i z e d Thiosphaera p a n t o t r o p h a i n r e l a t i o n t o oxygen p r o f i l e s . To b e p r e s e n t e d a t ECB 5, Coppenhague, 1 9 9 0 . A.A. v a n de G r a a f e t a l . , A n o x i c ammonium o x i d a t i o n . To b e p r e s e n t e d a t ECB 5 , Coppenhague, 1 9 9 0 . G . l e Puang: N i t r a t e s , a t t e n t i o n , d a n g e r , Le n o u v e l B c o n o m i s t e -1, 158-162, 1 9 8 9 . W . Kopp, B i o l o g i s c h e V e r f a h r e n z u r N i t r a t e n t f e r n u n g , W i s s e n s c h a f t u n d Umwelt 1. 2 3 - 3 1 . 1 9 8 7 . R.W. Okey a n d O.E. A l b e r t s o n : D i f f u s i o n ' s r o l e i n r e g u l a t i n g r a t e e n d mask n g t e m p e r a t u r e e f f e c t s i n f i x e d f i l m n i t r i f i c a t i o n , J o u r n a l WPCF, 61, 4 , 5 0 0 - 5 0 9 , 1 9 8 9 . P. H a r r e m o e s , The s i g n i f i c a n c e o f p o r e d i f f u s i o n t o f i l t e r den i t r i f i c a t i o n J o u r n a l WPCF, 4 8 , 3 7 7 - 3 8 1 , 1976. H. Sahm, Adv. i n B i o c h e m . E n g r g / B i o t e c h n o l . , 29, 84, E d . : A . F i e c h t e r , S p r n g e r - V e r l a g B e r l i n , H e i d e l b e r g , New Y o r k , T o k y o 1984. L . M o r v a i , P. M i h B l t z and L . Czakb: P a r t i c l e - s i z e d i s t r i b u t i o n o f a n a e r o b i c g r a n u l a r s l u d g e . P r o c . PORANAL ' 8 9 , Symp. P a r t i c l e S i z e A n a l . , Powder T e c h n o l . , 2 0 7 - 2 1 8 , Szeged, H u n g a r y , 1 9 8 9 . Y.C. Wu e t a l . , B i o t e c h n o l . B i o e n g . , 31, 2 5 7 , 1 9 8 8 . L. M o r v a i , P . M i h B l t z , L . Czakb, M. P B t e r f y a n d J. H o l l L : The i n f l u e n c e o f o r g a n i c l o a d on g r a n u l a r s l u d g e d e v e l o p m e n t i n a c e t a t e f e d system, Accepted f o r A p p l . M i c r o b i o l . B i o t e c h n o l . 1990. L . C z a k 6 , P. M i h 6 l t z a n d L . M o r v a i : B i o l . s u l p h a t e r e m o v a l i n t h e a c i d i c p h a s e o f a n a e r o b i c d i g e s t i o n , P r o c . 5 t h I n t . Symp. on A n a e r o . D i g e s t i o n , 833-837, B o l o g n a M o n d u z z i E d i t o r e , 1 9 8 8 . L . C z a k b , P . M i h B l t z , L . M o r v a i a n d J. H o l l b : D e v e l o p m e n t o f new n u t r a l i s a t i o n p r o c e d u r e o f h i g h l y a c i d i c w a s t e w a t e r s d u r i n g anaer. t r e a t m e n t . Proc. 4 t h Eur. Congr. B i o t e c h n o l . V I , 6 9 - 7 1 , E l s e v i e r , Amsterdam, 1 9 8 7 . L . Czakb, T . Cseh, P . M i h B l t z a n d J. T 6 t h : P r o c e s s f o r a n a e r o b i c b i o l . t r e a t m e n t o f t o x i c waste w a t e r s c o n t a i n i n g a c e t i c a c i d a n d f u r f u r a l , 1 9 6 . 0 4 3 . Hung. p a t e n t , 1 9 8 8 . As a b o v e . DE 3617 686 A 1 West German P a t e n t , 1 9 8 7 . T . Cseh, L. Czakb, J. T b t h a n d R . P . T e n g e r d y : Two-phase a n a e r o b i c f e r m e n t a t i o n o f l i q u i d s w i n e waste methane. B i o t e c h n o l . B i o e n g . 26, 1 4 2 5 - 1 4 2 9 , 1 9 8 4 . T. Cseh, L . Czakb, P. M i h B l t z a n d J. H o l l 6 : I n v e s t i g a t i o n b i o g a s v e r s u s y e a s t S C P p r o d u c t i o n f r o m p i g manure. P r o c . I n t . Symp. B i o t e c h n o l . Food I n d u s t r y , 617-623, Budapest, 1988. L . C z a k b , T . Cseh, P. M i h B l t z , J. G y b r y a n d F. N y i t r a i : P r o c e s s and equipment f o r two-phase a n a e r o b i c t r e a t m e n t o f s o l i d s cont a i n i n g w a s t e s , 200308 Hung. P a t e n t , 1 9 8 9 . F . C e c c h i e t a l . A n a n e r . d i g e s t i o n o f o r g a n i c f r a c t i o n o f munic i p a l s o l i d w a s t e s d i g e s t e r p e r f o r m a n c e , The s c i e n c e o f t h e t o t a l e n v i r o n m e n t , 56, 183-197, 1986. W . Stegmann: A n a e r o b i c B e h a n d l u n g v o n A b f a l l e n - Neue V e r f a h r e n , P r o c . UTEC ' 8 9 , 1 6 9 - 1 7 4 , T r e n d Commerz. GmbH V e r l a g , L i n z , 1 9 8 9 .

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A N A E R O B I C T R E A T M E N T OF EFFLUENT FROM E P O X Y R E S I N S P R O D U C T I O N U N I T S

P.

SAN N A ,

D ' A D D A R I O and A .

E.

E n i r i c e r c h e S.p.A

v i a E.

ROBERTIELLO

Ramarini,

32

-

M o n t e r o t o n d o (Roma)

-

ITALY

INTRODUCTION Anaerobic d i g e s t i o n has found a d e f i n i t i v e p l a c e i n w e s t e r n c o u n t r i e s a f t e r t h e 1973 e n e r g e t i c c r i s i s . T h i s t e c h n i q u e had been g e n e r a l l y r e g a r d e d as k i n e t i c a l l y s l o w and o n l y a p p l i c a b l e t o t h e d e c o n t a m i n a t i o n o f h i g h l y p o l l u t e d , inhibitory,

low

non t o x i c wastewater.

Therefore,

through the years,

i t h a s h a d an i n c r e a s i n g d i f f u -

s i o n i n t r e a t i n g a g r i c u l t u r a l and a g r o - i n d u s t r i a l I n t h e s e cases,

effluents.

t h e a n a e r o b i c t e c h n i q u e i s v e r y o f t e n more ad-

v a n t a g e o u s t h a n t h e a e r o b i c one, i s more f a v o r a b l e e c o n o m i c a l l y

and f o r l a r g e s c a l e a p p l i c a t i o n s ,

(1, 2 ) .

I n the present world energetic-economical

context,

the biogas

r e c o v e r y v e r y o f t e n p l a y s a m a r g i n a l r o l e when compared t o o t h e r a d v a n t a g e s o f f e r e d by a n a e r o b i c p r o c e s s e s ,

namely:

1. t r e a t m e n t o f h i g h p o l l u t e d w a s t e w a t e r ,

2.

low sludge production,

3- s m a l l o p e r a t i v e c o s t s . F o r t h e s e reasons huge r e s e a r c h e f f o r t s a r e p r e s e n t l y i n p r o gress t o enlarge the a p p l i c a t i o n o f the anaerobic d i g e s t i o n t o t h e t r e a t m e n t o f wastewater c o n t a i n i n g i n h i b i t o r y , l e s o r h a v i n g l o w o r g a n i c l o a d . F o r example,

r e c a l c i t r a n t molecu-

t h i s i s t h e case o f i n -

d u s t r i a l e f f l u e n t s and o f m u n i c i p a l wastewater. I n the f i r s t a p p l i c a t i o n chemical,

b i o c h e m i c a l and m i c r o b i o l o -

g i c a l p r o b l e m s must be f a c e d ( 3 1 , w h i l e i n t h e second,

r e s e a r c h ac-

t i v i t i e s on r e a c t o r i s t i c a n d e n g i n e e r i n g s u b j e c t s a p p e a r t o p l a y a more p r o m i n e n t r o l e . As an e x a m p l e o f t h e e x t e n t i o n o f t h e a n a e r o b i c t e c h n i q u e t o t h e treatment

o f i n d u s t r i a l wastewater,

anaerobic process for

t h i s w o r k a i m s t o show a new

t h e p u r i f i c a t i o n o f e f f l u e n t s o r i g i n a t i n g from

t h e epoxy r e s i n s p r o d u c t i o n .

-

48

-

O V E R V I E W AND W A S T E W A T E R C H A R A C T E R I S T I C S The e p o x y r e s i n s a r e p r o d u c e d f r o m t h e s y n t h e s i s b e t w e e n e p y s u c h as 4 , 4 ’

c h l o r o h y d r i n and d i f f e r e n t monomers, diphenol (bisphenol A),

4,4’-isopropylidene-bis

-

isopropylidene-

(tetrabromophenol).

The i n d u s t r i a l p r o c e s s e s a r e c h a r a c t e r i z e d b y o p e r a t i o n s c o n c e r n i n g b o t h t h e s y n t h e s i s and p u r i f i c a t i o n o f t h e raw p r o d u c t . Most o f t h e s e p r o c e s s e s o p e r a t e b a t c h w i s e . The e f f l u e n t o r i g i n a t i n g f r o m t h e s y n t h e s i s r e a c t i o n c o n t a i n s m o s t o f t h e p o l l u t i n g compounds, e n t COD) a n d f o r t h i s r e a s o n ,

(approx.

85 % o f t h e t o t a l e f f l u -

i n t h e p r e s e n t work o n l y t h e e f f l u e n t

was e x p e r i m e n t e d . Wastewater from t h e epoxy r e s i n s s y n t h e s i s c o n t a i n s :

1, b i o d e g r a d a b l e m o l e c u l e s ( e s s e n t i a l l y g l y c e r o l ) , 2.

b a c t e r i o s t s t i c - b a c t e r i c i d e compounds ( o r g a n i c s o l v e n t s a s i s o p r o p y l a l c o h o l and a c e t o n ,

sodium c h l o r i d e ,

etc.)

and,

3 . t o x i c m o l e c u l e s ( e p y c h l o r o h y d r i n and d e r i v a t i v e s o b t a i n e d from i t s p a r t i a l h y d r o l y s i s ) . The e f f l u e n t u s e d i n t h e e x p e r i m e n t s a n d d e s c r i b e d i n t h i s w o r k o r i g i n a t e s f r o m t h e s y n t h e s i s between e p y c h l o r o h y d r i n and b i s p h e no1 A.

I t h a s t h e c h a r a c t e r i s t i c s s u m m a r i z e d i n T a b l e 1. TABLE 1 C h a r a c t e r i s t i c s o f wastewater o r i g i n a t i n g f r o m epoxy r e s i n s p r o d u c t i o n ( d i s c o n t i n u o u s u n i t ; s y n t h e s i s : s t r e a m s 1-3; p u r i f i c a t i o n : s t r e a m s 4-6, n o t t e s t e d ) .

ECH

IPA

GLY

CHL

[%I

[x 1

L% 1

0.44

1.62

1.05

0.34

0.74

1.92

1.40

0.38

0.68

2.20

-

-

ix]

28.9 7.8 6

-

ECH:

Epichlorohydrin;

IPA:

Isopropyl alcohol;

CHL:

1.62

I

I I

0.12

-

Chloridrine;

TOL:

Toluene;

[!?;]

0.22

I

-

1

0.02

I I

-

GLYC:

GLY:

0.08

Glycerol

Glicidol

The numerous a p p r o a c h e s t o t h e r e c o v e r y o f t h e m a i n p r o d u c t c o n t a i n e d i n t h e “epoxy”

w a s t e w a t e r are i n d i c a t e d i n t h e c u r r e n t

literature.

Among t h e s e ,

49

-

those r e l a t e d t o g l y c e r o l recovery

(4,

5)

and t h o s e c o n c e r n i n g t h e sodium c h l o r i d e r e m o v a l ( 6 ) a r ? mentioned. M e t h o d s b a s e d on t h e u s e o f t h e a n a e r o b i c p r o c e s s a r e a l s o d e s c r i bed.

-

These m e t h o d s c o n s i d e r t-he f o l l o w i n g m a i n s t e p s ( 7 ) :

Pretreatment,

c a r r i e d o u t i n o r d e r t o remove r e s i n o u s r e s i d u a l

m a t e r i a l s and r e f r a c t o r y o r n o n - b i o x i d a b l e m o l e c u l e s ;

-

Strong d i l u t i o n with low p o l l u t i n g effluent;

-

Biological oxidation. T h i s a p p r o a c h a l l o w s good p e r f o r m a n c e s (81, b u t o f f e r s t h e

t y p i c a l disadvantages o f the aerobic treatment ( h i g h production o f sludge,

h i g h o p e r a t i v e c o s t s ) and,

quantities o f diluted effluents.

i n addition,

requires large

I n o r d e r t o overcome t h e s e draw-

b a c k s a new a l t e r n a t i v e p r o c e s s b a s e d on t h e a n a e r o b i c d i g e s t i o n was s t u d i e d ( 9 ) . I n t h e f o l l o w i n g ,

t h e main e x p e r i m e n t s p e r f o r m e d

i n order t o develop t h i s process are described,

and t h e p r o c e s s

i t s e l f i s showed and d i s c u s s e d .

EXPERIMENT The maximum c o n c e n t r a t i o n s o f e a c h t o x i c o r i n h i b i t o r y comp o u n d t o l e r a b l e b y a t y p i c a l a n a e r o b i c i n o c u l u m ( d i g e s t e d p i g manure;

T S 4,5

I;V S 3 , 5 %;

micro-reactors d i t i o n s (35

(Fig.

COO

4,8 g / l ) have been d e t e r m i n a t e d u s i n g

1, 6 0 m l t o t a l v o l u m e ) u n d e r m e s o p h i l i c c o n -

OC).

@ WATER BATH OMAGNETIC STIRRER @MICROREACTOR @ CYLINDER (GAS MEASURMENT) 0BOTTLE (WATER -SODIUM CHLORIDE, 10°/o 1 Fig.

1. M i c r o r e a c t o r s a r r a n g e m e n t . The e f f e c t o f e a c h o f t h e s e compounds on t h e a n a e r o b i c m i c r o

f l o r a was o b s e r v e d by a d d i n g t h e m d i r e c t l y t o t h e i n o c u l u m ,

as w e l l

as by u s i n g them i n m i x t u r e w i t h a p r o p e r s y n t h e t i c medium.

The

strongly t o x i c organo-chlorinated

50

-

compounds h a v e b e e n c o n v e r t e d t o

glycerol v i a alkaline h y d r o l i t i c reactions.

The h y d r o l y s i s c o n d i -

t i o n s have been s e l e c t e d s t u d y i n g t h e e f f e c t o f : t i o n (sodium hydroxide,

5-15

g/l),

a l k a l i concentra-

t e m p e r a t u r e (60-130

OC),

and t i -

me ( 5 - 6 h o u r s ) on t h e c h l o r i n a t e d p r o d u c t s r e s i d u a l c o n c e n t r a t i o n . The s o d i u m c h l o r i d e was r e m o v e d b y e v a p o r a t i o n / c r y s t a l l i z a t i o n , and by f i l t r a t i o n .

The p r e t r e a t e d e f f l u e n t ,

a f t e r a proper .dilution,

was d i g e s t e d u n d e r m e s o p h i l i c c o n d i t i o n s ( 3 5 filter

OC)

h a v i n g t h e f o l l o w i n g main c h a r a c t e r i s t i c s :

h e i g h t 2 m;

s o l i d support:

i n an a n a e r o b i c d i a m e t e r 1 0 cm;

small r i v e r stones (average diameter

5 cm). The a n a e r o b i c f i l t e r was i n o c u l a t e d w i t h t h e same e f f l u e n t u sed f o r t h e m i c r o - r e a c t o r s

tests.

I n order t o f a c i l i t a t e the support colonization,

the anaerobic

f i l t e r was s t a r t e d u s i n g a s y n t h e t i c medium s i m i l a r t o t h e one obtained a f t e r the pretreatment operations ( s p e c i f i c organic load 3 Kg C O D / m d ) . The a c c l i m a t i z a t i o n was c a r r i e d o u t s u b s t i t u t i n g

,

5-6

i n c r e a s i n g amounts o f t h e r e a l p r e t r e s t e d e f f l u e n t t o t h e s y n t h e t i c medium. Before the feeding, t h e wastewater.

p r o p e r amounts o f n u t r i e n t s were added t o

F o r t h i s purpose,

t e c h n i c a l g r a d e ammonium h y d r o x i -

d e a n d p h o s p o r i c a c i d were u s e d .

A t the beginning o f acclimatization,

t h e aame s p e c i f i c o r g a n i c

l o a d u s e d f o r t h e c o l o n i z a t i o n was m a i n t a i n e d . H o w e v e r ,

observed d u r i n g c o l o n i z a t i o n , t o a b o u t 4 KgCOD/m3,

d.

t h e s p e c i f i c o r g a n i c l o a d was r e d u c e d

After colonization,

i n order t o observe t h e

maximum p e r f o r m a n c e s o f t h e a n a e r o b i c f i l t e r , experiments

a t t h e end

i n o r d e r t o r e a c h t h e same l e v e l o f COD r e m o v a l

o f acclimatization,

6 months o f c o n t i n u o u s

a t i n c r e a s i n g f e e d i n g f l o w r a t e s were c a r r i e d o u t .

RESULTS A N D DISCUSSION The r e s u l t s o f t h e a n a e r o b i c d e g r a d a b i l i t y t e s t s o f e a c h comp o u n d c o n t a i n e d i n t h e e f f l u e n t a r e s u m m a r i z e d i n T a b l e 2. The r e a c t i o n s e q u e n c e r e l a t e d t o t h e a l k a l i n e h y d r o l y s i s o f organo-chlorinated shown

compounds a n d a t y p i c a l e x p e r i m e n t a l k i n e t i c s a r e

i n F i g u r e 2.

The s e q u e n c e o f t h e u n i t o p e r a t i o n s o f t h e s t u d i e d p r o c e s s i s s c h e m a t i z e d i n F i g u r e 3. The c h a r a c t e r i s t i c s (spprox.

o f t h e p r e t r e a t e d and d i l u t e d e f f l u e n t

1:4 w i t h tap water)

a r e s u m m a r i z e d i n T a b l e 3.

-

51

-

TABLE 2 Main r e s u l t s o f p r e l i m i n a r y " t o x i c i t y " Inhibition'

Recalcitrance'

Toxicity3

[PPd

[p pml

[PPml

75

150

Epichlorohydrin Glycidol

3 C1 1 - 2 p r o p a n e d i o l 1200

Isopropyl alcohol

tests.

-

600

-

-

2400

1200

1500

-

7200

-

1) Acclimatization required;

evidence o f biodegradation; other degradative processes scarcely influenced.

2) Acclimatization required;

no e v i d e n c e o f b i o d e g r a d a t i o n ; other degradative processes s i g n i f i c a n t l y retarded.

3 ) No e v i d e n c e o f a n a e r o b i c a c t i v i t y .

.GLYCEROL +

3 CL PROPANEDIOL GLYCIDOL

'iE

12

4 20

2

4

6

HOURS

Na OH

NaOH C+-CH-CYb CHz-CH-Cb I

OH Fig.

\ /

0

'

&i AH AH

2 . Alkaline hydrolysis o f organo-chlorinated The a n a e r o b i c f i l t e r s r e a c h e d s a t i s f a c t o r y

o r g a n i c l o a d (max.

6 . 5 Kg COD/m3,d)

as COD r e m o v a l (80-85

compounds.

values o f s p e c i f i c

and gave good p e r f o r m a n c e s b o t h

%) and as energy r e c o v e r y (30-35

3

Nm /m3

of

t r e a t e d wastewater). These p e r f o r m a n c e s were o b t a i n e d w i t h a r e s i d u a l c o n c e n t r a t i o n o f e p i c h l o r o h y d r i n and 3 - c h l o r o - p r o p a n e d i o l

(Table 2) t a n g i b l y lower

t h a n t h o s e o b s e r v e d f o r t h e p u r e compound ( T a b l e 3). T h i s c a n b e a s c r i b e d t o t h e combined e f f e c t o f t h e r e s i d u a l o r g a n o - c h l o r i n a t e d

-

-

52

compounds on t h e a n a e r o b i c m i c r o f l o r a .

From t h i s p o i n t o f v i e w i t

was o b s e r v e d t h a t r e s i d u a l c o n c e n t r a t i o n s o f e p i c h l o r o h y d r i n ,

gly-

c i d o l a n d s o d i u m c h l o r i d e a p p e a r t o p l a y t h e c r i t i c a l r o l e on t h e e f f i c i e n c y o f the acclimatized anaerobic microflora.

I n fact,

the

e x p e r i m e n t a l r e s u l t s showed t h a t t h e e f f i c i e n c y o f t h e p r o c e s s i s s t r o n g l y r e d u c e d for c o n c e n t r a t i o n s o f e p y c h l o r o h y d r i n , and sodium c h l o r i d e h i g h e r t h a n 5,

glycidol

1 2 0 0 a n d 1 5 0 0 ppm r e s p e c t i v e l y .

EFFLUENTS

1

HYDROLrjlS WATER

1

-

EVAPORATION CRYSTALLIZATION

ISOPROPYL ALCOHOL (RECCWW OPTIONAL)

MIXING -DILUTED

WASTEWATER

1

ANAEROBIC DIGESTION

BIOGAS Fig.

1

FINAL TREATMENT

3.

Treatment o f w a s t e w a t e r f r o m epoxy r e s i n s p r o d u c t i o n .

TABLE 3 Composition o f the e f f l u e n t feeding

the anaerobic f i l t e r .

Glycerol

8.5

g/l

Isopropyl alcohol

4.1

g/l

NaCl

12.5

g/l

X) H3P04 ( 8 5 X )

2.2

ml/l

1.1

ml/l

Epichlorohydrin

5

ppm max

Glycidol

800

ppm

3-chloro-propanediol

20

PPm

1-1 D i c h l o r i d r i n e

20

PPm

1-2 D i c h l o r i d r i n e

20

PPm

COD

19.8

g/l

PH

7

911

NH40H ( 3 0

- 53

-

i t m u s t be s t r e s s e d t h a t no s i g n i f i c a n t s l u d g e p r o -

Finally,

d u c t i o n waa o b s e r v e d ( t o t a l s o l i d s c o n c e n t r a t i o n i n t h e t r e a t e d e f f l u e n t are approximately equal t o the anaerobic f i l t e r i n f l u e n t ) .

CONCLUSIONS E f f l u e n t s o r i g i n a t i n g from t h e epoxy r e s i n s p r o d u c t i o n c o n t a i n i n h i b i t o r y a n d t o x i c m o l e c u l e s w h i c h do n o t a l l o w t h e i r d i r e c t b i o l o g i c a l treatment. F o r t h i s reason t h e c u r r e n t p u r i f i c a t i o n processes a r e based on chemical-

p h y s i c a l p r e t r e a t m e n t s f o l l r a e d by b i o l o g i c a l o x i d a t i o n

steps. The a n a e r o b i c s o l u t i o n d e s c r i b e d i n t h i s w o r k o v e r c o m e s t h e drawbacks o f these processes ( s t r o n g d i l u t i o n , e l e c t r i c power,

h i g h consumption o f

h i g h s l u d g e p r o d u c t i o n ) and o f f e r s t h e f o l l o w i n g

main advantages:

-

S t r o n g r e d u c t i o n o f t h e r e q u i r e d d i l u t i o n s ( i n l e t COD c o n c e n t r a t i o n f o r aerobic processes 0.5-1 f o r the experimented anaerobic

-

g/l, i n l e t COD c o n c e n t r a t i o n process spprox.

20 9 / 1 1 .

A l m o s t n e g l i g i b l e c o n s u m p t i o n o f e l e c t r i c p o w e r and s i g n i f i c a n t 3 p r o d u c t i o n o f b i o g a s ( 3 0 - 3 5 Nm / m 3 o f t r e a t e d w a s t e w a t e r , C H 4 60-65

-

X v).

Recovery o f sodium c h l o r i d e p u r i t y 95-97

76,

( 2 0 0 - 2 5 0 Kg/m3

o f treated effluent,

m a r k e t a b l e as b u l k p r o d u c t ) a n d t h e c o n s e q u e n t

e l i m i n a t i o n o f t h e l e g a l p r o b l e m s r e l a t e d t o t h e s a l t and c h l o r i d e c o n t e n t s i n t h e t r e a t e d wastewater.

REFERENCES

1 2

3

4 5 6

7 8 9

P . Sanna, Acqua A r i a , 4, 4 8 3 , 1 9 8 1 . P . Sanna, M. C a m i l l i a n d L . Degen, G l o b a l B i o c o n v e r s i o n s , Volume 2 n d , 49, 1 9 8 7 , C R C P r e s s D . L . Wise E d i t o r . C . H o l l i n g e r e t e l . , 5 t h I n t . Symp. on A n a e r o b i c D i g e s t i o n , Boloona i 9 8 8 . 211 E . V . > R u d n e k o ’ e t a l . , P l a s t . M a s s . , 1, 5 4 , 1 9 8 7 106-107405). N . V . Ozumedzei e t e l . , L a k o k r a s . M a t e r . I k h . P r i m e n . , 3 , 107-120489). 65, 1987 R . A . F r i m a n e t a l . , P l a s t . Massy., 3 , 5 6 , 1 9 8 2 &. 96-186735). T.F. Gumbatova e t e l . , K h i m . T e k h n o l . Vody, 4, 3 7 6 , 1 9 8 2 (Chem. Ab. 9 7 - 1 6 8 2 6 9 ) . G . A . B y s t r o v e t e l . , P l a s t . Massy., 2 , 4 7 , 1 9 8 4 (Chem. Ab. 100-161350). A . R o b e r t i e l l o e t a l . , A p p l i c a t i o n p a t e n t N . 22221A/88, 7 Oct. 1988, I t a l y .

(e. fi.

(e. m.

(w.

This Page Intentionally Left Blank

ANAEROBIC T R E A T M E N T OF EFFLUENTS:

U S E OF BIOINOICATORS FOR

PROCESS M O N I T O R I N G C.

SORLINI

D i p a r t i m e n t o d i Scienze e Tecnoloqie A l i m e n t a r i e M i c r o b i o l o g i c h e , Sezione M i c r o b i o l o g i a A g r a r i a , A l i m e n t a r e , E c o l o g i c a , U n i v e r s i t a d e g l i S t u d i d i M i l a n o , V i a C e l o r i a , 2, 20133 M i l a n o , I t a l y

Anaerobic treatment Anaerobic treatment, a n d d o m e s t i c sewage, i n origin,

used i n t h e p a s t o n l y f o r a n i m a l s l u r r i e s

has been a p p l i e d l a t e l y t o e f f l u e n t s v a r y i n g

p o l l u t i o n l e v e l s and c h e m i c a l c o m p o s i t i o n ,

w i t h interes-

t i n g r e s u l t s c o n c e r n i n g t h e decrease i n p o l l u t i o n and t h e product i o n o f methane.

Anaerobic d i g e s t i o n i s o f p a r t i c u l a r i n t e r e s t f o r

o l i v e o i l m i l l s waatewater, wood p r o c e s s i n g p l a n t s ,

d i s t i l l a r y sloaps,

cheese-whey,

Among t h e new t e c h n o l o g i e s , blanked digesters),

digesters,

h a v e become o f p r i m a r y i m p o r t a n c e . w i t h a m e t h a n e p r o d u c t i o n f r o m 0.3 1989).

These d i g e s t e r s c a n o p e r a t e a t t a l i k e whey,

t o 0.35

c a n r e a c h 95 %

mc/Kg C O D r e m

(Schroder

Good r e s u l t s h a v e a l s o b e e n o b t a i n e d e v e n i n

t h e case o f e f f l u e n t s ,

generally d i f f i c u l t t o treat,

o i l m i l l wastewater and p y r o l y a e w a t e r , microorganisms,

used a l s o on a r e a l s c a l e ,

u s e d on a n e x p e r i m e n t a l s c a l e ,

i n i n g a C O D r e m o v a l t h a t i n some c a s e s ,

a n d De H a a a t ,

etc.

a f t e r UASB ( u p f l o w a n a e r o b i c s l u d g e

f i x e d bed d i g e s t e r s ,

h y b r i d and expanded-bed

papermill effluents,

petrochemical wastewater,

s u c h aa o l i v e

r i c h i n t o x i c compounds f o r

where C O D r e m o v a l h a s r e a c h e d s a t i s f a c t o r y

values

r a n G i n g f r o m 7 5 t o 8 5 % a n d s p e c i f i c m e t h a n e y i e l d a b o u t o f 0,3 rnc/CgCODrem

(Laveni e t a l . ,

1984;

Andreoni

P h y s i c a l and c h e m i c a l p a r a m e t e r s (pH, t o t a l solids,

a l k a l i n i t y and methane y i e l d )

f o r the monitoring o f the digesters,

e t al.,

COD,

1989).

volatile solids,

a r e g e n e r a l l y used

w h i l e m i c r o b i o l o g i c a l and en-

zym<.tic parameters are u s u a l l y neglected ( S o r l i n i ,

B o n f a n t i 1989).

D e t e r m i n a t i o n o f t h e number o f m i c r o o r g a n i s m s c a n be done b y t r a d i t i o n a l t e c h n i q u e s s u c h a s c o u n t i n g o f c o l o n i e s on p l a t e s , d e t e r m i n a t i o n i n t h e l i q u i d medium ( S o r l i n i e t e l . ,

19831,

MPN

direct

c o u n t by o p t i c a l m i c r o s c o p e , o r ,

-

56

f o r methanogenic b a c t e r i a ,

by

f l u o r e s c e n t m i c r o s c o p y and by i m m u n o l o g i c a l t e c h n i q u e s based on t h e use o f monoclonal and p o l y c l o n a l a n t i b o d i e s 1988;

Koornneef e t al.,

1990).

Not a l l b a c t e r i a ,

i n f i x e d and e x p a n d e d - b e d d i g e s t e r s , ces;

(Dubouriger e t a l . , normally present

adhere t o t h e support m a t r i -

a c o n s i d e r a b l e number r e m a i n s s u s p e n d e d i n t h e c i r c u l a t i n g

f r a c t i o n and o t h e r s a r e d e p o s i t e d a t t h e b o t t o m o f t h e d i g e s t e r . The c a p a b i l i t y o f m e t h a n o g e n i c b a c t e r i a t o a d h e r e t o s u p p o r t s v a ries significantly;

f o r example,

M e t h a n o s p i r i l l u m h u n q a t e i JF1 has

a b e t t e r adherence t o h y d r o p h i l o u s s u r f a c e s ,

whereas M e t h a n o t h r i x

soehnqenii t o hydrophobic surfaces ( V e r r i e r e t al.,

I t h a s a l s o been n o t e d t h a t ,

1988).

i n anaerobic digesters,

contrary

t o what h a s b e e n o b s e r v e d i n rumen a n d mammalian i n t e s t i n e ,

aceto-

t r o p h i c methanogens a r e more abundant t h a n h y d r o g e n o t r o p h i c b a c t e r i a ( D o l f i n g and M u l d e r ,

1985).

I n v e s t i g a t i o n s on m i c r o b i a l p o p u l a t i o n i n d i g e s t e r s a n d i t s d i s t r i b u t i o n on s u p p o r t s u r f a c e a r e i m p o r t a n t t o about t h e p r o c e s s and f o r i t s o p t i m i s a t i o n .

deepen k n o w l e d g e

However, t h e d a t a o b t a -

i n e d f o r d i f f e r e n t r e a s o n s a r e n o t much u s e f u l f o r m o n i t o r i n g t h e running o f the digesters.

Coenzyme F420 a s b i o i n d i c a t o r o f m e t h a n o q e n i c p o t e n t i a l i t y I n an a t t e m p t t o s e l e c t b i o i n d i c a t o r s c a p a b l e o f r e v e a l i n g p r o m p t l y t h e p o t e n t i a l i t y f o r a s l u d g e t o produce methane, t e n t i o n h a s b e e n f o c u s e d on coenzyme F 4 2 0 a f a c t o r p r e s e n t , anaerobic microorganisms,

o n l y i n methanogenic b a c t e r i a .

the atamong

This fac-

t o r is r a p i d t o e x t r a c t a n d e a s y t o d e t e r m i n e by means o f a s p e c trofluorimeter.

I t c o n s i s t s o f a 7,8-didemethyl-8-hydroxi-5-deazo-

flavin-5-phosphate a c i d chain,

w i t h an N - ( N - L - l a c t y l -

t h a t a c t s as c o - f a c t o r

7-L-glutamy1)-L-glutamic

o f v a r i o u s enzymes f o r t h e i n -

c o r p o r a t i o n o f C O P i n t o o r g a n i c c a r b o n and p l a y s a key r o l e i n m e t h a n e p r o d u c t i o n as i t t r a n s f e r s e l e c t r o n s t o t h e CoM r e d u c t a s e s y s t e m i n t h e f i n a l s t e p s o f r e d u c t i o n o f C02 i n t o m e t h a n e . The r e s u l t s on t h e p r e d i c t a b l e r o l e o f f a c t o r F420 on m e t h a nogenic p o t e n t i a l i t y are s t i l l c o n t r a d i c t o r y . P u r e c u l t u r e o f Methanobacterium a r b o r i p h i l u s and Methanobacte-

r i u m thermoautotrophicum (Taya e t a l . ,

1 9 8 6 ) h a v e shown a s t r o n g

c o r r e l a t i o n b e t w e e n F420 c o n t e n t e n d m i c r o b i a l b i o m a s s . A c l o s e c o r r e l a t i o n was a l s o e v i d e n c e d i n p u r e c u l t u r e o f M e t h a n o b a c t e r i u m

-

m. b r i a n t i i

arboriphilus,

and

-

57

&.

b a r k e r i (Heine-Oobbernach

et

sl., 1 9 8 8 1 , b u t t h e r e was n o c o r r e l a t i o n b e t w e e n F 4 2 0 c o n t e n t a n d methane p r o d u c t i o n .

were c o n d u c t e d i n b a t c h .

These e x p e r i m e n t s

Other e x p e r i m e n t s c a r r i e d o u t w i t h mixed c u l t u r e s o b t a i n e d by i n o c u l a t i n g g r a n u l a r s l u d g e i n s y n t h e t i c media added s e p a r a t e l y w i t h d i f f e r e n t c a r b o n s o u r c e s h a v e shown a g o o d c o r r e l a t i o n b e t w e e n

F 4 2 0 a n d methane p r o d u c e d o n l y i n c u l t u r e a d d e d w i t h f o r m a t e , w i t h hydrogen, However,

a c e t a t e and e t h a n o l ( O o l f i n g end Mulder,

not

1985)

t h e r e s u l t s o b t a i n e d by t h e s e A u t h o r s c o u l d have been

i n f l u e n c e d by unfavourable c o n d i t i o n s i n the s t a t i o n a r y c u l t u r e l e a d i n g t o an e x h a u s t i o n o f t h e s u b s t r a t e and t o an i n c r e a s e o f catabolites,

I t can

c o n d i t i o n s n o t found i n c o n t i n u o u s d i g e s t e r s .

be presumed t h a t ,

i n such d i g e s t e r s ,

most o f t h e carbom d i o x i d e

t h a t i s reduced f o l l o w s t h e pathway o f b i o s y n t h e s i s l e a d i n g t o t h e p r o d u c t i o n o f a c e l l u l a r biomass,

n o t t h e way o f t h e m e t h a n e p r o -

The f i r s t s t e p s o f C02 r e d u c t i o n a r e ,

duction.

b o t h processes.

i n fact,

common t o

T h i s c o u l d e x p l a i n t h e l s c k o f c o r r e l a t i o n between

F 4 2 0 c o n t e n t and m e t h a n e p r o d u c t i o n . Other Authors (Zabranska e t a l . ,

1985) have a l s o n o t e d t h a t

F420 c o n t e n t i n b a t c h c u l t u r e s as w e l l i n semi-continuous

and con-

t i n u o u s l a b o r a t o r y d i g e s t e r s was d e p e n d e n t u p o n t h e c u l t u r e c o n d i t i o n s a d o p t e r and d i d n o t appear t o be s u i t a b l e t o e v a l u a t e t h e l e v e l o f methanogenic b a c t e r i a l a c t i v i t y .

However,it

c o u l d be used

A hig-

t o e v a l u a t e t h e number o f t h e s e b a c t e r i a i n m i x e d c u l t u r e s . h e r F420 c o n t e n t i n t h e s t a r t - u p

phase c o u l d be due t o t h e p r e s e n -

ce o f hydrogenotrophic b a c t e r i a ,

which p r e v a i l i n g d u r i n g t h a t pha-

se and w h i c h a r e r i c h e r i n F420 c o n t e n t t h a n a c e t o t r o p h i c b a c t e r i a p r e v a i l i n g d u r i n g t h e subsequent phase. E x p e r i m e n t s c a r r i e d o u t by o t h e r A u t h o r s w i t h s e m i c o n t i n u o u s a n d UASB d i g e s t e r s f e d w i t h d i f f e r e n t i n p u t s h a v e i n d i c a t e d , contrary,

on t h e

a s i g n i f i c a n t c o r r e l a t i o n between F 4 2 0 c o n t e n t and metha-

ne y i e l d e s p e c i a l l y d u r i n g t h e s t a r t - u p

phase.

A s l i g h t decrease

i n F420 c o n t e n t , evidenced d u r i n g t h e s t e a d y - s t a t e

phase i s i n con-

t r a d i c t i o n w i t h what i s p r e d i c t e d b y s t e a d y - a t a t e

k i n e t i c s and w i t h

q u a n t i t y o f methane produced.

I n s p i t e o f t h i s small discrepancy,

t h e A u t h o r s c o n s i d e r e d t h a t F420 c o n t e n t c o u l d be used as an i n d i c a t o r o f methanogenic a c t i v i t y ( O e l a f o n t a i n e e t e l . , e t el.,

1979;

Schulze

198’8).

Some o t h e r A u t h o r s ( v a n B e l e e n ,

1984) have found a p o s i t i v e

c o r r e l a t i o n between F420 c o n t e n t and methane p r o d u c t i o n depending

-

58

-

o n t h e t y p e o f p r o c e s s a p p l i e d . The F420 c o n t e n t h a s r e s u l t e d t o be p a r t i c u l a r l y s i g n i f i c a n t o f t h e methanogenic p o t e n t i a l i t y i n plug-flow

and h y d r a u l i c d i g e s t e r s (Withmore e t al.,

For a b e t t e r understanding o f t h i s subject,

1986). we h a v e c a r r i e d

o u t a a e r i e s o f e x p e r i m e n t s w i t h t h r e e 62 1 f i x e d - b e d d i g e s t e r s : up-flow,

down-flow

and down-up-flow.

The d i g e s t e r s w e r e p a c k e d w i t h P V C empty c y l i n d e r s 35/mm)

and f e d w i t h d i l u t e d s w i n e s l u r r y .

thane y i e l d ,

COD,

VS,

TS,

( 0 33,h

D i f f e r e n t p a r a m e t e r s (me-

pH) were c o n s t a n t l y m o n i t o r e d .

During

the steady-state

phase,

t h e t h r e e d i g e s t e r s had a d i f f e r e n t metha-

ne y i e l d (22.50,

33.20,

27.15

were

l/day,

fed under i d e n t i c a l conditions.

respectively),

although they

Supports were t h e r e f o r e drawn

a t e q u a l d e p t h s a n d F420 c o n t e n t a n d t h e number o f m e t h a n o g e n i c b a c t e r i a were d e t e r m i n e d on t h e biomass a t t a c h e d . The r e s u l t s o b t a i n e d i n d i c a t e d t h a t F420 c o n t e n t , t o volume u n i t ,

referred

i s r e l a t e d t o methane y i e l d f o r each d i g e s t e r ,

b u t n o t t o t h e number o f m e t h a n o g e n i c b a c t e r i a ( T a b l e 1 ) .

TABLE 1 F i x e d - b e d d i g e s t e r s ( 6 2 1) CH4 [l/day]

Param. Digesters

CH4 dig]

ji/i

F420 [Clmol/g SV]

F420 [pmol/l dig]

1 CH4/’m F420/day

Methan.Bact. [logN/gdw]

UP-FLOW

22.50

0.362

0.370

0.230

1.57

8.36

DOWN-FLOW

33.20

0.534

0.570

0.384

1.39

8.36

0.437

0.430

0.283

1.54

9.79

DOWN-UP FLOW I

27.15 I

I

The v a l u e s o f coenzyme F 4 2 0 a n d m e t h a n o g e n i c b a c t e r i a a r e t h e a v e r a g e o f t h r e e d i f f e r e n t samples.

Other e x p e r i m e n t s were c a r r i e d o u t w i t h a 1200 1 u p - f l o w bed p i l o t p l a n t packed w i t h PVC empty c y l i n d e r s .

fixed-

The same p a r a m e t e r s

were c o n s t a n t l y m o n i t o r e d . S u p p o r t s were drawn t h r e e t i m e s ,

(Tl,

T2,

T3)

a t t h e t h r e e months i n t e r v a l

a t t w o d i f f e r e n t d e p t h s ( 5 0 a n d 2 0 0 cm) a n d F 4 2 0 c o n -

t e n t a n d t h e number o f m a t h a n o g e n i c b a c t e r i a biomass a t t a c h e d .

w e r e d e t e r m i n e d on t h e

The d i g e s t e r was i n a s t e a d y - s t a t e

w o r k i n g phase

a t T 1 a n d T2 a n d i t was i n a f i n a l p h a s e w i t h i r r e g u l a r

feeding a t

73.

59

-

The r e s u l t s showed t h a t F420 c o n t e n t was h i g h e r i n t h r e e s u p -

p o r t s d r a w n a t 200 cm,

a c c o r d i n g t o Koeff-Bank

and S c h r a e w e r ( 1 9 8 7 )

and i n c o n t r a s t w i t h R e y n o l d s and C o l l e r a n f i n d i n g s ( 1 9 8 7 ) . more,

F420 c o n c e n t r a t i o n ,

a s w e l l a s methane p r o d u c t i o n ,

Further-

was h i g h e r

i n t h e s u p p o r t s d r a w n a t T2 w i t h r e s p e c t t o t h o s e d r a w n a t T 1 .

There

was a s h a r p d e c r e a s e i n m e t h a n e p r o d u c t i o n due t o d i s c o n t i n u o u s f e e d i n g a t T3,

w h e r e a s F420 c o n t e n t was h i g h e r .

The number o f m e t h a -

n o g e n i c b a c t e r i a were g r e a t e r a t t h e T2 t h a n a t 11 ( T a b l e 2 ) .

TABLE 2 F i x e d - b e d p i l o t d i g e s t e r s ( 1 2 0 0 1)

Param.

CH4

CH4

Phases

[l/dayl

[l/l d i g ]

F420 S”

T1

290

12

447

13

351

0.241

0.372

0.292

I

dig1

h+

0.085

I++

0.334

h

0.099

1

0.393

h

0.470

1

0.500

1

1 C H 4 / p n o l Methan-. B[ l oUg N. / g d w ]

F420

[ pmo 1e s / g ~flmoles’llF 4 2 0 / d a y 0.138

h+

1.74

8.36

1++ 9.36 0.159

2.33

h+

10.36

1++ 10.36 0.326

0.895

h+

7.79

1++ 8.20

The v a l u e s o f coenzyme F420 a n d m e t h a n o g e n i c b a c t e r i a a r e t h e a v e rage o f t h r e e d i f f e r e n t samples. h+

=

at a

50 cm d e p t h

1++ = a t a 200 cm d e p t h The r e s u l t s showed t h a t b o t h F420 c o n t e n t

(pmoles/l

dig)

and

t h e d a i l y p r o d u c t i o n o f methane ( 1 / 1 d i g ) a r e s l i g h t l y h i g h e r i n t h e t h r e e 62 1 d i g e s t e r s t h a n i n t h e p i l o t d i g e s t e r . The v a l u e s o b t a i n e d f o r those o f Delafontaine e t e l .

F420 ( p m o l e s / l (1979)

dig)

a r e comparable t o

and o f S c h u l z e e t a l .

b u t a r e l o w e r t h a n t h o s e o f van B e l e e n e t a l .

(1983)

(1988)

and t h e v a l u e s

f o r m e t h a n e p r o d u c t i o n a r e o f t h e same o r d e r o f m a g n i t u d e t h a n t h o se o f Zabranska e t e l .

lafontaine e t al.

(1985)

b u t s l i g h t l y h i g h e r t h a n t h o s e o f De-

(1979) and G o r r i a e t a l .

Other’experiments

(1988).

conducted w i t h a 6 1 expanded bed d i g e s t e r

packed w i t h m a r l have a l s o i n d i c a t e d a c o r r e l a t i o n between methane y i e l d a n d F420 c o n c e n t r a t i o n .

With t h i s d i g e s t e r ,

F420 c o n t e n t and

-

60

-

methane y i e l d r e f e r r e d t o volume o f d i g e s t e r as w e l l t o F420 moles a r e h i g h e r t h a n i n t h e f i x e d bed d i g e s t e r ,

a confirmation o f the

b e t t e r e f f i c i e n c y o f t h i s d i g e s t e r (Table 3).

TABLE 3 Expanded b e d r e a c t o r

Phase

I

F420

1

lCH4/,umol/

Meth.

[l/day]

[ l / lCdHi 4g ]

0.480

0.080

0.0343

2.330

3.846

0.641

0.1410

0.445

nd

2.040

0.340

0.1026

3 310

6.79

2.124

0.354

0.4300

0.820

7.79

4.842

0.807

0.5600

1.440

6 79

T4

[,umoles/ldigJ

F420/day

[logN/g

Bact. inert]

nd

The v a l u e s o f coenzyme F420 a n d m e t h a n o g e n i c b a c t e r i a a r e t h e average o f t h r e e d i f f e r e n t samples.

The r e s u l t s o b t a i n e d a r e i n a g r e e m e n t w i t h t h o s e i n d i c a t i n g F420 c o n t e n t o f s l u r r y a s a b i o i n d i c a t o r

capable o f p r e d i c t i n g

methanogenic p o t e n t i a l i t y .

Analogs o f F420 T o g e t h e r w i t h t h e s t u d i e s on t h e r o l e o f F 4 2 0 c o n t e n t a s a bioindicator

f o r methanogenic p o t e n t i a l i t y , i n v e s t i g a t i o n s were

done o n t h e c h e m i c a l s t r u c t u r e o f t h i s coenzyme i n v a r i o u s p h y s i o l o g i c a l groups o f methanogenic b a c t e r i a . e t el.,

1983;

G o r r i s and v a n d e r D r i f t ,

These s t u d i e s

1 9 8 7 ) h a v e l e d t o t h e c o n c l u s i o n t h a t F420-2

i s the only form o f

t h i s coenzyme f o u n d i n h y d r o g e n o t r o p h i c b a c t e r i a , t r o p h i c b a c t e r i a F420-5

i s p r e v a i l i n g ( 2 a n d -5

the glutamic acid residues i n the side chain).

i n hydrogenotrophic bacteria, but functionally

t h e c a r r i e r o f C1

identical

whereas i n a c e t o are indicating

I n addition t o that

while i n acetotrophic bacteria i s sarcinapterin, different

(van Beleen

1 9 8 6 : Peck a n d A r c h e r ,

i s methanopterin, which i s chemically

I

More r e c e n t s t u d i e s h a v e shown t h a t t h e a b o v e - m e n t i o n e d c o f a c t o r c o n t e n t i n g r a n u l a r s l u d g e can be used t o measure t h e methanogenic p o t e n t i a l o f hydrogenotrophic r i s e t al.

1988).

and a c e t o t r o p h i c b a c t e r i a (Gor-

I n a subsequently study,

61

-

Peck ( 1 9 8 9 )

showed t h a t t h e c o n c e n t r a -

t i o n o f v a r i o u s a n a l o g s o f F420 p r e s e n t i n t h e c e l l s o f and

&.

&. b a r k e r i

mazei v a r i e d i n f u n c t i o n o f g r o w t h s t a g e ( h i g h e r i n he f i r s t

phase) and t y p e o f s u b s t r a t e .

The F420 c o n t e n t i n t h e c e l l s o f d i f f e -

r e n t m e t h a n o g e n i c b a c t e r i a was e x t r e m e l y v a r i a b l e . However,

many p r o b l e m s a r e s t i l l t o b e s o l v e d .

The d e t e r m i n a -

t i o n o f F420 c o n t e n t i n h y d r o g e n o t r o p h i c a n d a c e t o t r o p h i c b a c t e r i a l g r o u p s s h o u l d b e e x a m i n e d more c l o s e l y ,

s i n c e r e c e n t s t u d i e s on va-

r i o u s s p e c i e s o f methanogens i n p u r e c u l t u r e have i n d i c a t e d t h a t t h e F420 c o n t e n t i n b o t h g r o u p s i s v e r y v a r i a b l e a n d t h a t t h e h y p o t h e s i s t h a t hydrogenotrophic

b a c t e r i a have a h i g h e r c o n t e n t appears t o

be d o u b t f u l .

I t remains,

however,

t h a t t h e F420 c o n t e n t i n s l u r r y o r i n p u r e

c u l t u r e s varies according t o the chemical c h a r a c t e r i s t i c s substrate.

o f the

I t i s t h e r e f o r e p r o b a b l e t h a t F420 c a n be u s e d a s a n i n -

d i c a t o r o f m e t h a n o g e n i c p o t e n t i a l i t y when t h i s f a c t o r i s i n v e s t i g a t e d i n d i f f e r e n t d i g e s t e r s f e d w i t h t h e same s l u r r y or i n one d i g e a t e r d u r i n g d i f f e r e n t phases b u t n o t i n d i g e s t e r f e d w i t h d i f f e r e n t inputs.

The F420 c o n t e n t h a s t h e r e f o r e a r e l a t i v e ,

not absolute,

predicting role. F i n a l l y i t has t o be u n d e r l i n e d t h a t t h e e x t r a c t i o n methods used by d i f f e r e n t A u t h o r s a r e n o t s t a n d a r d i s e d and o f t e n t h e i r res u l t s c a n n o t be compared (Reynolds and C o l l e r a n ,

1987).

Given t h e importance o f anaerobic d i g e s t i o n f o r t h e t r e a t m e n t of

sludges,

more a t t e n t i o n s h o u l d b e p a i d t o a n a e r o b i c d i g e s t e r a i n

order t o improve t h e i r performance t o reach t h e l e v e l o f h i g h technology. gas,

The e f f l u e n t s f r o m t h e s e d i g e s t e r s t r e a t e d w a t e r s a n d b i o -

do n o t h a v e a h i g h m a r k e t v a l u e ,

but they c o n t r i b u t e s i g n i f i -

c a n t l y t o p r o t e c t t h e e n v i r o n m e n t w h i c h i s a r e s o u r c e b e y o n d any market value.

REFERENCES

1

2

3

.

V. Andreoni, P B o n f a n t i , D. D s f f o n c h i o , G. R a n a l l i , C . S o r l i n i , M. V i l l a , A n a e r o b i c d i g e s t i o n o f s w i n e s l u r r y a n d a g r o i n d u s t r i a l w a s t e s i n f i x e d - b e d up f l o w d i g e s t e r s . I n : T e c h n i c a l a d v a n c e s i n b i o f i l m r e a c t o r s . N i c e 4 - 6 a p r i l 1 9 8 9 , 553-554. A . A v e n i , B i o g a s r e c o v e r y f r o m o l i v e - m i l l w a s t e w a t e r b y anaer o b i c d i g e s t i o n . I n A n a e r o b i c d i g e s t i o n and c a r b o h y d r a t e h y d r o l y s i s o f w a s t e (C.L. F e r r e r o , M . P . F e r r a n t i , H. Nouveau Eds.) E l s . A p p l . Sc. P u b l . ( U . K . ) 1984. M . J . D e l a f o n t a i n e , H . P . Naveau, E . J . Nyna, F l u o r i m e t r i c m o n i t o r i n g o f methanogenesis i n anaerobic d i g e a t o r s . Biotech. L e t t . , -1, 71-74 ( 1 9 7 9 ) .

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-

J.

D o l f i n g , J.W. M u l d e r , C o m p a r i s o n o f m e t h a n e p r o d u c t i o n r a t e a n d coenzyme F 4 2 0 c o n t e n t o f m e t h a n o g e n i c c o n s o r t i a i n a n a e r o b i c granular s l u d g e . A p p l . E n v i r o n . M i c r o b i o l . 49, 1 1 4 2 1145 ( 1 9 B 5 ) . H . C . D u b o r g i e r , D.B. A r c h e r , G. A l b a g n a c , G. P r e n s i e r , S t r u c t u r e a n d m e t a b o l i s m o f m e t h a n o g e n i c m i c r o b i a l c o n g l o m e r a t e s . I n : AnaH a l l and P . N . Hobson E d s . ) , May 22-26, e r o b i c d i g e s t i o n (E.R. 1988, 1 3 - 2 3 . L.G. G o r r i s , T.M. De K o k , B.M. K r o o n , C . Van d e r D r i f t , G.D. Vog e l s , R e l a t i o n s h i p between Methanogenic C o f a c t o r C o n t e n t and Maximum S p e c i f i c M e t h a n o g e n i c A c t i v i t y o f A n a e r o b i c G r a n u l a r S l u d g e s , A p p l . E n v i r o n . M i c r o b i o l . 54, 1 1 2 6 - 1 1 3 0 ( 1 9 8 8 ) . L.G. G o r r i s , C . Van d e r D r i f t , M e t h a n o g e n i c c o f a c t o r s i n p u r e c u l t u r e o f m e t h a n o g e n s i n r e 1 a t i o . n ~t o s u b s t r a t e u t i l i z a t i o n . Ouborguier e t a l . I n B i o l o g y o f A n a e r o b i c B a c t e r i a . Eds. H.C. E l s e v i e r Sc. P u b b l . , pp. 1 4 4 - 1 5 0 , Amsterdam 1986. E. H e i n e D o b b e r n a c k , S . M . S c h o b e r t , H. Sahm, R e l a t i o n s h i p o f i n t r a c e l l u l a r coenzime F 4 2 0 c o n t e n t t o g r o w t h and m e t a b o l i c a c t i v i t y o f M e t h a n o b a c t e r i u m b r y a n t i i a n d M e t h a n o s a r c i n a &A p p l . E n v i r o n . M i c r o b i o l , 54, 454-459 ( 1 9 8 s ) . H . J . Koepp-Bank, R . S c h r a e w e r , A n a e r o b i c d i g e s t i o n o f c h e e s e whey t o m e t h a n e i n f i x e d - b e d , f i x e d - b e d - l o o p and f i x e d - f i l m r e a c t o r s . I n : Technology o f B i o l o g i c a l Processes-Safety i n B i o t e c h n o l o g y . A p p l i e d G e n e t i c E n g i n e e r i n g 5 t h Dchema. A n n u a l a M e e t i n g o f B i o t e c h n o l o g i s t s . p p . 4 0 3 - 4 0 8 , May 1 2 - 1 3 1 9 8 7 E. K o o r n n e e f , A.J.L. M a c a r i o , J.T.C. G r o t e n h u i s , E.C. de M a c a r i o , Methanogens r e v e a l e d i m m u n o l o g i c a l l y i n g r a n u l e s f r o m f i v e upf l o w a n a e r o b i c s l u d g e b l a n k e d b i o r e a c t o r s g r o w n on d i f f e r e n t s u b s t r a t e s . Fems M i c r o b i o l . E c o l o g y , 73, 225-230 ( 1 9 9 0 ) M.W. P e c k , Changes i n c o n c e n t r a t i o n s o f c o e n z i m e F 4 2 0 a n a l o g s d u r i n g b a t c h growth o f Methanosarcina b a r k e r y and Methanos a r c i n a m a z e i A p p l . a n d E n v i r o n . M i c r o b i o l o g y , 55, 9 4 0 - 9 4 5

m.

(19a97112

13

14 15

16

17 18 19

M.W. P e c k , D.B. A r c h e r , Improved assay o f coenzime F420 analogues from methanogenic b a c t e r i a . Biotechn. Techniques, 279-284 (1987). P.J. Reynolds, E . C o l l e r a n , E v a l u a t i o n o f improvement methods f o r coenzyme F 4 2 0 a n a l y s i s i n a n a e r o b i c s l u d g e s . J o u r n . o f M i c r o b i o l . Methods, 7, 115-130 (1987). E.W. S c h r o d e r , J . 6 e H a a s t , A n a e r o b i c d i g e s t i o n o f d e p r o t e i n a t e d c h e e s e whey i n an u p - f l o w s l u d g e b l a n k e d r e a c t o r . J. o f D a i r y R e s e a r c h , 59, 1 2 9 - 1 3 9 ( 1 9 B 9 ) . 0. S c h u l z e , M. Menkhaus, R . F i e b i g , H. D e l l w e g , A n a e r o b i c t r e a t ment o f p r o t e i n - c o n t a i n i n g w a s t e - w a t e r s : c o r r e l a t i o n b e t w e e n coenzyme F 4 2 0 a n d m e t h a n e p r o d u c t i o n . A p p l . M i c r o b i o l . B i o t e c h 506-510 ( 1 9 B 8 ) . nol., 2, C. S o r l i n i , P. B o n f a n t i , D i g e s t i o n o f a n i m a l s l u r r y . T e c h n o l o g i c a l , c h e m i c a l , m i c r o b i o l o g i c a l and m a n a g e r i a l a s p e c t s . I n B i o l o g i c a l Waste T r e a t m e n t ( A . M i z r a h i e d . ) , 1 9 8 9 , 2 0 3 - 2 3 4 , A l a n R . L i a s , I n c . New Y o r k . C . S o r l i n i , M.E. Cosmai, A . F e r r a r i , P r e v a l e n c e o f o x i g e n i n t o l e r a n t methanogenic b a c t e r i a i n f e c e s and a n a e r o b i c wastes. C u r r e n t M i c r o b i o l o g y , 2 , 355-358 ( 1 9 8 3 ) M. Taya, N. Aoky, T . K o b a y a s h y , K i n e t i c e v a l u a t i o n a n d m o n i t o r i n g o f methanogen c u l t u r e b a s e d u p o n f l u o r i m e t r y . A p p l . E n v i r o n . M i c r o b i o l . , 2 3 , 342-347 ( 1 9 8 6 ) . P. Van B e l e e n , A . C . D i j k s t r a , G.D. Vogels, Q u a n t i t a t i o n o f coenzyme F420 i n m e t h a n o g e n i c s l u d g e b y t h e u s e o f r e v e r s e d p h a s e h i g h performance l i q u i d chromatography and a f l u o r e s c e n c e detect o r . E u r . J. A p p l . M i c r o b i o l . B i o t e c h n o l . , l8, 67-69 ( 1 9 8 3 ) .

I,

20

21 22

63

-

D. V e r r i e r , B. M o r t i e r , H.C. D o u b o u r q i e r , G. A l b a g n a c , A d h e s i o n o f a n a e r o b i c b a c t e r i a t o i n e r t s u p p o r t s and development o f H a l l and m e t h a n o g e n i c b i o f i l m a . I n : A n a e r o b i c d i g e s t i o n (E.R. P . N . Hobson E d a . ) , May 22-26 1 9 8 8 , 61-67. T . N . W h i t m o r e , S . P . E t h e r i d g e , D.A S t a f f o r d , U.E.A. Leroff, 0. Hughes, The e v a l u a t i o n o f a n a e r o b i c d i g e s t e r p e r f o r m a n c e b y coenzyme F420 a n a l y s i s , B i o m a s s , 2 , 29-35 ( 1 9 8 6 ) . J. Z a b r a n s k a , K. S c h e i d e r o v a , M. Dohanyos, R e l a t i o n o f coenzyme F420 t o t h e a c t i v i t y o f m e t h a n o g e n i c m i c r o o r g a n i s m a . B i o t e c h ; L e t t . , 1, 545-552 ( 1 9 8 5 ) .

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- DENITRIFICATION I N ACTIVATED SLUDGE P R O C E S S COMBINED W I H BIOFILM CULTIVA I O N SIMULTANEOUS NITRIFICATION

M.

DRTIL,

J. TiLGVESSY and I. B O D f K

Department o f e n v i r o n m e n t a l c h e m i s t r y and t e c h n o l o g y , Chemical-technological f a c u l t y , Slovak t e c h n i c a l u n i v e r s i t y B r a t i s l a v a , CSFR

A c t i v a t e d s l u d g e p r o c e s s combined w i t h b i o f i l m p r o c e s s i s used i n two main m o d i f i c a t i o n s :

1. Suspended b i o m a s s a n d a t t a c h e d g r o w t h b i o m a s s a r e i n s e p a r a t e r e a c t o r s and b i o f i l m r e a c t o r s a r e m o s t l y preceded.

2.

B o t h f o r m s o f b i o m a s s a r e i n one r e a c t o r .

B i o f i l m s u p p o r t mate-

t i a l i s e i t h e r p l a c e d i n an a c t i v a t e d s l u d g e t a n k

f i r m l y or i s

kept i n f r e e motion together with activated sludge. many t e c h n o l o g i c a l e.g.

submerged p l a s t i c m e d i a o r n e t s ,

s m a l l p a r t i c l e s made o f

p l a s t i c abd p o r o u s m a t e r i a l d i s p e r s e d i n a t a n k , g i c a l contactors,

o f biomass,

r e p r e s e n t systems w i t h two k i n d s

which d i f f e r i n t h e i r main parameter

ex.

rotating biolo-

etc.

A l l r e a c t o r s mentioned-above t i o n time

I t i s obvious,

-

s o l i d s reten-

t h a t t h i s combination i s s u i t a b l e f o r

t h e t r e a t m e n t o f f a s t a n d s l o w d e g r a d a b l e compounds.

A typical e-

xample i s t h e i m p r o v e m e n t o f n i t r i f i c a t i o n e f f i c i e n c y . t o this effect,

There a r e

systems u t i l i z i n g t h i s biomass c o m b i n a t i o n ,

I n addition

a l l s y s t e m s e x h i b i t good s e t t l i n g p r o p e r t i e s and

suppress f i l a m e n t o u s microorganisms growth.

P r e s e n c e o f s u p p o r t ma-

t e r i a l w i t h b i o m a s s a l s o r e s u l t s i n an i n c r e a s e o f t h e t o t a l amount o f biomass and a decrease o f a c t i v a t e d s l u d g e l o a d i n g w i t h o u t

nega-

t i v e e f f e c t s on f i n a l c l a r i f i e r s 11, 2 1 . The m a i n a i m o f f o r the treatment of

t h i s s t u d y was t o s p e c i f y o p t i m a l c o n d i t i o n s i n d u s t r i a l waste water c o n t a i n i n g hexamethyle-

n e t e t r a m i n e (HMT) a n d h i g h c o n c e n t r a t i o n s o f NH; p r o c e s s w i t h combined biomass.

i n a c t i v a t e d sludge

HMT a s a n o n - b i o d e g r a d a b l e

organic

compound c a n b e r e m o v e d f r o m w a s t e w a t e r a f t e r a c i d h y d r o l y s i s :

-

(CH2)6

+

4H+

66

+ 6H20

-

4NHl

+ N COH

(1)

The p r o d u c t o f t h i s h y d r o l y s i s i s a b i o d e g r a d a b l e f o r m a l d e h y d e . T h e r e a r e t w o ways t o c r e a t e an a c i d pH. t i o n o f inorganic n i t r i f i c a t i o n o f NH; hydrolysis.

The f i r s t one i s t h e a d d i -

a c i d t o t h e w a s t e w a t e r a n d t h e s e c o n d one i s t h e p r e s e n t e d i n t h e w a s t e w a t e r a n d c r e a t e d by

I t i s w i d e l y known,

t h a t n i t r i f i c a t i o n i s inhibited i n

a c i d media and so t h e n i t r i f i c a t i o n r a t e s a r e v e r y low. h e r hand, pH

t h e s c t i v i t y o f n i t r i f y i n g b a c t e r i a was o b s e r v e d e v e n a t

= 4 131.

i n systems, l o w e r pH.

On t h e o t -

These f a c t s i n d i c a t e ,

t h a t i t i s neccessary t o maintain

a s u f f i c i e n t concentration o f n i t r i f y i n g bacteria a t a

I n t h i s s t u d y we h a v e o b s e r v e d t h e e f f e c t s o f

and t h e

a d d i t i o n o f b i o f i l m s u p p o r t s on n i t r i f i c a t i o n a n d on pH c r e a t e d b y t h e n i t r i f i c a t i o n . P i e c e s o f p o r o u s media - cubes from p o l y u r e t h a 3 served as f i x e d f i l m biomass s u p p o r t (system n e foam (1 cm )

-

LINPOR)

1 4 1 . We h a v e d e c i d e d t o a p p l y t h i s s y s t e m b e c a u s e o f i t s

technological

simplicity.

I n a compressed a i r a e r a t i o n t a n k ,

the

only step i s the i n s t a l l a t i o n o f a sieve a t the o u t l e t o f t h e tank.

EXPERIMENTAL The s u b s t r a t e c o n t a i n e d 480 mg.1-' c y c l o h e x a n o l and cyclohexanon.

o f HMT,

1 5 0 mg.1-'

The t o t a l C O D was 4 0 0 0 mg.1-l.

The l a b o r s t o r y s y s t e m s o p e r a t e d s e m i c o n t i n u o u s l y as c o m p l e t e l y mixed r e a c t o r .

o f NH;

and c o n t i n u o u s l y

Technological parameters are given i n

t a b l e 1. TABLE 1 T e c h n o l o g i c a l parameters o f models Parameter Volume

iemicontinual

111

Volumetric

94

l o a d i n g on COD [ k g . ~ n - ~ d - l

Recirculation r a t i o Solids retention time

[dl

Solids retention time only

5,

Continual

4.4

2

Hydraulic retention time [ h l

1

80

1.02

1.2

1

1

15,

25,

35

25

f o r suspended biomass.

K i n e t i c c o n s t a n t s o f n i t r i f i c a t i o n were d e t e r m i n e d b y b a t c h feed t e s t s .

A l l c h e m i c a l a n a l y s e s were c a r r i e d o u t a c c o r d i n g t o

t h e Czechoslovak S t a n d a r d Methods.

-

67

-

RESULTS A N D DISCUSSION The p o s i t i v e e f f e c t o f Elx a n d t h e p r e s e n c e o f f i x e d f i l m b i o mass i s c l e a r l y d e m o n s t r a t e d i n t a b l e 2.

Nitrification kinetic

t e s t s c a r r i e d o u t w i t h b o t h forms o f biomass h a v e shown, t h a t i n s p i t e o f h i g h Elx o f s u s p e n d e d b i o m a s s , occurred mainly i n biofilm.

(Fig.

1).

active nitrifying bacteria T h e pH o f a c t i v a t e d s l u d g e

m i x t u r e i n a c o n t i n u o u s l y o p e r a t e d c o m p l e t e l y mixed r e a c t o r v a r i e d from 3.9 t o 4.7.

Average r e s u l t s c a r r i e d out d u r i n g a p e r i o d o f

3 weeks o f o p e r a t i o n ( a f t e r 2 months of a d a p t a t i o n )

i n t a b l e 3.

are displayed

T h e e x t r e m e l y l o w pH v a l u e s were t h e o b j e c t o f t h e

following research.

The aim o f n e x t e x p e r i m e n t s was t o e x p l a i n t h e

mechanism o f n i t r i f i c a t i o n p r o c e s s e s i n b i o m a s s f i x e d i n p l a s t i c cubes.

TABLE 2 Influence of

ex

a n d f i x e d f i l m b i o m a s s o n pH a n d H M T r e m o v a l

Absence o f b i o f i l m O X

[dl

PH

-

Presence

HMT I m q . 1-’1

of biofilm

HMT

PH [d

-

I

[ mq.l-’l

5

7.4

470

5

6.6

418

15

6.9

460

15

6.2

400

25

6.6

422

25

5.3

365

35

6.4

406

35

4.2

270

E f l u e n t s from semicontinuously operated models, s t r a t e i n Experimental,

pH o f s u b s t r a t e 7 . 1 ,

composition of sub-

alkalinity o f substra-

te 5

TABLE 3 C o n t i n u o u s l y o p e r a t e d c o m p l e t e l y mixed r e a c t o r Psrameter

COD

HMT

NO;-N

NO;-

N

NH~-N

PH

mg. 1-11

Kinetic tests with continuously fed biomass confirned,

too,

t h a t o n l y a low p o r t i o n o f n i t r i f y i n g b a c t e r i a was i n s u s p e n d e d

form. H o w e v e r , t h e r e was n o n i t r i f i c a t i o n o b s e r v e d a t a pH b e l o w 5 , 3 i n b i o m a s s s q u e e z e d from s u p p o r t m a t e r i a l .

I t is v i s i b l e from

t a b l e 3,

68

-

t h a t n i t r i f i c a t i o n o c c u r r e d e v e n a t a pH l o w e r t h a n 5,3.

K i n e t i c t e s t s c a r r i e d o u t w i t h o n l y f i x e d f i l m b i o m a s s h a s shown t h s t t h e b i o m a s s f i x e d i n s u p p o r t m a t e r i a l p r o d u c e d NO;

a n d NO;

at

a l o w e r pH (4,O) t h a n t h e same b i o m a s s s q u e e z e d f r o m p l s s t i c c u b e s

(5,5). These r e s u l t s c o n f i r m e d t h e e x i s t e n c e o f m e d i a i n d e e p e r p a r t s o f support meterial,

w h e r e t h e pH i s h i g h e r t h a n i n t h e s u r -

rouding activsted mixture.

E x i s t e n c e o f t h i s media i s connected

with simultaneous d e n i t r i f i c a t i o n . h e r l o c a l c o n c e n t r a t i o n o f biomass,

I n biofilm,

according t o a hig-

organic substrate i s u t i l i z e d

i n an o x i c a n d i n d e e p e r p a r t s i n a n a n o x i c way.

i s c o n n e c t e d w i t h t h e p r o d u c t i o n o f OHo f m e d i a w i t h a h i g h e r pH.

Anoxic r e s p i r a t i o n

i o n s and w i t h t h e g e n e r a t i o n

To c o n f i r m t h i s t h e o r y ,

we h a v e m e a s u r e d

d e n i t r i f i c a t i o n r a t e s a t s e v e r a l d i s s o l v e d oxygen c o n c e n t r a t i o n s . F i g u r e 2 c l e a r l y demonstrates,

t h s t simultaneous d e n i t r i f i c a t i o n

3

t a k e s p l a c e i n b i o m a s s f i x e d i n p l a s t i c c u b e s ( 1 cm ) e v e n a t h i g h 0

concentrations.

2

PH

Fig.

-

8,

-

-

1. N i t r i f i c a t i o n r a t e s r N a t v s r i o u s pH. o suspended biomass, b i o f i l m fixed i n support, x b i o f i l m squeezed from s u p p o r t , 25 days ( o n l y f o r suspended biomass).

-

CONCLUSIONS

1. The p o s i t i v e e f f e c t confirmed.

o f f i x e d f i l m b i o m a s s o n n i t r i f i c a t i o n waa

A t a l o w e r pH,

n i t r i f i c a t i o n o c c u r r e d o n l y i n biomass

f i x e d i n support m a t e r i a l ( p l a s t i c cubes).

This fact

i s connec-

t e d w i t h t h e e x i s t e n c e o f m e d i a w i t h a h i g h e r pH i n d e e p e r p a r t s o f biofilm.

2.

A h i g h e r pH i s c r e a t e d b y OHnitrification.

i o n s p r o d u c e d b y s i m u l t a n e o u s de-

T h i s p r o c e s s was c o n f i r m e d i n 1 cm3 c u b e s w i t h a

f i x e d b i o m a s s e v e n a t O 2 c o n c e n t r a t i o n s o f 6.5

.

mg.1- 1

-

69

-

e

F i g . 2 . D e n i t r i f i c a t i o n r a t e s rD a t v a r i o u s 0 2 c o n c e n t r a t i o n s . Cuadded w i t h b e s w i t h b i o m a s s m i x e d w i t h a i r a n d n i t r o g e n b u b b l e s NO; s u b s t r a t e a t t h e b e g i n n i n g o f t h e t e s t , a l l y l t h i o u r e a added t o p r e 1 0 mg.1 vent n i t r i f i c a t i o n , c o n c e n t r a t i o n o f biomass i n cubes cube-', volume o f cubes = 1 5 X o f t o t a l r e a c t o r volume.

3.

The a c t i v a t e d s l u d g e p r o c e s s c o m b i n e d w i t h b i ' o f i l m c u l t i v a t i o n c s n be u t i l i z e d f o r HMT r e m o v a l f r o m i n d u s t r i a l w a s t e w a t e r cont a i n i n g h i g h e r c o n c e n t r a t i o n s o f NH;.

Non-

b i o d e g r a d a b l e HMT i s

h y d r o l y z e d i n a c i d media c r e a t e d by n i t r i f i c a t i o n t o biodegradab-

l e formaldehyde.

When t h e v o l u m e o f s u p p o r t m a t e r i a l o f f i x e d

f i l m b i o m a s s was 1 5 X o f t h e t o t a l a c t i v a t e d s l u d g e t a n k , c r e a t e d a n d m a i n t a i n e d i n t h e r e a c t o r v a r i e d f r o m 4.0

-

t h e pH

4.7.

( S u p p o r t s w e r e 1 cm3 c u b e s f r o m p o l y u r e t h a n e f o a m ) .

LITERATURE

1 2

3 4

3. Wanner, K . Kucman a n d P. Grau, W a t e r r e s . 22, 1 9 8 8 , 2 0 7 . W . Hegeman a n d E. Englmann, GWF Wasser - Abwasser 1 2 4 , 1 9 8 3 , 233. D . S . M a v i n i c and D.A. K o e r s , JWPCF 54, 1 9 8 2 , 342. W. Hegemann, W a t e r S c i . Tech. 1 6 , 1 9 8 4 , 1 1 9 .

-

This Page Intentionally Left Blank

HEXAMETHYLENETETRAAMINE REMOVAL I N SINGLE

J. DERCO'

I.B O O I K ,

a n d M.

-

SLUDGE A C T I V A T I O N S Y S T E M

DRTIL'

I n s t i t u t e o f Biotechnology, Slovak Technical U n i v e r s i t y , 81237 B r a t i s l a v a , Czecho-Slovakia ' F a c u l t y o f Chemistry and Chemical Technology, Slovak T e c h n i c a l U n i v e r s i t y , 81237 B r a t i s l a v a , C z e c h o - S l o v a k i a

H e x a m e t h y l e n e t e t r a a m i n e (HMT) stance which i s s o l u b l e i n water t l y b a s i c aqueous s o l u t i o n s . tics,

explosives,

i s a c o l o r l e s s c r y s t a l l i n e sub-

(813 g . l - l ,

12

OC)

and forms s l i g h -

HMT i s used i n t h e p r o d u c t i o n o f p l a s -

d r u g s and d i s i n f e c t a n t s .

The H M T m o l e c u l e h a s

A

s t a b l e s y m m e t r i c a l s t r u c t u r e t h a t causes

t h e r e s i s t e n c e o f HMT i n t h e p r o c e s s e s o f w a s t e w a t e r t r e a t m e n t . The d e g r e e o f HMT b i o l o g i c a l d e g r a d a t i o n i s r e p o r t e d i n l i t e r a t u r e i n t w o d i f f e r e n t ways ( 1 - 4 ) .

The a u t h o r s h a v e f o u n d e.g.

necessity o f long-term adaptation o f a c t i v a t e d sludge, ce o f a c i d h y d r o l y s i s on HMT removal e f f i c i e n c y , hydrolysis r a t e etc.

I n general,

the

the influen-

and l o w s p e c i f i c

t h e w o r k s d e a l i n g w i t h HMT d e g r a -

d a t i o n can be d i v i d e d i n t o s t u d i e s o f HMT b i o l o g i c a l d e g r a d a t i o n i n a c i d s o l u t i o n s a n d s t u d i e s o f HMT b i o l o g i c a l d e g r a d a t i o n i n n e u t r a l solutions. The s t u d i e s o f HMT b i o l o g i c a l d e g r a d a t i o n i n n e u t r a l m e d i a (ph

E

7)

i n d i c a t e t h a t t h e HMT m o l e c u l e s a r e n o t decomposed b y a c -

t i v a t e d sludge microorganisms.

HMT c a n b e r e m o v e d f r o m w a s t e w a t e r s

o n l y a f t e r c h e m i c a l h y d r o l y s i s i n a c i d media:

(CH2l6N4

+

4 H+

where t h e p r o d u c t s ,

+

6 H20

-

4 NH;

+

ammonia a n d f o r m a l d e h y d e ,

6 HCHO

(1)

a r e b i o d e g r a d a b l e com-

pounds.

A d e c r e a s e o f pH v a l u e s by t h e a d d i t i o n o f a c i d s ,

or using the

c a t a l y s t e f f e c t s o f some i n o r g a n i c or o r g a n i c compounds ( e . g . aldehydes,

g l y c o l etc.)

t h e HMT r e m o v a l .

HN02,

i n c r e a s e s t h e h y d r o l y s i s r a t e and improves

However,

these methods a r e connected w i t h problems

-

72

-

o f h i g h concentrations o f s a l t s i n waters.

These m e t h o d s a r e a v a i -

l a b l e f o r t h e t r e a t m e n t o f w a s t e w a t e r s w i t h h i g h HMT c o n c e n t r a t i o n s (more t h a n 500 mg.1-l)

and f o r s h o r t h y d r a u l i c r e t e n t i o n t i m e o f

t r e a t e d water. The a i m o f t h i s w o r k was t o d e t e r m i n e t h e p o s s i b i l t y o f c r e a t i n g a c i d media by n i t r i f i c a t i o n processes. N i t r i f i c a t i o n i s t h e b i o l o g i c a l o x i d a t i o n o f ammon a a n d i s c h a r a c t e r i z e d b y H+ i o n s p r o d u c t i o n :

N H + ~ 2

-

o2

P r o d u c e d H+ i o n s s u p p o r t a n d a t t h e same t i m e ,

+

NO;

2 H+

+

H20

t h e a c i d h y d r o l y s i s o f HMT ( e q u a t i o n 1)

t h e HMT h y d r o l y s i s p r o d u c t ,

t h e n i t r i f i c a t i o n process.

NH;

ions,

support

From t h e e q u a t i o n s 1 a n d 2 i t i s c l e a r ,

t h a t 1 mmol o f H M T ( 1 4 0 mg H M T )

f o r m s 7 2 mg NH;

t r i f i c a t i o n 4 mmol H+ i o n s a r e p r o d u c e d . t h e t o t a l a l c a l i n i t y o f waste water,

i o n s and a f t e r n i -

T h i s p r o c e s s d e p e n d s on

BOO5,

HMT c o n c e n t r a t i o n and

n i t r i f y i n g bacteria activity. The n i t r i f i c a t i o n p r o c e s s i s i n h i b i t e d a t pH v a l u e s b e l o w 6 , 5 . the l i t e r a t u r e (5

Hcwever,

v e d e v e n a t pH

=

4.0.

-

8) shows t h a t n i t r i f i c a t i o n was o b s e r -

Low s p e c i f i c n i t r i f i c a t i o n r a t e s i n a c i d m e -

d i a can be compensated b y h i g h e r s o l i d s r e t e n t i o n t i m e ( S R T , values,

ex)

which support t h e existence o f slowly growing n i t r i f y i n g

bacteria.

EXPERIMENT The H M T b i o l o g i c a l d e g r a d a t i o n was s t u d i e d i n l a b o r a t o r y s c a l e semi-continuous

and c o n t i n u o u s models w i t h p a r a m e t e r s i n Table 1

and Table 2 . These p a r a m e t e r s w e r e s e t up t o s i m u l a t e r e a l p a r a m e t e r s i n t h e w a s t e w a t e r t r e a t m e n t p l a n t i n c h e m i c a l e n t e r p r i s e CHEMKO S t r A f s k e (Czecho-slovakia).

A n a l y t i c a l methods used i n t h i s work were c a r r i e d

o u t a c c o r d i n g t o Czecho-slovak

s t a n d a r d m e t h o d s ( 9 ) . The H M T c o n c e n -

t r a t i o n was d e t e r m i n e d b y t h e s p e c t r o p h o t o m e t r i c m e t h o d w i t h c h o rotropic acid after acid d i a t i l a t i o n /lo). RESULTS AND D I S C U S S I O N

1. S e m i - c o n t i n u o u a

System

The HMT c o n c e n t r a t i o n was g r a d u a l l y rimente.

i n c r e a s e d d u r i n g t h e expe-

The t i m e o f a d a p t a t i o n was 1 0 0 d a y s .

The u s e d c o n c e n t r a t i o n s

- 73

-

Substrate

Model parameters parameter

amount

peptone

3.0

9.1-1

starch

0.5

g . 1-1

-

KH2P04

0.05

g.1-l

PH

7.2

COD

4.2

9.1-l

5.5

mmol.1

2 1

e

SRT2e

concentration

100 h

volume V

HRT'

componen t

5

50 d

recirculation

1

ratio R

total alcalinity

Model parameters

-__

parameter

-1

Substrate amount

component

concentration

! 1

SRT'

4

V

volume

HRT'

e e

1

120 h 50 d

recirculation ratio

3

R

cyclohexanole

150

mg.1-'

HMT

225

mg.1"

NH4C1

ZOO

mg.1-l

KH 2P04

150

mg.1-I

COD

550

mg.1-l

PH

7.0

total 6.5

alcalinity

... ...

HRT SRT

mmol ..l-l

hydraulic r e t e n t i o n time s o l i d s r e t e n t i o n time

o f HMT h a v e n o t e x h i b i t e d any a f f e c t s o n b i o m a s s . t i n u o u s s y s t e m s t h e S R T v a l u e s were 5 ,

10,

15,

I n s i x semi-con-

2 0 , 3 0 a n d 50 d a y s .

Some m e a s u r e d d a t a c a n b e f o u n d i n T a b l e 3 .

I t i s c l e a r t h a t n i t r i f y i n g a c t i v i t y c o n n e c t e d w i t h H+ i o n p r o d u c t i o n i m p r o v e s t h e HMT d e g r a d a t i o n . lue o f

8,

= 5

F o r example,

d any pH d e c r e a s e a n d any NO;

was n o t o b s e r v e d ,

i n such systems.

a n d NO;

when t h e vaproduction

t h a t means any n i t r i f i c a t i o n d o e s n o t t a k e p l a c e When

8,

was i n c r e a s e d t o t h e v a l u e s h i g h e r t h a n

10 days,

-

n i t r i f i c a t i o n ( d e c r e a s e o f pH, p r o d u c t i o n o f NO;

HMT d e g r a d a t i o n )

l y s i s rate.

8,

a n d NO;,

was o b s e r v e d .

The c r e a t i o n o f NH; 1 for

74

i o n s i s connected w i t h a h i g h e r HMT hydro-

Time d e p e n d e n c e o f m e a s u r e d p a r a m e t e r s i s shown i n F i g . 50 days.

I n spite of inhibited nitrification,

t h e pH c r e a t e d a n d m a i n -

t a i n e d d u r i n g t h i s p r o c e s s i s a v a i l a b l e f o r HMT r e m o v a l .

PH

O X

(d)

2.

COD

[?;I

5

8.5

46.4

10

6.6

56.2

‘b

HMT

tXI

[g

1.1

.1-’3

0.7

18.2

1.7 3.0

15

5.8

61.4

25.5

20

5.7

67.4

33.6

3.9

30

5.5

70.0

41.3

5.7

50

5.2

73.0

52.5

1.0

C o n t i n u o u s System The p r o b l e m s c o n n e c t e d w i t h t h e s e m i - c o n t i n u o u s

c y c l e can be

s o l v e d i n c o n t i n u o u s system ( T a b l e 2 ) . Long h y d r a u l i c r e t e n t i o n t i me 8 a n d s o l i d s r e t e n t i o n t i m e e x c a u s e a h i g h e f f i c i e n c y o f HMT r e moval, 5.7.

t h a t was more t h a n 7 0 X w i t h t h e pH i n t h e r a n g e o f 5.4

The c o n s e q u e n c e o f n i t r i f i c a t i o n was t h e f o r m a t i o n

-

o f NO;

a n d NH+* i o n s .

CONCLUSIONS I n t h i s work,

t h e e f f e c t o f t h e n i t r i f i c a t i o n p r o c e s s o n HMT

r e m o v a l f r o m w a s t e w a t e r s was o b s e r v e d .

H i g h e r v a l u e s o f SRT and

o x y g e n c o n c e n t r a t i o n a r e an a s s u m p t i o n f o r n i t r i f i c a t i o n p r o c e s s e s i n aystema.

The a c i d m e d i a , t h a t i s c a u s e d b y n i t r i f i c a t i o n i n wa-

t e r s w i t h low b u f f e r q u a l i t i e s ,

s u p p o r t s t h e a c i d h y d r o l y s i s o f HMT.

The r e s u l t o f a l l t h e s e p r o c e s s e s i s t h e i n c r e a s e o f HMT r e m o v a l efficiency without

t h e a d d i t i o n o f a c i d s t o t h e system.

-

75

-

6

-*-+

0

12

2L

36 t [hl

F i g . 1. Time d e p e n d e n c e o f m e a s u r e d p a r a m e t e r s d u r i n g one a e r a t i o n - HMT’ Bx = 50 d a y s (1 - pH, 2 - COD, 3 c cycle for NO;? C NO;

-

-

LITERATURE

1 2 3 4 5 6

E . Gomolka a n d B. Gomolka, R e s i s t e n c e o f H e x a m e t h y l e n e t e t r a a r n i n e t o B i o d e g r a d a t i o n i n A e r a t e d M u n i c i p a l Sewage. E n v i r o n . P r o t . Eng., 1 0 ( 1 9 8 4 ) 29-43. A . S m i t h and K.O. Colquhoun, B i o d e g r a d a b i l i t y o f Hexamethylenet e t r a a m i n e . Chernosphere, 16 ( 1 9 8 7 ) 1 5 5 5 - 1 5 5 6 . H . Tada, D e c o m p o s i t i o n R e a c t i o n o f Hexearnine b y A c i d . J. Am. Chem. S O C . 82 ( 1 9 6 0 ) 255-263. V . 6 e r e f n ) i a n d J. M i S k o v c o v B , S t u d y o f t h e B i o d e g r a d a b i l i t y o f H e x a m e t h y l C n e t e t r a a m i n e . V o d n f h o s p o d e r s t v f , 39 8 ( 1 9 8 9 ) 1 0 6 - 1 0 9 . D.S. M a v i n i c a n d D.A. Koera, F a t e o f N i t r o g e n i n A e r o b i c Sludge D i g e s t i o n . J. Wat. P o l l u t . C o n t r o l Fed. 5 4 ( 1 9 8 2 ) 3 4 2 - 3 6 0 . D.S. Bharqava and M.T. D a t a r , N i t r i f i c a t i o n D u r i n a A e r o b i c D i gestion A c t i v a t e d 5ludge:Effluent Wet. T r e a t . - J . , 24 (1984) 352-355.

OF

7 8 9 10

76

-

E . K o w a l s k i a n d Z . L e w a n d o w s k i , N i t r i f i c a t i o n P r o c e s s i n a Pac h e d Bed R e a c t o r w i t h a C h e m i c a l l y A c t i v e Bed. Wat. Res., 17 ( 1 9 8 3 ) 157-160. Z . Lewadowski, N i t r i f i c a t i o n P r o c e s s i n A c t i v a t e d Sludge w i t h Suspended M a r b l e P a r t i c l e s . Wet. Res., 1 9 ( 1 9 8 5 ) 535-539. M. H o r A k o v 6 , P. L i s c h k e a n d A . G r u n w a l d , C h e m i c k e a f y z i k h l n f m e t o d y a n a l f z y vod. SNTL A l f a , P r a h a 1 9 8 8 . Anon.: J e d n o t n i m e t o d y c h e m i c k e h o r o z b o r u v o d . SNTL, P r a h a 1965.

E X P E R I M E N T A L A N D M A T H E M A T I C A L M O D E L L I N G OF A C T I V A T E D SLUDGE PROCESS P . F A R K A S O V A l , J . DERCO'

and M.

KRALIK'

Department o f E n v i r o n m e n t a l Chemistry and Technology, S l o v a k Technical Univeraity, Bratislava Department o f Organic Technology, Slovak Technical U n i v e r s i t y , Bratislava SUMMARY T h i s p a p e r d e a l s w i t h e x p e r i m e n t a l i n v e s t i g a t i o n a n d m at h em at i c a l m o d e l l i n g o f a c t i v a t e d s l u d g e p r o c e s s . T h r e e m a t h e m a t i c a l mod e l s f o r p r o c e s s d e s c r i p t i o n were d e r i v e d , e x p e r i m e n t a l l y v e r i f i e d f o r a t r a n s i e n t s u b s t r a t e c o n c e n t r a t i o n l o a d a n d t h e r e s u l t s were c o m p a r e d . The f i r a t t w o m o d e l s a r e b a s e d o n s u b s t r a t e a n d b i o m a s s m a t e r i a l b a l a n c e s a r o u n d a n a e r a t e d t a n k . The t h i r d o n e i s a d i s c r e t e s t a t i s t i c a l m o d e l . The b e s t f i t b e t w e e n e x p e r i m e n t a l a n d c s l c u l a t e d v a l u e s was o b t a i n e d u s i n g t h e s t a t i s t i c a l m o d e l . PREFACE C u r r e n t l y , t h e moat economic t r e a t m e n t o f w a s t e w a t e r s c o n t a i n i n g b i o d e g r a d a b l e o r g a n i c c o m p o u n d s i s a c h i e v e d by t h e u t i l i z a t i o n o f b i o c h e m i c a l r e a c t i o n s m e d i a t e d by m i c r o o r g a n i s m s . One o f t h e moat w i d e l y u s e d b i o l o g i c a l t r e a t m e n t methods t h a t r e q u i r e s a n a e r o b i c environment f o r e f f e c t i v e o p e r a t i o n is t h e a c t i v a t e d s l u d g e pro-

cess 1 1 1 . Because o f time v a r i a t i o n a n d c h a n g e i n t h e f l o w r a t e a n d t h e composition of t h e wastewater, t h e process is usually operated under highly variable losding conditions with variability of effluent q u a n t i t y . Many d e s i g n e n g i n e e r s d o n o t a p p r e c i a t e t h e d y n a m i c c h a m r a c t e r of t h e p r o c e s s , and s t e a d y - s t a t e p r o c e d u r e s are used.

Tran-

s i e n t l o a d i n g s t u d i e s o f a c t i v a t e d s l u d g e p r o c e s s e s a r e of i n t e r e s t b o t h t o m i c r o b i o l o g i s t s and e n g i n e e r s . M i c r o b i o l o g i a t s have been i n t e r e s t e d i n t h e dynamics o f m i c r o b i a l r e s p o n s e from t h e p o i n t o f

.

view t h a t i n f o r m a t i o n o b t a i n e d c o u l d b e h e l p f u l t o u n d e r s t a n d i n g t h e mechanisms i n v o l v e d i n m i c r o b i a l growth and r e p r o d u c t i o n .

Practical

i n t e r e s t o f e n g i n e e r s is focussed on d e s i g n and c o n t r o l o f t h e a c t i vated sludge process.

The i n c r e a s i n g a v a i l a b i l i t y o f c o m p u t e r s e n -

c o u r a g e s t h e uae o f m a t h e m a t i c a l models, which are a p p l i c a b l e b o t h

-

-

78

i n d e s i g n and o p e r a t i o n o f w a s t e w a t e r

treatment plants.

A l a r g e amount o f l i t e r a t u r e h a s a p p e a r e d i n t h e l a s t t w e n t y y e a r s on t h e d y n a m i c m o d e l l i n g o f a c t i v a t e d s l u d g e p r o c e s s e s . o f the transient

Many

l o a d i n g s t u d i e s have been p e r f o r m e d u s i n g t h e c o n t i -

nuous f l o w s t i r r e d t a n k r e a c t o r w i t h o u t s l u d g e r e c y c l e s t a t e o f wastewater

t r e a t m e n t system c o n t r o l ,

f2-81.

The

d y n a m i c m o d e l s a n d i-

d e n t i f i c a t i o n a p p l i c a t i o n s h a v e b e e n r e v i e w e d b y Andrews [ 9 1 a n d Olson (101.

Dynamic b e h a v i o u r o f a c t i v a t e d s l u d g e has been s t u d i e d

u s i n g c o m p u t e r s i m u l a t i o n b y B u s b y 1111. A t t i r

1 1 2 1 has d e s c r i b e d

t h e r e s u l t s o f a s i m u l a t i o n s t u d y o f t h e dynamics and c o n t r o l o f t h e a c t i v a t e d sludge process,

u s i n g a model o f dynamics o f c o n t i n u o u s

s e d i m e n t a t i o n t h a t a c c o u n t s f o r s e v e r a l d i s t i n c t models o f operation.

A dynamic model o f n i t r i f i c a t i o n i n t h e a c t i v a t e d s l u d g e p r o -

c e s s has been developed,

a n d an e x p e r i m e n t a l a n d s i m u l a t i o n s t u d y

h a s b e e n p r e s e n t e d b y P o d u s k a 1131.

T h k r i e n I 1 4 1 h a s p r e s e n t e d a mo-

d e l f o r p r e d i c t i n g t h e dynamics o f oxygen u t i l i z a t i o n i n t h e a c t i v a t e d sludge process.

M o d e l s m e n t i o n e d above u s e u s u a l l y f o r p r o c e s s

d e s c r i p t i o n Monod t y p e k i n e t i c e q u a t i o n s f o r a m i c r o b i a l g r o w t h a n d Michaelis-Menten type k i n e t i c equations f o r t h e b i o l o g i c a l o x i d a t i o n o f organic substrate.

These t y p e s o f m o d e l s c o n t a i n a l a r g e s e t o f

d i f f e r e n t i a l e q u a t i o n s i n which t h e k i n e t i c c o n s t a n t s appear.

The

v a l u e o f t h e s e k i n e t i c parameters i s sometimes d i f f i c u l t t o d e t e r m i ne w i t h p r e c i s i o n .

From t h e p r a c t i c a l p o i n t o f view t h e s t a t i s t i c a l

model c o u l d a l s o be a v a i l a b l e f o r t h e a c t i v a t e d s l u d g e p r o c e s s control.

Berthouex

1151 h a s d e v e l o p e d s t a t i s t i c a l t e c h n i q u e s t o s t u d y

t h e d a t a from m u n i c i p a l wastewater

treatment plants.

died the impact o f operating conditions,

Hansen I 1 6 1 s t u -

waste c o n t r i b u t i o n s ,

e n v i r o n m e n t a l f a c t o r s on t r e a t m e n t o f i n d u s t r i a l wastewater

and

evaluated

using a s t a t i s t i c a l technique. The p u r p o s e o f t h i s w o r k was t o i n v e s t i g a t e t h e r e s p o n s e t o quantitative

s h o c k l o a d i m p o s s e d u p o n t h e p r o c e s s a n d t o e x a m i n e a-

v a i l a b i l i t y o f t h r e e d e r i v e d m a t h e m a t i c a l models f o r p r o c e s s desc r i p t i o n under t r a n s i e n t

conditions.

MATHEMATICAL MODEL DESCRIPTION

F o r t h e m a t h e m a t i c a l p r o c e s s d e s c r i p t i o n we d e r i v e d t h r e e models.

The f i r s t two a r e k i n e t i c models,

t h e t h i r d one i s a s t a t i s t i -

c a l model. The s u b s t r a t e a n d b i o m a s s m a t e r i a l b a l a n c e f o r a c o m p l e t e l y m i x e d b i o r e a c t o r i n k i n e t i c m o d e l s may be d e s c r i b e d i n e q u a t i o n s (1, 2 ) .

-

79

-

Substrate balance

(Symbols a r e e x p l a i n e d a t t h e end o f t h i s p a p e r ) . Biomass b a l a n c e dX R9

-

= dt

qx

Michaelis-Menten

(2)

'a

t y p e r e l a t i o n s h i p was u s e d f o r d e s c r i p t i o n

o f s u b s t r a t e u t i l i z a t i o n r a t e rs

r

x.5

= - - pmax

8

K~

'obs

+

Monod r e l a t i o n s h i p was u s e d f o r d e s c r i p t i o n o f maximum m i c r o o r g a n i s m s g r o w t h r a t e pmax

5 "max

Model No.

(4)

KS

+

2 i s b a s e d on t h e same p r i n c i p l e s ,

l a t i o n s h i p i s extended,

b u t t h e Monod r e -

and s l s o t a k e s i n t o a c c o u n t t h e decay o f

which can be d e s c r i b e d as

microorganisms,

S

p

=

%ax

Ks

+

The e x p l i c i t f o u r t h - o r d e r

-

(5)

kd.X

S

Runge-Kutta-Merson

m e t h o d [ 1 7 1 was

used f o r s o l v i n g t h e s e t o f d i f f e r e n t i a l equations.

The s i m p l e x o p -

t i m i z a t i o n t e c h n i q u e 1 1 8 1 waa u s e d t o d e t e r m i n e t h e v a l u e o f b i o k i n e t i c p a r a m e t e r s by m i n i m i z a t i o n o f t h e f u n c t i o n

To r e d u c e t h e c o m p u t a t i o n a l t i m e , r i z a t i o n u s i n g t h e T a y l o r s e r i e s 1191.

we u s e d t h e m e t h o d o f l i n e a -

-

80

-

The t h i r d m o d e l i s a d i s c r e t e s t a t i s t i c a l m o d e l w i t h one i n p u t a n d one o u t p u t ,

and c a n b e d e s c r i b e d a s

... an,

The p a r a m e t e r s al

...

bo

bm w e r e e s t i m a t e d u s i n g l i n e a r

[ 19 1 .

regression

EXPERIMENTAL The e x p e r i m e n t s w e r e c a r r i e d o u t i n l a b o r a t o r y a p p a r a t u s c o n s i s t i n g o f a c o m p l e t e l y m i x e d b i o r e a c t o r and s e t t l e r . had a volume o f a p p r o x i m a t e l y f e e d f l o w r a t e o f 3.06

4.4

ml.min-l.

The b i o r e a c t o r

1 and was s u p p l i e d w i t h a c o n s t a n t T h r e e a i r d i f f u s e r s a t r i g h t an-

g l e s a t the bottom o f the bioreactor provided aeration o f the biomass.

S l u d g e was c o n d i t i o n e d t o a f e e d c o n t a i n i n g c y c l o h e x a n o l e

( c = 0.56 (c

=

0.33

dehyde ( c

k ~ j . m - ~ ) c, y c l o h e x a n o n e ( c

= 0.56

k g . n ~ - ~ ) ,p e n t a e r y t r i t o l

k g . ~ n - ~ ) ,h e x a m e t y l e n e t e t r a a m i n e ( c

=

0.0375

0.48

k g . ~ n - ~ ) ,as s o u r c e s o f c a r b o n ,

a m o u n t s o f m i n e r a l s a l t s a c c o r d i n g t o t h e r a t i o C:N:'P The a v e r a g e COD v a l u e was 3.894

k g . n ~ - ~ ) ,f o r m a l -

besides the proper o f 100:5:1.

k g . ~ n - ~ .The b i o d e g r a d a b l e p o r t i o n

o f s u b s t r a t e corresponds t o 0.5.

The s e t t l e d s l u d g e was r e c y c l e d t o

t h e b i o r e a c t o r u s i n g a p e r i s t a l t i c pump a n d t h e s l u d g e was w a s t e d f r o m t h e a e r a t i o n t a n k i n o r d e r t o m a i n t a i n t h e mean s l u d g e age Qx

= 10

days.COD,

suspended s o l i d s ,

NH;

and f o r m a l d e h y d e c o n c e n t r a -

t i o n a n a l y s i s were p e r f o r m e d f o l l o w i n g s t a n d a r d m e t h o d s b y 1201. pH and d i s s o l v e d o x y g e n m e a s u r e m e n t s w e r e p e r f o r m e d u s i n g a R a d e l k i s Aquacheck. R E S U L T S AND D I S C U S S I O N

Before s t a r t i n g t h e s t e p shock l o a d , under steady-state

conditions.

t h e s y s t e m was o p e r a t e d

A f t e r reaching the steady-state,

( o u t p u t C O O and M L S S v a l u e s were o s c i l a t i n g a r r o u n d s t a t i o n a r y

va-

l u e s ) t h e s t e p t w o f o l d d e c r e a s e i n s u b s t r a t e c o n c e n t r a t i o n was i m possed t o t h e system.

The d i s s o l v e d o x y g e n c o n c e n t r a t i o n

r e a c t o r was a l w a y s m a i n t a i n e d a b o v e t h e g r o w t h - l i m i t i n g i.e. the

above 2 m g . 1 - l .

i n the bio-

conditions,

The a v e r a g e C O D s u b s t r a t e c o n c e n t r a t i o n a f t e r

s t e p c h a n g e was 1 8 0 0 m g . 1 - l .

The C O O c o n c e n t r a t i o n r e s p o n s e o f

t h e a c t i v a t e d s l u d g e r e a c t o r i s shown i n F i g .

1. One c a n o b s e r v e

oscillations

COO v a l u e s .

-

r a n g i n g a b o v e a n d b e l o w new s t e a d y - s t a t e

possed change. steady-state

81

after

an i m -

R e l a t i v e l y s m o o t h t r a n s m i t i o n s w e r e made t o t h e new

i n t h e case o f biomass c o n c e n t r a t i o n i n comparison w i t h

I n l e s s t h a n 1 0 d a y s a f t e r s h o c k was i m p o s e d , c h a n g e s o f

biomass c o l o u r and f l o c s m a c r o s c o p i c appearance were observed. new s t e a d y - s t a t e ved,

was r e a c h e d ,

b u t no changes i n m a g n i t u d e o f

o f the transient

After

t h e i n i t i a l b i o m a s s c o l o u r was o b s e r s l u d g e f l o c s had occured.

Some

r e s p o n s e d a t a were u s e d t o o b t a i n t h e b i o k i n e t i c

parameters f a r t h e m a t h e m a t i c a l models.

The c a l c u l a t e d COD v a l u e s

d u r i n g t h e s t e p s h o c k t e s t u s i n g t h e Monod and m o d i f i e d Monod equat i o n are a l s o p l o t t e d i n F i g .

l. F r o m t h i s F i g . l one c a n s e e t h a t

a b e t t e r f i t b e t w e e n e x p e r i m e n t a l a n d c a l c u l a t e d d a t a was o b t a i n e d

n 0 u

T O

5

1;

1;

2b

215

o;

3;j

Lb

L; t IDAYSl

F i g . 1. C O O c o n c e n t r a t i o n r e s p o n s e o f a c t i v a t e d s l u d g e p r o c e s s t o twofold decreasing o f feed concentration experimental - - - - - - c a l c u l a t e d u s i n g Monod e q u a t i o n . . . . . . . . . . . c a l c u l a t e d u s i n g m o d i f i e d Monod e q u a t i o n .

-

u s i n g t h e m o d i f i e d Monod e q u a t i o n .

The g r e a t e s t d i f f e r e n c e s between

e x p e r i m e n t a l a n d c a l c u l a t e d v a l u e s w e r e o b s e r v e d i n t h e c a s e o f COD d u r i n g t h e f i r s t stage o f t h e experimental run.

One c a n c o n c l u d e t h a t

an i n s t a n t a n e o u s c h a n g e i n e p e c i f i c g r o w t h r a t e d i d n o t o c c u r a s p r e d i c t e d b y t h e Monod e q u a t i o n . w o r k o f Mor 1 2 1 1 .

These r e s u l t s a r e c o n s i s t e n t w i t h t h e

I t was o b s e r v e d t h a t t h e t i m e d e l a y f o r t h e s y s t e m

t o r e s p o n d t o t h e s h o c k was d e p e n d e n t o n t h e m a g n i t u d e o f t h e s p e c i f i c growth rate.

I t s h o u l d be n o t e d t h a t s a t u r a t i o n c o n s t a n t s c a l c u -

l a t e d ( f o r t h e Monod m o d e l

-

82

K s = 0 . 7 8 4 kg.m-3

a n d f o r m o d i f i e d Monod

K s = 0.786 k g . ~ n - ~ )a r e s i g n i f i c a n t l y h i g h e r t h a n t h o s e p u b l i s h e d e l s e w h e r e 1 2 2 1 . We assume t h a t t h i s i s b e c a u s e o f a r e l a t i v e l y h i g h p o r t i o n o f b i o l o g i c a l l y r e s i s t e n t HMT compound i n t h e f e e d o f t h e a c t i v a t e d s l u d g e p r o c e s s o p e r a t e d u n d e r pH v a l u e s a b o u t 7 .

Comparing

c a l c u l a t e d a n d e x p e r i m e n t a l v a l u e s one c a n s e e t h a t t h e M o n o d - t y p e k i n e t i c e q u a t i o n i s n o t adequate f o r t h e p r e d i c t i o n o f t r a n s i e n t b e h a v i o u r o f an a c t i v a t e d s l u d g e p r o c e s s . S i m i l a r r e s u l t s w e r e o b t a i n e d a l s o u s i n g l i n e a r i z e d Monod a n d m o d i f i e d Monod e q u a t i o n s . E x p e r i m e n t a l a n d c a l c u l a t e d v a l u e s o f COD c o n c e n t r a t i o n u s i n g s t a t i s t i c a l m o d e l s a r e shown i n F i g .

2.

2 one c a n c o n f i r m

From F i g .

t h a t t h e d i s c r e t e s t a t i s t i c a l m o d e l i s more s u i t a b l e f o r d e s c r i b i n g a c t i v a t e d sludge process dynamics.

The c o m p u t a t i o n s 1 t i m e f o r t h e

m o d e l p a r a m e t e r s e s t i m a t i o n was s i g n i f i c a n t l y s h o r t e r i n c o m p a r i s o n t o b o t h n o n l i n e a r and l i n e a r i z e d k i n e t i c models.

mL

0.6

l5

Y

I

0

0

0.5

OA

0.3

0.2

(

5

10

15

20

25

30

35

40

45 t [ OAYSl

F i g . 2. C O D c o n c e n t r a t i o n r e s p o n s e o f a c t i v a t e d s l u d g e p r o c e s s t o twofold decreasing o f feed concentration experimental ------ c a l c u l a t e d u s i n g d e s c r e t e s t a t i s t i c a l model.

CONCL US I ON5 The m a j o r o b j e c t i v e o f t h i s w o r k was t o e x a m i n e t h e e x p e r i m e n t a l l y dynamic b e h a v i o u r o f a c t i v a t e d s l u d g e process.

Previously the

-

83

-

o b t a i n e d e x p e r i m e n t a l d a t a was u s e d f o r v e r i f i c a t i o n o f t h r e e d e r i ved m a t h e m a t i c a l models.

From c a l c u l a t i o n s we c a n assume,

that the

Monod-type k i n e t i c models a r e n o t a c c u r a t e i n t h e p r e d i c t i o n o f t r a n s i e n t behaviour o f t h e a c t i v a t e d sludge process. r i f i e d model

-

d i s c r e t e s t a t i s t i c a l model

-

The t h i r d ve-

provides a better f i t

between e x p e r i m e n t a l and c a l c u l a t e d v a l u e s . The n e x t s t e p o f o u r work i s t o v e r i f y a s t a t i s t i c a l m o d e l u s i n g more i n p u t s . SYMBOLS ai,

bi

parameters o f d i s c r e t e s t a t i s t i c a l model

J

COD kd KS

c h e m i c a l o x y g e n demand endogenous decay c o e f f i c i e n t substrate saturation constant

M

number o f m e a s u r e m e n t s

m

number o f p a r a m e t e r s b .

n

number o f p a r a m e t e r s a

9

feed flow r a t e

qX

rs R

9

RS

S

J

i

biomass wastage r a t e substrate u t i l i z a t i o n rate microbial growth r a t e substrate removal r a t e e f f l u e n t substrate concentration

=o

feed substrate concentration

t

time

A t

time

U

i n p u t i n s t a t i s t i c a l model

Ut "a

X 'obs Y

yY L1

Pmax

interval

value o f u i n time t a e r a t i o n t a n k volume mixed l i q u o r suspended s o l i d s ( M L S S ) observed y i e l d c o e f f i c i e n t o u t p u t i n s t a t i s t i c a l model value o f y i n time t s p e c i f i c growth r a t e maximum s p e c i f i c g r o w t h r a t e

w

minimization

e

s l u d g e age

function

-

84

-

REFERENCES

1 2 3 4 5 6 7 8 9 10

11 12 13 14 15 16 17 18 19 20 21 22

M e t c a l f Eddy, W a s t e w a t e r E n g i n e e r i n g , T r e a t m e n t , d i s p o s a l , r e u s e , M c G r a w - H i l l I n c . , New Y o r k , 1972. K . K o m o l r i t a n d A.F. Gaudy, J o u r n a l WPCF, 3 8 , 85, 1 9 6 6 . K. K o m o l r i t a n d A.F. Gaudy, J o u r n a l WPCF, 3 8 , 1 2 5 9 , 1 9 6 6 . J.R. M o r a n d A. F i e c h t e r , B i o t e c h . a n d B i o e n g . , X , 7 8 7 , 1968. J.W. G i l l e y a n d H.R. Bungay, B i o t e c h . a n d B i o e n g . , I X , 6 1 7 , 1967. J.W. G i l l e y a n d H.R. Bungay, B i o t e c h . a n d B i o e n g . , X, 99, 1968. T.B. Young e t a l , B i o t e c h . a n d B i o e n g . , X I I , 747, 1970. S. Koga a n d A.F. Humprey, B i o t e c h . a n d B i o e n g . , I X , 3 7 5 , 1 9 6 7 . J.F. Andrews, W a t e r Res., 8, 261, 1974. G. O l s o n , A I C h E Symp. S e r . N. 1 5 9 , 72, 52, 1976. J.B. B u s b y a n d J.F. Andrews, J o u r n a l WPCF, 47, 1055, 1 9 7 5 . V . A t t i r a n d M.M. Denn, A I C h E J . 24,, 6 9 3 , 1978. R.A. P o d u s k a , J.F. Andrews, J o u r n a l WPCF, 4 7 , 2599, 1 9 7 5 . N. T h 6 r i e n a n d S. P e r d i e u x , J o u r n a l WPCF, 53, 576, 1 9 8 1 . P . M . B e r t h o u e x e t a l l . , W a t e r Res., 1 0 , 6 8 9 , 1976. J.L. Hansen, A.E. F i o k and J.C. H o v i o u s , J o u r n a l WPCF, 52, 1 9 6 6 , 1980. M. K u b f E e k , N u m e r i c k 6 a l g o r i t m y GeSeni c h e m i c k o - i n f e n f r s k f c h d l o h , SNTL P r a h a 1 9 8 3 . D.A. P i e r r e , O p t i m i z a t i o n t h e o r y w i t h a p p l i c a t i o n s , J . W i l l e y , New Y o r k 1969. R e k t o r y s a k o l . , P F e h l e d u f i t b m a k e m a t i k y SNTL P r a h a 1 9 8 8 . M. HorBkovB, P. L i s c h k e a n d A . G r u n w a l d , M e t o d y c h e m i c k b a n a l y z y vod, SNTL P r a h a , 1981. J.R. Mor a n d A. F i e c h t e r , B i o t e c h . a n d B i o e n g . , X, 7 8 7 , 1 9 6 8 . G. T c h o b a n o g l o u s , W a s t e w a t e r E n g i n e e r i n g , M c G r a w - H i l l , I n c . , New Y o r k , 1977.

AEROBIC THERMOPHILIC SLUDGE TREATMENT

M . BOMIO Department o f B i o t e c h n o l o g y , Swiss F e d e r a l I n s t i t u t e o f Technology, 8093 Z i r i c h , S w i t z e r l a n d

1

INTRODUCTION

1.1 S l u d g e t r e a t m e n t A c h a r a c t e r i s t i c o f most i n d u s t r i a l i z e d c o u n t r i e s i s c o m b i n e d t r e a t m e n t o f domestic and i n d u s t r i a l l i q u i d wastes.

The p r i m a r y ob-

j e c t i v e o f w a s t e w a t e r t r e a t m e n t p l a n t s i s a h i g h degree o f w a t e r pur i f i c a t i o n with r e l a t i v e l y l e s s a t t e n t i o n directed t o the primary byproduct,

sewage s l u d g e .

The e c o n o m i c a l v a l u e o f sewage s l u d g e i s

amongst t h e l o w e s t o f a l l m a t e r i a l s a v a i l a b l e on e a r t h ,

b u t even so

t h e sludge w i l l c o n t i n u e t o be produced i n very l a r g e q u a n t i t i e s i n t h e f u t u r e a n d i t s e f f e c t i v e t r e a t m e n t w i l l b e one o f t h e m o a t i m p o r t a n t problems t o solve. I n Switzerland,

where 95 X o f t h e p o p u l a t i o n i s c o n n e c t e d t o

one o f t h e 1 0 0 9 w a s t e w a t e r t r e a t m e n t p l a n t s , sewage s l u d g e i s 4 000 000 m 3 a - l o r 250 000 t corresponding t o 0 , l

kg d - l p e r s o n - '

t h e p r o d u c t i o n o f wet 8-l

dry matter.

p f dry matter, This high quantity

o f sewage s l u d g e i s t y p i c a l f o r i n d u s t r i a l i z e d c o u n t r i e s a n d w i l l even i n c r e a s e i n t h e f u t u r e ,

p a r t i c u l a r l y i n t h o s e c o u n t r i e s where

w a s t e w a t e r t r e a t m e n t i s ' s t i l l e x p a n d i n g (BUWAL 1 9 9 0 ) . I n Switzerland,

culture,

t h e sludge produced i s p r e s e n t l y used i n a g r i -

d e p o s i t e d i n l a n d f i l l s i t e s or i n c i n e r a t e d .

The amount r e -

30 % o f t h e t o t a l p r o d u c t i o n ;

cycled i n a g r i c u l t u r e i s approximately a percentage t h a t v a r i e s from r e g i o n t o

region.

A b o u t 40-50

s l u d g e p r o d u c e d i s d e p o s i t e d i n l a n d f i l l s and 10-30

% o f the

X incinerated.

The m a g n i t u d e o f t h e s l u d g e p r o b l e m c a n b e i l l u s t r a t e d b y t h e s i t u a t i o n t h a t e x i s t a t t h e . l a r g e s t waatewater t r e a t m e n t p l a n t i n S w i t z e r land,

the Werdh6lzli p l a n t i n Zirich

t h e near f u t u r e , of

dry matter.

T h i s p l a n t w i l l produce,

an e s t i m a t e d a n n u a l s l u d g e v o l u m e o f 1 6 0 0 0 t

in 8-l

Aa o n l y a v e r y s m a l l amount c a n b e r e c y c l e d i n a g r i -

- 86

culture,

-

t h e a u t h o r i t i e s a r e planning t o e i t h e r i n c i n e r a t e or export

t h e sludge for disposal! The u s e o f s e w a g e s l u d g e i n a g r i c u l t u r e i s d e c l i n i n g :

a t the

b e g i n n i n g o f t h e 1980s t h e amount u s e d was 5 0 I o f t h e t o t a l s l u d g e production,

b u t r e a l i s t i c e s t i m a t e s s u g g e s t t h i s q u a n t i t y w i l l re-

d u c e t o 2 0 % o r 5 0 0 0 0 t a-'

o f d r y m a t t e r i n t h e n e x t few y e a r s

( S c h w e i z e r 1 9 8 8 ) . O n l y i f waste s l u d g e c a n b e p r o c e s s e d t o g i v e a p propriate bacteriological,

chemical and p h y s i c a l p r o p e r t i e s , w i l l

any e x p a n s i o n o f sewage s l u d g e u t i l i z a t i o n a s f e r t i l i z e r b e p o s s i b le. Bioprocesses f L . t h e t r e a t m e n t o f t h e sewage s l u d g e have been implemented a t 766 wastewater t r e a t m e n t p l a n t s i n Switzerland.

Until

now t h e m o s t p o p u l a r b i o l o g i c a l t r e a t m e n t t e c h n i q u e s were m e s o p h i l i c anaerobic s t a b i l i z a t i o n a t l a r g e p l a n t s and a e r o b i c c o l d s t a b i l i z a tion a t small plants.

The a e r o b i c t h e r m o p h i l i c p r o c e s s r e p r e s e n t s

a new t e c h n o l o g y t h a t c a n e i t h e r b e i n t e g r a t e d i n t h e t r a d i t i o n a l treatment sequence or s u b s t i t u t e d f o r t h e anaerobic process giving h y g i e n i z a t i o n w i t h much i m p r o v e d t r e a t e d s e w a g e s l u d g e q u a l i t y .

Tab-

l e 1 summarizes t h e t y p e s o f s l u d g e b i o p r o c e s s i n g used i n Switzerl a n d (BUWAL 1 9 9 0 ) .

p l a n t s number

percentage

anaerobic mesophilic

575

7 5 A:

aerobic cold

139

1 8 ?A

sludge treatment

hy g i e n i z a t i o n total

52 766

I n t h e decade 1980-1990,

7

x

1 0 0 7;

20 p l a n t s w i t h a n a e r o b i c t h e r m o p h i -

l i c s l u d g e t r e a t m e n t s t e p were c o n s t r u c t e d i n S w i t z e r l a n d , b u t t h i s

s i t u a t i o n was n o t a c o n s e q u e n c e o f t e c h n o l o g i c a l contrary,

innovation.

On t h e

i t w a s c a u s e d by l e g i s l a t i o n c o n c e r n i n g t h e u t i l i z a t i o n

o f sewage s l u d g e i n a g r i c u l t u r e . T h i s l e g i s l a t i o n r e q u i r e s t h a t on-

l y 100 E n t e r o b a c t e r i a c e a e g - l and,

should be detected i n treated sludge

f u r t h e r , s l u d g e m u s t a l s o b e f r e e o f p a r a s i t i c worm e g g s b e f o -

r e i t i s a c c e p t a b l e f o r s p r e a d i n g o n a g r i c u l t u r a l l a n d ( F e d e r a l De-

partment o f I n t e r i o r 1981).

87

-

B e t w e e n 1975 a n d 1985 t h e d e v e l o p m e n t o f

t h e a e r o b i c t h e r m o p h i l i c s l u d g e t r e a t m e n t t e c h n o l o g y was c h a r a c t e r i zed by r a p i d p r o g r e s s i o n from t h e o r y t o p r a c t i c e w i t h o u t

t h e deve-

l o p m e n t o f any s o l i d b a c k g r o u n d . The m a j o r i t y o f t h e s t u d i e s c a r r i e d o u t enhanced t r e a t m e n t p e r f o r m a n c e i n e x i s t i n g p l a n t s and sought t o demonstrate the e f f i c i e n c y o f these,plants plant constructor

responsible.

However,

and t h e a b i l i t i e s o f t h e

v i r t u a l l y no a t t e m p t s were

made t o e x a m i n e t h e p h y s i o l o g y o f t h e m i c r o o r g a n i s m s i n v o l v e d i n a e robic thermophilic

t r e a t m e n t u s i n g sewsge s l u d g e a s s u b s t r a t e .

Most

authors f i r s t considered small scale bioreactors using c l a s s i c a l b i o t e c h n o l o g i c a l approaches t o determine t h e performance l i m i t s o f the process.

One r e a s o n f o r t h i s was t h a t t h e a u t h o r i t i e s r e s p o n s i b -

l e f o r e n f o r c i n g l e g i s l a t i o n were t o be s o l v e d very q u i c k l y .

f a c e d w i t h a new p r o b l e m t h a t h a d

Further,

i n Switzerland,

m u n i c i p a l was-

t e w a t e r t r e a t m e n t p l a n t s a r e managed b y p u b l i c a u t h o r i t i e s .

T h i s has

an i m p o r t a n t e f f e c t on t h e d e v e l o p m e n t o f new s o l u t i o n s f o r t h e t r e atment o f b o t h wastewater

a n d sewage s l u d g e .

The t s r g e t s o f n o n - p r o -

f i t o r g a n i z a t i o n s d i f f e r from t h o s e o f c o m m e r c i a l companies.

1.2

P o t e n t i s l o f a bioprocess

A b i o t e c h n o l o g i c a l approach t o t h e study o f sludge t r e a t m e n t i n v o l v e s i n v e s t i g a t i o n s o f microorganisms as b i o c a t a l y s t s i n t h e process environment.

However, t h e p r e s e n t proposed p r o c e s s f o r s l u d -

ge b i o t r e s t m e n t d i f f e r s f r o m m o s t b i o t e c h n o l o g i c a l p r o c e s s e s i n t h e f o l l o w i n g ways:

. . . .

a p p l i c a t i o n o f mixed populations complex and i l l - d e f i n e d

substrates

i r r e l e v a n t commercial value o f the f i n a l product presently low technology

M o s t c o m m e r c i a l b i o t e c h n o l o g i c s l p r o c e s s e s a r e c a r r i e d o u t usinr

s p e c i f i c m i c r o b i a l s t r a i n s i n c o n t r o l l e d c h e m i c a l and p h y s i c a l

environments. peptides,

H i g h v a l u e p r o d u c t s such as a n t i b i o t i c s ,

hormones,

enzymes a n d o p t i c a l l y p u r e c h e m i c a l s o b t a i n e d b y b i o t r a n s -

formation f o r medical, representative

a n a l y t i c a l or i n d u s t r i a l a p p l i c a t i o n s are

o f biotechnology

.

Such h i g h c o s t b i o t e c h n o l o g i c a l

products are t y p i c a l l y produced a t low concentrations.

Most f u t u r e

developments i n b i o t e c h n o l o g y w i l l p r o b a b l y concern improvements i n c u l t i v a t i o n techniques,

g e n e t i c s t s b i l i t y and m a n i p u l a t i o n o f

pro-

c e s s m i c r o b e s a n d i m p r o v e m e n t s i n down s t r e a m p r o c e s s t e c h n o l o g y .

-

88

-

Few new b u l k b i o t e c h n o l o g i c a l p r o d u c t s c a n b e e x p e c t e d i n t h e n e x t decade. The s t u d y o f t h e r m o p h i l i c m i c r o o r g a n i s m s f o r b i o t e c h n o l o g i c a l processes i s a w e l l developed f i e l d o f research a c t i v i t y ,

but u n t i l

now o n l y a few l a r g e s c a l e i n d u s t r i a l a p p l i c a t i o n s h a v e b e e n d e v e l o p e d ( S o n n l e i t n e r 1983). amylases,

nucleases a r e examples o f ganisms.

T h e r m o s t a b l e enzymes s u c h a s p r o t e a s e s ,

g l u c o s e i s o m e r a s e , DNA p o l y m e r a s e a n d r e s t r i c t i o n endoproducts produced by t h e r m o p h i l i c microor-

Other a p p l i c a t i o n s a r e under development ( B e r g q u i s t e t a l .

1987). The t r e a t m e n t o f sewage s l u d g e w i t h a e r o b i c t h e r m o p h i l i c

mic-

roorganisms i s a genuine p r a c t i c a l a p p l i c a t i o n o f t h e r m o p h i l i c process microbes. zerland,

M o s t d e v e l o p m e n t w o r k h a s b e e n d o n e i n Germany,

Sweden,

UK,

Swit-

R e p u b l i c o f S o u t h A f r i c a a n d USA.

A e r o b i c b i o p r o c e s s e s f o r t h e t r e a t m e n t o f sewage s l u d g e a t h i g h t e m p e r a t u r e s o f f e r many a d v a n t a g e s ,

The b e n e f i t s o f t h e t e c h -

n o l o g y a l l o w t h e p r o d u c t t o be s a f e l y u s e d as a n a g r i c u l t u r a l f e r t i l i z e r a n d when d i s p o s a l i s t h e f a v o u r e d o p t i o n , t h e t r e a t e d s l u d g e can a l s o be improved.

the quality o f

Advantages can be summari-

zed as f o l l o w s :

. . . .

b i o d e g r a d a t i o n o f n o r m a l l y b i o d e g r a d a b l e o r g a n i c components a t high r a t e s b i o d e g r a d a t i o n o f r e c a l c i t r a n t compounds improvement o f t h e p h y s i c a l c h a r a c t e r i s t i c s s t a b i l i t y a n d r e l i a b i l i t y o f t h e p r o c e s s due

o f t h e sludge t o t h e uniform

m i c r o f l o r a employed ( s e l e c t i v i t y p r e s s u r e o f t h e temperatu-

.

re 1 hygienization o f the sludge ( i n a c t i v a t i o n o f pathogenic organisms)

The h y g i e n i z a t i o n o f s l u d g e c a n a l s o b e a c h i e v e d u s i n g n o n - b i o l o g i c a l thermal technology,

p h y s i c a l processes such as i r r a d i a t i o n

o r c h e m i c a l p r o c e d u r e s s u c h as t h e a d d i t i o n o f l i m e .

However,

such

p r o c e s s e s do n o t i n c o r p o r a t e t h e f u l l r a n g e o f b e n e f i t s t h a t c a n be achieved with bioproceasing. The t r e a t m e n t o f t h e sewage s l u d g e w i t h t h e r m o p h i l i c m i c r o o r g a n i s m s r e p r e s e n t s an e x a m p l e o f a l o w t e c h n o l o g y b i o p r o c e s s ( B e r g quist e t el.

1987);.

The n o n - p r o f i t

n a t u r e o f wastewater

industry i n Switzerland i n h i b i t s innovation,

treatment

b u t l e g i s l a t i o n can

g i v e t h e n e c e s s a r y p o s i t i v e i m p u l s e t o change t h e s t a t u s quo.

2

89

-

MATERIAL AND M E T H O D S

A d e t a i l e d d e s c r i p t i o n o f m a t e r i a l and methods can be f o u n d elsewhere

3

( B o m i o 1990).

RESULTS AND D I S C U S S I O N

3 . 1 G r o w t h l i m i t a t i o n s and a c t i v i t i e s Growth i s g e n e r a l l y under t h e c o n t r o l o f e x t r a c e l l u l a r chemic a l f a c t o r s s u c h as c a r b o n e n e r g y s o u r c e , and g r o w t h f a c t o r a v a i l a b i l i t i e s . r e s p e c t t o such a v a i l a b i l i t i e s .

nutrient,

t r a c e element

Sewage s l u d g e . i s i l l d e f i n e d w i t h

T h e r e f o r e e x p e r i m e n t s t h a t sough

t o i d e n t i f y m e t a b o l i z a b l e c a r b o n s o u r c e s and l i m i t i n g f a c t o r s f o r thermophilic 3.1.1

b a c t e r i a d u r i n g t h e t r e a t m e n t were performed.

Limitinq factors The n a t u r e o f l i m i t a t i o n s r e s p o n s i b l e f o r d e c r e a s e s i n b o t h

t h e o x y g e n u p t a k e r a t e and t h e t o t a l d e h y d r o g e n a s e a c t i v i t y d u r i n g g r o w t h were d e t e r m i n e d b y p u l s i n g t h e c u l t u r e w i t h d i f f e r e n t c a r b o n energy s u b s t r a t e s ,

salts,

t r a c e e l e m e n t s and v i t a m i n s .

Figure 1

100

--$

0” a

80

60

40

20

0 0

4

8

12

16

20

24

TlME [ h l F i g . 1. Oxygen u p t a k e r a t e ( ) and oxygen p a r t i a l p r e s s u r e p 0 2 ( m d u r i n g a f e d b a t c h c u l t i v a t i o n a t 6 5 O C , 1 5 0 0 m i n - 1 , vvm a n d pH 7 w i t h f r u c t o s e p u l s e e x p e r i m e n t i n t h e c a r b o n l i m i t e d g r o w t h phase. The e x p o n e n t i a l i n c r e a s e o f t h e OUR i m m e d i a t e l y a f t e r p u l s e shows t h a t t h e f r u c t o s e p u l s e d was l i m i t i n g .

)

-

90

-

shows a t y p i c a l r e s p o n s e a f t e r a c a r b o n s u b s t r a t e p u l s e :

the increa-

s e o f t h e oxygen u p t a k e r a t e and t h e s i m u l t a n e o u s decrease o f t h e oxygen p a r t i a l p r e s s u r e i n t h e s l u d g e , fructose,

immediately a f t e r p u l s i n g

shows t h a t c a r b o n l i m i t a t i o n was o c c u r i n g .

no r e a c t i o n ,

I f t h e r e wss

t h e c u l t u r e s were n o t l i m i t e d by t h e s u b s t a n c e p u l s e d .

The p u l s e e x p e r i m e n t s were d o n e a t t h e e n d o f f e d b a t c h c u l t u -

r e s and r e s u l t s a r e summarized i n T a b l e 2.

.

They c l e a r l y show t h a t

b i o d e g r a d a b l e c a r b o n e n e r g y s o u r c e a v a i l a b i l i t y was the only l i m i t a t i o n

.

salts,

t r a c e e l e m e n t s and v i t a m i n s w e r e n o t l i m i t i n g .

Oxygen was d e f i n i t e l y n o t l i m i t i n g b e c a u s e p 0 2 was a l w a y s m o r e t h a n 80

X d u r i n g these pulse experiments.

TABLE 2 C a r b o n s o u r c e s o u l s e d a t a c o n c e n t r a t i o n o f 4 k o m-3 a n d s a l t s . t r a c e e l e m e n t s and' v i t a m i n s p u l s e d a t t h e c o n c e n t r e t i o n s d e s c r i b e d ( K ( K e n k e l and T r e l a 1979) t o t h e r m o p h i l i c c u l t u r e s g r o w i n g on s l u d g e d u r i n g t h e c a r b o n l i m i t e d phase (phase V ) . An i n c r e a s e o f OUR i s m a r k e d a s + r e s p o n s e ; n o c h a n g e o f O U R i s response. marked as

-

~~

Glucose,

fructose

Maltose Sucrose, Xylose,

lactose ribose

Pyruvate,

glycerol

Glutamic acid Valine Casein Soya p r o t e i n s Gelatine :I

t a r ch

Pect i n e Cellulose Olive o i l Micrococcua l u t e u s Salts Trace elements

V it a m i n a

~~

OUR i n c r e a s e i m m e d i a t e l y after pulsing

substance

1

-

':suspension

3.1.2

o f 10.2

kg

91

-

wet w e i g h t .

A c t i v i t i e s i n sewaqe s l u d q e a n d p u r e c u l t u r e s The p r e v i o u s e x p e r i m e n t s i n d i c a t e d p r o t e o l y t i c a c t i v i t y b e c a u -

se o f g r o w t h i m m e d i a t e l y protein.

a f t e r pulses o f casein,

g e l a t i n e and soya

T h i s was d e m o n s t r a t e d b y m e a s u r i n g t h e a c t i v i t y o f t o t a l

p r o t e o l y t i c enzymes a n d a l s o b y i s o l a t i n g a m i x e d s u b - p o p u l a t i o n o f t h e r m o p h i l e s w h i c h g r e w on s e m i - s y n t h e t i c m e d i a w i t h s o y a p r o t e i n a s t h e s o l e c a r b o n s o u r c e (Bomio e t a l .

1989)

P u l s e e x p e r i m e n t s showed t h a t

.

e x t r a c e l l u l a r proteaseswere the only polymer degrading enzymes p r o d u c e d d u r i n g c u l t i v a t i o n o f t h e p r o c e s s m i c r o b e s on sewage s l u d g e .

These r e s u l t s were c o n f i r m e d b y m e a s u r e m e n t s o f e n z y m a t i c a c t i v i t i e s i n t h e sludge.

Other polymers than p r o t e i n s normally pre-

s e n t i n sewage s l u d g e r e p r e s e n t p o t e n t i a l s u b s t r a t e s f o r t h e r m o p h i -

l e s , b u t c a n n o t be h y d r o l i z e d b y t h e p r o c e s s c u l t u r e .

The a c t i v i t i e s

t e s t e d a r e s u m m a r i z e d i n T a b l e 3.

TABLE 3 E x t r a c e l l u l a r enzymes a c t i v i t i e s i n sewage s l u d g e d e t e r m i n e d b y p u l s e s o f t h e p o l y m e r s and d e t e r m i n a t i o n o f t h e e n z y m a t i c a c t i v i t y . I n c r e a s e o f OUR a f t e r a pulse experiment

Enzymatic a c t i v i t

+

+

-

-

Lipase

-

Pectinase

-

-

Protease Amylase Cellulase Keratinase

-

The p r o t e o l y t i c a c t i v i t y c o r r e l a t e d w i t h t h e i n c r e a s e o f t h e o x y g e n u p t a k e r a t e d u r i n g b o t h b a t c h and f e d b a t c h c u l t i v a t i o n s o f a e r o b i c t h e r m o p h i l i c m i c r o o r g a n i s m s on sewage s l u d g e i n d i c a t i n g a The p r o t e o l y t i c a c t i v i t y c o r r e l a t e d w i t h t h e i n c r e a s e o f t h e g r o t ef o l y tbi ac t cenzymes o x y g e nr ouwpttha kaes sr oa ct iea tdeudr ipnrgo dbuoct thi obna toc fh pand ed h c u l t i v (aFtiigounrse o2 f) . T h i s a l s o c o r r e l a t e d w i t h a n i n c r e a s e i n f r e e ammonium a e r o b i c t h e r m o p h i l i c m i c r o o r g a n i s m s on sewage s l u d g e i ncdoi nc ca et n i nt rga tai o n i n the c u l t u r e . g r o w t h a s s o c i a t e d p r o d u c t i o n o f p r o t e o l y t i c enzymes

(Figure 2).

This

a l s o c o r r e l a t e d w i t h a n i n c r e a s e i n f r e e ammonium c o n c e n t r a t i o n i n the c u l t u r e .

-

7

i

P c n Y

I

92

-

4.5 4.0 3.5

[L

3 0

3.0 2.5 2.0

1.5 1 .o

0.5

I

4

PROTEOLYTIC ACTIVITY

I mu rni-11

F i g . 2. L i n e a r c o r r e l a t i o n between p r o t e o l y t i c a c t i v i t y and oxygen u p t a k e r a t e d u r i n g a b a t c h c u l t i v a t i o n o n s e w a g e s l u d g e a t 2500 m i n - l , 6 5 O C , 0 . 8 vvm a n d pH 7 . C o r r e l a t i o n c o e f f i c i e n t r = 0.935. The a c t i v i t y was d e t e r m i n e d r e l a t i v e t o p r o t g i n a s e K ( S i g m a P-0390, t y p e X I ) s t a n d a r d s m e a s u r e d i n v i t r o a t 8 0 C . The c o r r e l a t i o n i n d i c a t e s a growth a s s o c i a t e d p r o d u c t i o n o f e x t r a c e l l u l a r p r o t e a a e by t h e p r o cess microbes.

-

c

50

! d

E 3

E

I

EI

45 40

35

I-

U

a

30

u c

25 20 15 10 5

0 4 0

I

1

2

3

4

5

6

7

8

9

1

0

TIME h l

F i g . 3. P r o t e o l g t i c a c t i v i t y i n t h e s l u d g e ( m ) a n d s u p e r n a t a n t ( 0 ) m e a g u r e d a t 80 C a n d pH 7 d u r i n g a s l u d g e c u l t i v a t i o n a t 2 5 0 0 m i n - 1 , 65 C , 0 . 8 vvm a n d pH 7 . The a c t i v i t y was c o m p a r e d t o p r o t e i n a s e K s t a n d a r d s . The a r r o w a h o w a t h e e n d o f t h e l a g p h a s e . The a c t i v i t y measured i n t h e s u p e r n a t a n t i s n o t i n c r e a s e d a f t e r t h e l a g phase, because p r o t e o l y t i c enzymes are absorbed t o s l u d g e p a r t i c l e .

-

93

-

The R Q v a l u e f o r sewage s l u d g e c u l t i v a t i o n s h a d an a v e r a g e val u e o f 0.82

which i s t y p i c a l f o r o x i d a t i v e metabolism o f p r o t e i n s

(Lentner 1981).

C u l t i v a t i o n s on s y n t h e t i c medium showed t h a t t h e

p r o t e o l y t i c a c t i v i t y was n o t c e l l w a l l b o u n d b e c a u s e h i g h a c t i v i t y was f o u n d i n t h e c e l l f r e e s u p e r n a t a n t .

On t h e o t h e r hand,

analyses

i n t h e s u p e r n a t a n t o f c u l t i v a t i o n s w i t h sewage s l u d g e a s s u b s t r a t e gave l o w a c t i v i t y .

T h i s i n d i c a t e s t h a t t h e enzymes a r e e x c r e t e d i n t o

t h e medium b u t may b e i m m e d i a t e l y a b s o r b e d b y p a r t i c l e s i n t h e c a s e o f sludge c u l t i v a t i o n s ,

a s shown i n F i g u r e 3 .

The R Q v a l u e o f a l l c u l t i v a t i o n s on sewage s l u d g e showed an a v e r a g e v a l u e o f 0.82 d u r i n g b o t h e x p o n e n t i a l a n d o x y g e n l i m i t e d g r o w t h phase.

T h i s v a l u e r o s e t o 1.1 i n t h e c a r b o n l i m i t e d p h a s e ,

i n d i c a t i n g changes i n t h e o v e r a l l metabolism. semi-synthetic

l e c a r b o n s o u r c e showed an a v e r a g e r e s p i r a t o r y which i s t y p i c a l f o r

.

C o n t r o l c u l t u r e s on a

m e d i a w i t h t h e same i n o c u l u m b u t w i t h g l u c o s e a s s o -

RQ values,

c o e f f i c i e n t o f 1.05

f u l l o x i d a t i v e g r o w t h on c a r b o h y d r a t e s .

proteolytic activity,

the pulse experiments

w i t h p r o t e i n s u b s t r a t e s and t h e g r o w t h a s s o c i a t e d p r o d u c t i o n o f f r e e ammonium s t r o n g l y i n d i c a t e t h a t t h e t h e r m o p h i l i c p o p u l a t i o n s p r e f e r t o u s e p r o t e i n a c e u s mat e r i a l as t h e i r carbon source.

Determinations o f lipase,

amylase,

c e l l u l a s e and p e c t i n a s e a c -

t i v i t i e s gave n e g a t i v e r e s u l t s w i t h b o t h t h e o r i g i n a l methods desc r i b e d and a f t e r m o d i f i c a t i o n and a d a p t i o n o f t h e a n a l y t i c a l methods f o r use i n complex media.

T h i s happened a l t h o u g h t h e r m o p h i l i c popu-

l a t i o n s i n sewage s l u d g e a r e p o t e n t i a l l y s b l e t o m e t a b o l i z e s u b s t r a t e s such as s t a r c h and l i p i d s i n complex media ( B a i e r 1987).

Figu-

re 4 shows t h e t i m e c o u r s e f o r p r o t e o l y t i c a c t i v i t y a n d t h e r e s p i r a t o r y q u o t i e n t o f one r e p r e s e n t a t i v e sewage s l u d g e c u l t u r e . D u r i n g t h e l a g phase t h e r e i s n o p r o d u c t i o n o f p r o t e a a e s , even i n t h i s phase, t e o l y t i c enzymes.

After

t h e l a g phase,

which l a s t e d 6 h i n t h e c u l -

t i v a t i o n f o r w h i c h t h e r e s u l t s a r e p l o t t e d i n f i g u r e 4,

thermophilic

p o p u l a t i o n s began t o grow on s l u d g e and t h e R Q i m m e d i a t e l y t o 0.81.

The p r o t e o l y t i c a c t i v i t y m e a s u r e d i n v i t r o a t 8 0

7 rose r a p i d l y , i n t o t h e medium. a n d pH 7 ,

but

t h e s l u d g e c o n t a i n s a s i g n i f i c a n t amount o f p r o -

increased OC

a n d pH

i n d i c a t i n g t h a t thermophilic proteaaes are excreted These p r o t e a s e s a r e l e s s a c t i v e i n v i t r o a t 6 5 O C

so t h a t t h e v a l u e s measured a t t h i s t e m p e r a t u r e d i d n o t

c c;

94

-

. 0.9 5 0

60

L

E

. 0.8

3

-E

50 '

t

0 . 7 ;3

a

! I

f

-EE

. 0.6

40

V

Q '

3

z: &

30

0.5

. 0.4

. 0.3

20

[L

LL

0 -I

E

10

-

0.2

-

0.1

- 0

0

1

2

5

4

3

7

6

8

9

10

TIME 1 h

1

F i g . 4. P r o t e o l y t i c a c t i v i t y d e t e r m i n e d a t 80 O C i n t h e s l u d g e ( ) and s u p e r n a t a n t ( * ) and r e s p i r a t i o n q u o t i e n t ( ) as a function o f t i m e d u r i n g a b a t c h c u l t i v a t i o n o f sewage s l u d g e a t 6 5 O C , 0 , 5 vvm, 1 5 0 0 m i n - l a n d pH 7 . A f t e r a l a g p h a s e o f 6 h, t h e r e s p i r a t o r y q u o t i e n t i n c r e a s e and p r o t e a s e a c t i v i t y measured a t 8 0 OC a l s o i n c r r a sed d e m o n s t r a t i n g t h e a c t i v i t y o f t h e r m o p h i l i c microbes. 1.4

05

1.2 0 4 1 .o

03

0.8 0.6

02

0.4 0 1

0.2

0.0

00 50

55

60

65

70

75

80

85

90

TEMPERATURE [ "C1

Temperature dependence o f p r o t e o l y t i c a c t i v i t y (Abs 3 7 6 ) f o r r a w - ( * ) a n d A T S - s l u d g e ( 0 ) a n d p r o t e a s e K ( m ) a t pH 7. The o p t i m u m f o u n d a t 8 0 OC f o r A T S s l u d g e a n d t h e i n s i g n i f i c a n t a c t i v i t y f o u n d i n raw s l u d g e a t t h i s t e m p e r a t u r e , a l l o w s t h e d e t e r m i n a t i o n o f thermophilic microbes a c t i v i t y w i t h the protease t e s t .

Fig. 5.

increase.

95

-

The h y d r o l y s i s o f p r o t e i n a c e o u s m a t e r i a l s w i t h s u b s e q u e n t

m e t a b o l i z a t i o n o f amino a c i d s

c a u s e d ammonium t o be r e l e a s e d i n t o

t h e medium. F i g u r e 5 shows t h e t e m p e r a t u r e d e p e n d e n c e o f p r o t e o l y t i c a c t i v i t y compared w i t h t h e commercial p r o t e a s e K . The t e m p e r a t u r e d e p e n d e n c e o f p r o t e o l y t i c a c t i v i t y was measur e d e i t h e r i n c u l t u r e s g r o w i n g on r a w s l u d g e a s c a r b o n e n e r g y s o u r ce d u r i n g a e r o b i c t h e r m o p h i l i c

semi-synthetic available. similar

t r e a t m e n t o r i n c o n t r o l c u l t u r e s on

media w i t h s o j a p r o t e i n as o n l y carbon energy source

The t e m p e r a t u r e d e p e n d e n c e f o r p r o t e o l y t i c a c t i v i t y was

f o r c u l t i v a t i o n s on b o t h m e d i a .

t h e r m o p h i l e s h a d an o p t i m u m a t 80 O C s l u d g e p r o t e a s e s were n o t a c t i v e .

.

The p r o t e o l y t i c a c t i v i t y o f

b u t a t h i s t e m p e r a t u r e raw

Therefore:

M i c r o b i o l o g i c a l a c t i v i t y and p r o c e s s performance can be f o l l o w e d by m e a s u r i n g p r o t e o l y t i c a c t i v i t y i n v i t -

r o a t 80 OC. T h i s t e m p e r a t u r e dependence i s o f i n t e r e s t compared t o t h a t o f t h e c o m m e r c i a l enzyme p r o t e a s e K ,

w h i c h shows a maximum a t 6 0

OC.

P o p u l a t i o n s e n r i c h e d f r o m a e r o b i c t h e r m o p h i l i c s l u d g e s showed a c t i v i t i e s u p t o 60 mU m 1 - l .

The t e m p e r a t u r e d e p e n d e n c e a t d i f f e r e n t pH

,

v a l u e s w i t h t h e a z o c a s e i n m e t h o d i s shown i n f i g u r e 6.

-,

0.45

I

l n rW

m

%

0.40 0.35

0.30 0.25

0.20 0.15 0.10 0.05

0.00 50

55

60

65

70

75

80

85

90

TEMPERATURE [ "C 1

F i g 6. P r o t e o l y t i c a c t i v i t y o f a e r o b i c t h e r m o p h i l i c t r e a t e d s l u d g e a t d i f f e r e n t t e m p e r a t u r e s i n v a r i o u s b u f f e g s a t pH 5 ( o 1, pH 7 ( m ) a n d pH 9 (I). The o p t i m u m i s f o u n d a t 8 0 C a n d pH 7. A t pH 9 a c t i v i t y i a d e t e c t a b l e a t l o w e r t e m p e r a t u r e s , w h i l e a t pH 5 o n l y v e r y l i t t l e a c t i v i t y i s measured.

The t h e r m o p h i l i c

-

96

p r o t e a s e s m u s t b e c l a s s i f i e d as n e u t r a l p r o -

t e a s e s a t t e m p e r a t u r e s u p t o 8 0 O C , b e c a u s e o f l o w a c t i v i t y a t pH 9 a n d p r a c t i c a l l y n o a c t i v i t y a t pH 5.

The i d e n t i c a l a c t i v i t y f o u n d

a t 65 O C f o r pH v a l u e s o f 7 a n d 9 , e x p l a i n a m i n i m a l e f f e c t o f t h i s f a c t o r d u r i n g a e r o b i c t h e r m o p h i l i c sewage s l u d g e t r e a t m e n t i n t h e pH r a n g e f r o m 6.8

t o 8.8.

3 . 2 I n a c t i v a t i o n o f pathoqenic microorqanisms A t t h e b e g i n n i n g o f t h e 1970s t h e i n a c t i v a t i o n o f p o t e n t i a l l y p a t h o g e n i c o r g a n i s m s i n sewage s l u d g e became an i m p o r t a n t q u e s t i o n i n Switzerland.

I t r e s u l t e d i n t h e i n t r o d u c t i o n o f d i f f e r e n t compe-

t i n g technologies.

After

disastrous experience w i t h p a s t e u r i z a t i o n

u n i t s i n s t a l l e d a f t e r the anaerobic d i g e s t i o n step,

ATS processes

were i n t r o d u c e d as p r e t r e a t m e n t p r o c e s s e s (Bomio 1 9 8 8 ) .

Although the

i n a c t i v a t i o n o f p o t e n t i a l l y p a t h o g e n i c o r g a n i s m s was r e q u i r e d b y Swiss law,

few k i n e t i c d a t a f o r m i c r o b i a l l y m e d i a t e d t h e r m o p h i l i c

p r o c e s s e s were a v a i l a b l e .

Whilst

the influence o f the thermophilic

m i c r o f l o r a d u r i n g t h e p r o c e s s was f r e q u e n t l y d i s c u s s e d ,

scientific

d a t a were l a r g e l y u n a v a i l a b l e .

3.2.1

Heat i n a c t i v a t i o n o f p a t h o q e n i c microorqanisms I n order t o investigate the heat i n a c t i v a t i o n k i n e t i c s of

h o g e n i c m i c r o b e s i n sewage s l u d g e ,

pat-

t h r e e s t a n d a r d t e s t o r g a n i s m s we-

r e e m p l o y e d f o r i n a c t i v a t i o n i n v e s t i g a t i o n s : E. c o l i , 5 . a u r e u s a n d P . Aeruqinosa. Theywere p u l s e d i n t o a e r o b i c t h e r m o p h i l i c s l u d g e c u l t u r e s i n d e f i n e d c o n c e n t r a t i o n s d u r i n g d i f f e r e n t . phases o f t h e p r o c e s s a n d i n a c t i v a t i o n was f o l l o w e d b y d e t e r m i n a t i o n o f t h e v i a b l e c o u n t o f t h e t e s t b a c t e r i a on s e l e c t i v e m e d i a .

5. a u r e u s was v e r y g o o d w i t h a z i d e a g a r

The s e l e c t i v i t y f o r

and f o r P .

aeruqinosa,

m i l a r l y good o n a g a r s u p p l e m e n t e d w i t h c e n t r i m i d e - n a l d i x i n s e l e c t i v e d e t e r m i n a t i o n o f E.

Therefore,

t h e ac-

c o l i was a l s o i d e n t i f i e d w i t h s u b c u l t u r i n g on f l u o r o -

cult-laurylsulphate development,

The

c o l i on e i t h e r e o s i n methylene b l u e

a g a r o r v i o l e t r e d b i l e a g a r was u n s a t i s f a c t o r y . t i v i t y o f E.

si-

acid.

b r o t h a t 44 OC t o g e t h e r w i t h d e t e c t i o n o f gas

f l u o r e s c e n c e and a p o s i t i v e i n d o l e r e a c t i o n .

The p r o b -

lem o f t h e a c t u a l d e t e c t i o n method f o r E n t e r o b a c t e r i a c e a e has a l s o b e e n r e c o g n i z e d by t h e S w i s s l e g i s l a t i v e a u t h o r i t i e s Department o f

the I n t e r i o r ,

Bern),

(BUWAL,

Federal

and a program f o r method impro-

vement h a s b e e n e s t a b l i s h e d ( W i t t e k i n d 1 9 9 0 ) .

-

97

-

The p u l s e e x p e r i m e n t s w e r e c a r r i e d o u t i n a 5 0 1 C O L O R b i o r e a c t o r d u r i n g g r o w t h o f t h e t h e r m o p h i l i c p r o c e s s c u l t u r e on sewage s l u d ge a t l o w a n d h i g h a e r a t i o n r a t e s a n d i n n u t r i e n t b r o t h i n o r d e r t o compare i n a c t i v a t i o n k i n e t i c s w i t h d e f i n e d m e d i a .

The r e s u l t s o f t h e

p u l s e e x p e r i m e n t s a r e s u m m a r i z e d i n T a b l e 4. TABLE 4 Compsrison o f h e a t i n a c t i v a t i o n and c o n d i t i o n s f o r p u l s e e x p e r i m e n t s . C o n s t a n t o p e r a t i n g c o n d i t i o n s were t e m p e r a t u r e o f 6 5 O C , pH 7, s t i r r e r s p e e d 1 0 0 0 min'l,

s

Medium

Aeration

Sludge

0.3

vvm

l a g phase

Sludge

0.3

vvm

e x p o n e n t i a l g r o w t h phase

none

vvm

carbon l i m i t e d phase

none

Synergis 1 effect

Pulse

-

Sludge

0.3

Sludge

0.05

vvm

Sludge

0.05

vvm

oxygen l i m i t e d phase

none

nutrient broth

0.15

vvm

e x p o n e n t i a l g r o w t h phase

none

nutrient broth

0.15

vvm

carbon l i m i t e d phase

none

nutrient broth

no aeration

-

l a g phase

-

medium s t e r i l e

d e c i m a l r e d u c t i o n t i m e s compared w i t h e q u i v a l e n t l a g phase

I n a c t i v a t i o n k i n e t i c s were s i m i l a r

values i n the

for a l l conditions tested

a n d t h e p u l s e e x p e r i m e n t s show t h s t t h e r e i s n o d i f f e r e n c e i n t h e heat i n a c t i v a t i o n a t low or h i g h aeration r a t e s i n the sludge. reover,

Mo-

the i n a c t i v a t i o n k i n e t i c s during experiments a t d i f f e r e n t

g r o w t h phases demonstrated t h a t t h e r m o p h i l i c

g r o w t h does n o t have

any e f f e c t o n t h e i n a c t i v a t i o n o f t e s t o r g a n i s m s . p i c a l i n a c t i v a t i o n p u l s e i s p l o t t e d f o r P.

I n f i g u r e 7,

a e r u q i n o s a and

a ty-

S. a u r e u s

i n sewage s l u d g e a t a l o w a e r s t i o n r a t e . The l o g a r i t h m i c p l o t o f c o l o n y

f o r m i n g u n i t s on s e l e c t i v e c u l -

t i v a t i o n m e d i a w i t h r e s p e c t t o t i m e showed, ment,

t h a t f o r each e x p e r i -

a s i m i l a r time course with a r a p i d d e c l i n e o f the mesophilic

t e s t organisms i m m e d i a t e l y a f t e r p u l s i n g , f o l l o w i n g by a s l o w e r dec r e a s e i n s second phase o f t h e i n a c t i v a t i o n .

Immediately a f t e r p u l -

s i n g t h e i n a c t i v a t i o n was s i m i l a r t o t h a i i n n u t r i e n t b r o t h ,

indi-

c s t i n g t h a t t h e m i c r o o r g a n i s m s were s u b j e c t t o t h e h e a t i n a c t i v a t i o n w i t h o u t any i n f l u e n c e o f t h e s l u d g e .

I n t h e second phase,

which a l -

-

98

-

ways o c c u r e d 4 t o 5 m i n u t e s a f t e r p u l s i n g , slower.

t h e i n a c t i v a t i o n was much

This s t r o n g l y s u g g e s t s t h a t s l u d g e components and p a r t i c l e s

p r o v i d e a p r o t e c t i v e e f f e c t f o r t h e t e s t organisms.

T h i s e f f e c t was

a l s o d e s c r i b e d w i t h S a l m o n e l l a sp a s t h e t e a t o r g a n i s m (Hammel 1 9 8 3 , Wassen 1 9 7 5 ) .

Average d e c i m a l r e d u c t i o n t i m e s f o r a l l e x p e r i m e n t s

a r e s u m m a r i z e d i n T a b l e 5. not obtained,

V a l u e s f o r E.

c o l i i n sewage s l u d g e w e r e

because o f t h e l o w s e l e c t i v i t y and u n r e l i a b l e . d e t e r m i -

n a t i o n on e i t h e r VRBD o r EMB c u l t i v a t i o n m e d i a .

1 lo9 1 loB 1 1 0 ~ 1 lo6 1 105 1 1 0 4 1 lo3

J 0

i

20

10

30

40

50

60

70

80

90

100

TIME [min 1

F i g . 7. R e s i d u a l v i a b l e c e l l s o f P. a e r u g i n o s a ( m ) a n d 5. a u r e u s ( A ) i n sewage s l u d g e a t 0.05 vvm, 6 5 O C , s t i r r e r s p e e d 1 0 0 0 m i n - l a n d pH 7 . The i n a c t i v a t i o n k i n e t i c w i t h t w o p h a s e s i s t y p i c a l f o r i n a c t i v a t i o n e x p e r i m e n t s w i t h p r o t e c t i v e e f f e c t s o f t h e medium.

TABLE 5 A v e r a g e d e c i m a l r e d u c t i o n t i m e a i n aewage s l u d g e a n d n u t r i e n t b r o t h a t T = 65 O C o b t a i n e d a t d i f f e r e n t a e r a t i o n r a t e s . D-Values

Test organism on n u t r i e n t broth

[ min

1

directly after pulse

i n t h e second

-

phase

E.

coli

0.3

-

P.

aeruginosa

0.8

1.7

22

1.7

2.6

62

5. a u r e u s E.

c o l i a n d P.

a e r u q i n o a a (Gram

-1

are l e s s heat r e s i s t a n t than

t h e Gram

+ o r g a n i s m 5. a u r e u s i n e i t h e r d e f i n e d medium o r i n sewage

sludge.

The d e c i m a l r e d u c t i o n r a t e s i n t h e s e c o n d p h a s e o f t h e i n a c -

-

99

-

t i v a t i o n , which a r e r e p r e s e n t a t i v e f o r t h e e f f e c t i v e i n a c t i v a t i o n i n the sludge, i n d i c a t e t h a t even

.

2 h o u r s h e a t t r e a t m e n t a t 6 5 OC i s i n s u f f i c i e n t

for the

complete hygienization of t h e sludge, although allowing t h e i n a c t i v a t i o n o f commonly u s e d i n d i c a t o r b a c t e r i a . In 2 hours it is only possible t o achieve a reducti0.n of only 2 o r d e r s o f magnitude

f o r 5.

aureus.

The c o n s i d e r a t i o n o f E n t e r o b a c t e r i a c e a e c e l l c o u n t s a s a n i n d i c a t o r f o r t h e s u c c e s s f u l h y g i e n i z a t i o n o f sewage s l u d g e is quest i o n a b l e . As a m a t t e r o f f a c t a n i n d i c a t o r f o r a n y i n a c t i v a t i o n s h o u l d f u l f i l a t l e a s t two r e q u i r e m e n t s :

.

i t s h o u l d b e more r e s i s t a n t t o t h e t r e a t m e n t t h a n a l l

.

other pathogenic organisms

i t s h o u l d be s t a b l e ( n o f u r t h e r growth or i n a c t i v a t i o n ) during storage after the treatment.

C o n s i d e r i n g t h e r e s u l t s f o r E.

c o l i i n t h i s work a n d r e s u l t s

o f o t h e r w o r k e r s ( K a b r i c k a n d Jewel1 1 9 8 2 , L a n g e l a n d e t a l .

S t r a u c h e t a l . 1 9 8 5 , Camp e t a l .

1985,

1 9 8 1 , F e a c h e m e t a l . 1983, E r a l p

a n d F a r r e l 1 9 8 6 ) , t h e s e r e q u i r e m e n t s a r e n o t met b y E n t e r o b a c t e r i a ceae. 3.2.2

S y n e r q i s t i c e f f e c t s o f t h e r m o p h i l e s on t h e i n a c t i v a t i o n Some a u t h o r s h a v e s u g g e s t e d t h e e x i s t e n c e o f e i t h e r a n t i b i o t i c

or a n t a g o n i s t i c e f f e c t s o f t h e r m o p h i l i c p o p u l a t i o n s o n p o t e n t i a l l y p a t h o g e n i c m i c r o o r g a n i s m s ( N e b i k e r 1 9 8 1 , Hammel 1 9 8 3 ) . H o w e v e r , t h e i n a c t i v a t i o n e x p e r i m e n t s showed t h a t t h e r e i s no i n f l u e n c e of t h e t h e r m o p h i l e s on t h e i n a c t i v a t i o n o f p o t e n t i a l p a t h o g e n s i n s l u d g e . E x p e r i m e n t a l d e t e r m i n a t i o n o f p o s s i b l e a n t i m i c r o b i a l s u b s t a n c e s was c a r r i e d o u t w i t h t h e MIC (Minimal I n h i b i t o r y C o n c e n t r a t i o n ) t e s t and conclusively confirmed t h e absence of such e f f e c t s . T o t a l m e s o p h i l i c microorganisms, i s o l a t e d from sewage s l u d g e , E. c o l i a n d S . a u r e u s were c u l t i v a t e d o n i s o s e n s i t e s t b r o t h w i t h a d d i t i o n of d i f f e r e n t amounts o f raw and a e r o b i c t h e r m o p h i l i c trea t e d s l u d g e . The r e s u l t s o f t h e t e s t f o r t o t a l m e s o p h i l i c m i c r o o r g a n i s m s , i s o l a t e d from s e w a g e s u l d g e a r e shown i n F i g u r e 8 S i m i l a r r e s u l t s o f t h e M I C t e s t were o b t a i n e d w i t h E . c o l i a n d S, aureus.

-

100

-

The a d d i t i o n o f b o t h r a w a n d t r e a t e d s l u d g e i n c r e a s e d t h e g r o w t h o f m e s o p h i l i c m i c r o o r g a n i s m s i n s t e a d o f i n h i b i t i n g them ( i n c r e a s i n g

OD546 w i t h p r o g r e s s i v e i n c r e a m e n t s o f s l u d g e ) .

The s l o p e o f t h e ab-

s o r p t i o n v a l u e s i s p o s i t i v e and s i g n i f i c a n t l y h i g h e r f o r a e r o b i c thermophilic

t h a n f o r t h e raw sludge;

t h i s t y p i c a l response o f t h e

M I C e x p e r i m e n t s shows c l e a r l y t h a t :

t h e r e i s no p r o d u c t i o n o f a n t i m i c r o b i a l agents d u r i n g aerobic thermophilic sludge treatment.

0

0.05

0.15

0.1

0.2

0.25

0.3

0.35

0.4

SLUDGE VOLUME FRACTION I rnt m i 4 1 F i g . 8. R e l a t i v e a b s o r p t i o n a t 5 4 6 n m o f t h e c u l t u r e a f t e r 18 h o f i n c u b a t i o n a t 37 OC w i t h d i f f e r e n t raw ( m ) and a e r o b i c t h e r m o p h i l i c s l u d g e ( 0 ) c o n c e n t r a t i o n s . The p o s i t i v e s l o p e o b t a i n e d i n d i c a t e t h a t t h e r e a r e no i n h i b i t o r y e f f e c t s i n b o t h raw and A T S s l u d g e . T r e a t e d s l u d g e even a c c e l e r a t e t h e g r o w t h o f p o t e n t i a l pathogens (higher slope).

S i m i l a r r e s u l t s were o b t a i n e d w i t h t h e r m o p h i l i c l a t e d f r o m sewage s l u d g e a t t e m p e r a t u r e s o f 45

OC

populations iso-

(Wassen 1 9 7 5 ) .

T h e r m o p h i l i c p o p u l a t i o n s i s o l a t e d f r o m sewage s l u d g e a r e a b l e t o grow i n a s e m i - s y n t h e t i c s u b s t r a t e (Mason e t e l .

medium u t i l i z i n g i n t a c t y e a s t c e l l s a s

1986,

o f a l y t i c enzyme f r o m 6 .

Hamer a n d Mason 1 9 8 7 ) .

The p r o d u c t i o n

atearothermophilus cultures a f t e r induction

by m i t o m y c i n C has a l s o been d e s c r i b e d (Welker

and Campbell 1966).

These o b s e r v a t i o n s s t i m u l a t e d t h e i n v e s t i g a t i o n o f t h e p r o d u c t i o n o f

-

101

l y s o z y m e by m i x e d p o p u l a t i o n s o f B.

s t e a r o t h e r m o p h i l u s s t r a i n s grown

o n sewage s l u d g e . Sewage s l u d g e c o n t a i n s a b o u t u n i t s per m l .

Naturally,

7 1 0 -10'

mesophilic colony forming

a c e r t a i n number o f n o n - a c t i v e c e l l s w i t h

i n t a c t c e l l w a l l s can a l s o be found i n t h e sludge. be u t i l i z e d by t h e t h e r m o p h i l e s as s u b s t r a t e . degradation o f c e l l s ,

for the bio-

t h e f i r s t obstacle i s the degradation o f the

c e l l w a l l a n d membrane. be lysozyme,

These c e l l s c o u l d

However,

The r e s p o n s i b l e enzyme w o u l d b e e x p e c t e d t o

e i t h e r a m u r a m i d s s e o r an endo-

t h e maximum c o n t e n t o f

13-glucanase.

c e l l s i n sewage s l u d g e t o b e

lo9

which i s a t l e a s t 1 o r d e r o f magnitude g r e a t e r t h a n c f u ' s b u t a l l o w s f o r dead c e l l s ,

Assuming

per m l , found,

an a v e r a g e p a r t i c l e s i z e o f 1 x 5 p m a n d

20 ?A d r y m a t t e r , t h e t o t a l b i o m a s s i s 0.8

kg

One may f u r t h e r

assume t h a t t h i s b i o m a s s c a n b e c o m p l e t e l y u t i l i z e d b y t h e t h e r m o p h i l e s d u r i n g growth.

Thus,

t h i s smount c a n n o t s i g n i f i c a n t l y c o n -

t r i b u t e a s a s u b s t r a t e when c o m p a r e d t o an a v e r a g e c o n t e n t o f 3 0 k q

m

-3

o f o r g a n i c m a t e r i a l i n sewage s l u d g e . Three d i f f e r e n t methods were u s e d i n o r d e r t o d i m i s h t h e un-

c e r t a i n t y o f t h e l y t i c a c t i v i t y d e t e r m i n a t i o n due t o t h e c o m p l i c a t e d

A l l m e t h o d s p e r m i t t e d t h e d e t e c t i o n o f m i n i m a l amounts

substrate.

o f lysozyme w i t h standards.

The e x t r a c t i o n o f l y s o z y m e f r o m c u l t u r e s

o f sewage s l u d g e showed a r e c o v e r y o f 8 5 - 9 0 % when s t a n d a r d enzyme L y t i c a c t i v i t y c o u l d n o t b e d e t e c t e d i n sewa-

s o l u t i o n s w e r e added.

i n s u p e r n a t a n t s o f c u l t u r e s o r i n e x t r a c t s o f sewage s l u d -

ge s l u d g e ,

ge f r o m a l l p h a s e s i n v o l v i n g t h e r m o p h i l i c

growth.

The m e t h o d c h a r a c -

t e r i z e d b y t h e f l u o r o g e n i c s u b s t r a t e c o u l d be u s e d o n l y a f t e r d i l u t i o n o f t h e sample because o f t h e s t r o n g background f l u o r e s c e n c e o f t h e r a w sewage s l u d g e .

Thesincrease o f 4-methylbelliferone

release

was i n s i g n i f i c a n t . L y t i c a c t i v i t y was m e a s u r e d d u r i n g t h e p u l s e e x p e r i m e n t s w i t h

E.

coli,

P.

a e r u g i n o s a a n d 5. a u r e u s ,

h o g e n i c c e l l a was a c h i e v e d .

u n t i l inactivation o f the pat-

The g r e a t e s t

increase o f l y t i c a c t i v i t y

was f o u n d when t h e p u l s e s w e r e a p p l i e d i n t h e c a r b o n l i m i t e d g r o w t h phase,

r a t h e r t h a n i n any o t h e r p h a a e .

mely low.

B u t g e n e r a l l y t h i s was e x t r e -

During these pulse experiments d i f f e r e n t populations o f

on

aerobic thermophilic

s t r a i n s were i s o l a t e d and grown a t 65

n u t r i e n t agar.

i s o l a t i o n o f the i n d i v i d u a l s t r a i n s these pure

After

OC

s t r a i n s were s c r e e n e d f o r l y a o z y m e p r o d u c t i o n u s i n g t h e l y s o - p l a t e agar technique.

Amongst 43 d i f f e r e n t

a positive result.

a t r a i n a i s o l a t e d o n l y one g a v e

This i n d i c a t e d t h e presence o f s thermophilic

-

-

102

b a c t e r i a l s t r a i n during sludge treatment w i t h a p o t e n t i a l f o r releas i n g lysozyme.

However,

t h e a c t i v i t y was s o l o w t h a t i t c o u l d n o t

e v e n b e d e t e c t e d a f t e r e x t r a c t i o n o f t h e l y s o z y m e f r o m sewage s l u d g e . These e x p e r i m e n t s s u g g e s t t h a t : i n a c t i v a t i o n o f pathogenic microorganism i s a p u r e l y thermal process without the thermophilic

3.2.3

any s y n e r g i s t i c e f f e c t s f r o m

process culture.

Recontamination w i t h pathoqenic microorqanisms R e c o n t a m i n a t i o n e x p e r i m e n t s were c a r r i e d o u t a t 20 OC i n a 50 1

C O L O R r e a c t o r b y p u l s i n g d e f i n e d c o u n t s o f E. a n d S.

aureus i n t o t h e r e a c t o r .

ated aerobic thermophilic

coli,

P.

aeruqinosa

The p u l s e s w e r e added t o f u l l y t r e -

s l u d g e a n d t o s l u d g e w h i c h was p a r t i a l l y

b i o d e g r a d e d . The b i o d e g r a d a t i o n was d e f i n e d b y m o n i t o r i n g t h e o x y gen u p t a k e r a t e o f a e r o b i c t h e r m o p h i l i c c a r b o n l i m i t e d p h a s e was a t t a i n e d ,

t r e a t e d sludge:

when t h e

t h e s l u d g e was d e f i n e d a s t o t a l l y

biodegraded by the aerobic t h e r m o p h i l i c process. A f t e r 4 days o f i n c u b a t i o n c o l o n y f o r m i n g u n i t s o f a l l t e s t organisms i n c r e a s e d i n b o t h f u l l y and p a r t i a l l y b i o d e g r a d e d s l u d g e .

T h e i n c r e a s e was g r e a t e r i f t h e s l u d g e was o n l y p a r t i a l l y b i o d e g r a ded,

b u t t h e r e s u l t s d e m o n s t r a t e t h a t even a t o t a l b i o d e g r a d a t i o n

w i t h aerobic thermophilic

t r e a t m e n t cannot p r e v e n t subsequent growth

o f m e s o p h i l i c b a c t e r i a i n t h e s l u d g e ( F i g u r e 9). When t h e s l u d g e i s t r e a t e d o n l y by an a e r o b i c t h e r m o p h i l i c

pro-

a r e c o n t a m i n a t i o n w i t h p o t e n t i a l pathogens microorganisms i s

cess,

s t i l l possible,

e s p e c i a l l y i f t h e s l u d g e i s s t o r e d i n open v e s s e l s

before a p p l i c a t i o n t o a g r i c u l t u r a l land. I d e n t i c a l e x p e r i m e n t s were c a r r i e d o u t w i t h t h e same t e s t o r ganisms w i t h a e r o b i c t h e r m o p h i l i c / a n a e r o b i c ge.

t h e r m o p h i l i c sewage s l u d -

The a n a e r o b i c t r e a t m e n t was c a r r i e d o u t o n t h e l a b o r a t o r y s c a l e .

R e c o n t a m i n a t i o n d a t a w i t h t h i s d o u b l e t r e a t m e n t a r e shown i n f i g u -

re 10. The p u l s e e x p e r i m e n t s w i t h t h e s e o r g a n i s m s c l e a r l y show t h a t g r o w t h o f p a t h o g e n s a f t e r r e c o n t a m i n a t i o n i s p r e v e n t e d when t h e s l u d ge i s s u b m i t t e d t o a n a e r o b i c t r e a t m e n t a f t e r t h e a e r o b i c t h e r m o p h i l i c pretreatment. These r e s u l t s s u g g e s t t h a t t h e c o n c e p t o f h y g i e n i z a t i o n t h a t i n v o l v e s o n l y t h e i n a c t i v a t i o n o f p o t e n t i a l l y pathogens i s uncomplete and s h o u l d be r e f o r m u l a t e d .

-

103

-

.=- 1000 1

-

‘ L

E

i?

) ‘

STANDARD

100

10

1

0 0

20

80

.60

40

100

120

TIME [ h 1 F i g . 9. Number o f c o l o n y f o r m i n g u n i t s i n t r e a t e d s l u d g e ( f u l l symb o l s ) and p a r t i a l l y t r e a t e d s l u d g e ( o p e n s y m b o l s ) f o r E. c o l i ( s q u a r e s ) a n d S. a u r e u s ( t r i s n g l e s ) . E v e n i f c a r b o n l i m i t a t i o n i s achieved d u r i n g ATS treatment, substrates f o r p o t e n t i a l pathogenic microbes a r e s t i l l p r e s e n t i n t h e s l u d g e and a l l o w g r o w t h i n case t h a t a contamination take place.

-E

7

1000

L

ii

”

100

10

1

1 I

0 4 0

. . . . . . . . . . , . . 20

40

60

80

100

120

T

140

TIME h 1 F i g . 10. Number o f c o l o n y f o r m i n g u n i t s o f a e r o b i c t h e r m o p h i l i c / a n s e r o b i c t h e r m o p h i l i c b i o d e g r a d e d s l u d g e f o r E. c o l i ( s q u a r e s ) a n d s . 0 u r e u s ( t r i s n g l e s ) . The l o w r e d o x p o t e n t i a l o f t h e s l u d g e a f t e r t h e a n a e r o b i c t r e a t m e n t i n h i b i t s t h e grow o f p o t e n t i a l pathogens p u l s e d t h e t r e a t e d sludge.

-

.

104

-

Recontamination t e s t s w i t h selected p o t e n t i a l l y pathog e n i c microorganisms s h o u l d be i n t e g r a t e d i n t o t h e concepts f o r sludge hygienization tests.

3.3

D e w a t e r i n q c h a r a c t e r i s t i c s o f sewaqe a l u d q e I n a d d i t i o n t o h y g i e n i z a t i o n o f sewage s l u d g e ,

thermophilic se,

the aerobic

treatment g i v e s c e r t a i n o t h e r advantages.

f o r m u l a t e d a s an o b j e c t i v e f u n c t i o n o f t h i s w o r k ,

vement o f t h e d e w a t e r i n g c h a r a c t e r i s t i c s

o f t h e sludge.

ment o f t h e s l u d g e d e w a t e r i n g c h a r a c t e r i s t i c s f u l l y a e r o b i c and d u a l a e r o b i c / a n a e r o b i c ses.

Such enhancement

One o f t h e i s t h e improEnhance-

i s valid for both

sludge treatment proces-

i s sometimes c a l l e d s l u d g e c o n d i t i o n i n g .

Due t o t h e s l u d g e c o m p o s i t i o n a n d s t r u c t u r e ,

the water

frac-

t i o n c a n be d i v i d e d i n t o t h r e e p a r t s ( M g l l e r 1 9 6 6 ) :

. . .

f r e e water

70 X

( i n t e r s t i t i a l water)

r e t a i n e d water

+

c a p i l l a r y water

i n t r a c e l l u l a r water

+

22

adsorbed water

Sludge can be s e p a r a t e d f r o m t h e f i r s t

X

8 % f r a c t i o n by s e d i m e n t a -

from t h e second e i t h e r by c e n t r i f u g a t i o n o r p r e s s u r e f i l t r a -

tion,

t i o n and f r o m t h e t h i r d ,

by d r y i n g .

F o r measurement o f t h e s e p a r a -

t i o n o f water from t h e s o l i d f r a c t i o n , technique

(8aaerga 1986) i s o f t e n used.

compared u s i n g t h i s t e c h n i q u e .

a c a p i l l a r y suction time D i f f e r e n t s l u d g e s can be

Lower c a p i l l a r y s u c t i o n t i m e s i n d i -

c a t e enhanced d e w a t e r i n g c h a r a c t e r i s t i c s .

E x p e r i m e n t s w i t h b o t h ae-

r o b i c t h e r m o p h i l i c and raw s l u d g e s gave i n c o n s i s t e n t r e s u l t s f o r measurements o f t h e CST v a l u e s . red ten-timea

I n spite of this, hence,

F o r each sample,

t h e C S T was measu-

and t h e t w o l o w e s t a n d t w o h i g h e s t v a l u e s d i s c a r d e d . t h e r e l a t i v e s t a n d a r d d e v i a t i o n was 8 . 1

was u n a c c e p t a b l e .

% and,

A comparison o f d i f f e r e n t b a t c h e s o f raw

s l u d g e a n d A T S s l u d g e i s p r e s e n t e d i n f i g u r e 11. A l t h o u g h d a t a a r e e f f e c t e d by a r e l a t i v e h i g h s t a n d a r d d e v i a t i o n ,

the trend of

results

show t h a t a e r o b i c t h e r m o p h i l i c

t r e a t m e n t had a n e g a t i v e i n f l u e n c e

on t h e c a p i l l a r y s u c t i o n t i m e

c o n f i r m i n g p r e v i o u s works (Jewel1

e t al.

1982,

Morgan e t a l .

1984).

The e x p e r i m e n t s c a r r i e d o u t f o r t h e d e t e r m i n a t i o n o f s e t t l i n g c h a r a c t e r i s t i c s gsve r e s u l t s t h a t a r e c o n t r o v e r s i a l w i t h t h e CST values (Figure 12).

CST v a l u e s o f 80.1

s f o r r a w a n d 245.4

s for

-

105

-

n

BATCH 2

BATCH 1

BATCH 3

F i g . 11. C a p i l l a r y s u c t i o n t i m e b e f o r e ( m ) thermophilic treatment f o r d i f f e r e n t sludge c u l t i v a t i o n c o n d i t i o n s . The v a l u e s o b t a i n e d d i c a t e a d e c r e a s e o f d e w a t e r a b i l i t y w i t h an t h i s c o n c l u s i o n i s n o t r e l i a b l e , because o f t i c a l v a l i d i t y o f t h e C S T method.

BATCH L

n BATCH 5

and a f t e r ( 0 1 a e r o b i c batches a t d i f f e r e n t w i t h t h i s method i n A T S t r e a t m e n t . However, unsatisfactory analy-

100

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v,

90

80

70 60

50 40 30

! 0

i

10

20

30

40

50

60

70

80

90

100

TIME 1 h 1 F i g . 12. Time c o u r s e f o r t h e s e t t l e d v o l u m e o f r a w s l u d g e ( m ) a n d a e r o b i c t r e a t e d s l u d g e ( o 1. A v e r a g e v a l u e s o f t h r e e b a t c h f r o m t h e 7 1 b i o r e a c t o r . The s e t t l i n g c h a r a c t e r i s t i c s a r e i m p r o v e d by t h e ATS treatment.

t r e a t e d sludge,

106

i n d i c a t e d t h a t poor dewatering c h a r a c t e r i s t i c f o r

t r e a t e d s l u d g e s h o u l d be expected, contrary.

-

b u t s e t t l i n g data demonstrate the

S i m i l a r u n c e r t a i n c o r r e l a t i o n s between s e t t l i n g t i m e and

r e s u l t s o f CST-tests

are reported f o r aerobic thermophilic sludges

( B r e i t e n b G c h e r 1985,

B r e i t e n b i c h e r 1983). F o r t h i s reason,

termination o f settling characteristics

t h e de-

was p r e f e r r e d t o t h e C S T -

t e s t f o r process optimization.

X

D a t a c o n c e r n i n g m e t a b o l i c a c t i v i t y showed t h a t 70-80 microbiological activity

o f the

r e s u l t s from degradation o f p a r t i c u l a t e

matter,

s o t h a t a change i n s l u d g e p a r t i c u l a t e m a t t e r i s t o be e x -

pected.

The i n t e r a c t i o n b e t w e e n i m p r o v e d s e t t l i n g c h a r a c t e r i s t i c s

a n d s l u d g e s o l i d s d e g r a d a t i o n was d e m o n s t r a t e d b y d e t e r m i n a t i o n o f the p a r t i c l e size d i s t r i b u t i o n during the treatment.

F i g u r e 1 3 shows

t h e changes i n t h e r e l a t i v e p a r t i c l e s i z e d i s t r i b u t i o n f o r d i f f e r e n t size classes o f s o l i d s during aerobic thermophilic

growth,

whilst

F i g u r e 1 4 shows t h e r e l a t i v e c h a n g e s i n t h e m e a s u r e d s i z e r a n g e .

4.0

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TIME [ h l F i g . 1 3 . Oxygen u p t a k e r a t e ( 0 ) a n d r e l a t i v e p a r t i c l e s i z e d i s t r i b u t i o n f o r 3 d i f f e r e n t p a r t i c l e c l a s s e s ( 4 9 0 p m : m ; 3 1 p m : o ; 12 pm: + ) during a fed batch c u l t i v a t i o n i n the laboratory scale bioreact o r under o p t i m a l c o n d i t i o n : s t i r r e r speed 1500 m i n - 1 , a e r a t i o n r a The c h a n g e s i n t h e d i s t r i t e 1.5 vvm, pH 7 a n d t e m p e r a t u r e 6 5 OC. bution correlate with microbial metabolic a c t i v i t y , i n d i c a t i n g the a c t i v e a n d s e l e c t i v e d e g r a d a t i o n o f t h e non s o l u b l e f r a c t i o n .

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PRACTICLE SIZE I ,urn 1 F i g . 1 4 . Changes o f p a r t i c l e s i z e d i s t r i b u t i o n r e l a t i v e t o r a w s l u d ge a t d i f f e r e n t c u l t i v a t i o n t i m e s ( 5 . 5 h:.; 8 . 0 h : o ; 10.5 h : + ) during a fed batch c u l t i v a t i o n i n the laboratory scale bioreactor u n d e r o p t i m a l c o n d i t i o n : s t i r r e r s p e e d 1 5 0 0 min-1, a e r a t i o n r a t e 1 . 5 v v m pH 7 a n d t e m p e r a t u r e 6 5 OC. P a r t i c l e s w i t h a l o w d i a m e t e r a r e a t t a c k e d by t h e r m o p h i l e s and a g g l o m e r a t e t o p a r t i c l e s w i t h a large diameter.

The p a r t i c l e s j z e d i s t r i b u t i o n c h a n g e d d u r i n g t h e c u l t i v a t i o n from small t o larger particles. l a g phase o f t h e t h e r m o p h i l i c

Some c h a n g e s e v e n o c c u r e d d u r i n g t h e

culture,

supporting the hypothesis

t h a t enzymes w h i c h a r e n o t d e r i v e d f r o m t h e r m o p h i l i c a l s o present i n the sludge, f i c a t i o n process.

However,

but

t h e most i m p o r t a n t c h a n g e s w e r e d e t e c t e d

during growth o f thermophiles. a t t a c k e d by t h e r m o p h i l e s

microbes,

p l a y an i m p o r t a n t r o l e i n t h e s i z e m o d i P a r t i c l e s i n t h e range 1 t o 2 0 . m

are

and p a r t i a l l y m e t a b o l i z e d w i t h a n i n c r e a s e

o f t h e p a r t i c l e s b e t w e e n 5 0 and 6 0 0 p m due t o a g g l o m e r a t i o n .

As c o n -

sequence o f t h e p r o g r e s s i o n t o l a r g e r s i z e d p a r t i c l e s ,

.

s e t t l i n g c h a r a c t e r i s t i c s o f t h e s l u d g e a r e improved by the aerobic thermophilic treatment

Another

i m p o r t a n t consequence i s t h e change i n t h e r e l a t i v e s u r -

face area o f the p a r t i c u l a t e matter,

as d e s c r i b e d i n F i g u r e 15.

-

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10

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25 TIME [ h I

F i g . 15. S p e c i f i c s u r f a c e a r e a ( m ) a n d o x y g e n u p t a k e r a t e ( 0 ) dur i n g a fed batch c u l t i v a t i o n i n the laboratory scale bioreactor at o p t i m u m c o n d i t i 0 n : s t i r r e r s p e e d 1 5 0 0 m i n - 1 , a e r a t i o n r a t e 1 . 5 vvm, pH 7 a n d t e m p e r a t u r e 6 5 OC. The d e c r e a s e o f t h e s p e c i f i c s u r f a c e i s a consequence o f t h e s h i f t e d p a r t i c l e s i z e d i s t r i b u t i o n .

Owing t o t h e c h a n g i n g s i z e d i s t r i b u t i o n , o f t h e p a r t i c u l a t e m a t t e r decreased by 16

the specific surface

Z d u r i n g t r e a t m e n t . The

d e c r e a s e was m o s t s i g n i f i c a n t d u r i n g t h e a c t i v e p h a s e o f t h e r m o p h i l i c microorganism growth.

The r e d u c e d s p e c i f i c s u r f a c e a r e a c o r r e s -

ponds w i t h t h e i n c r e a s e d C S T o b t a i n e d d u r i n g a e r o b i c t h e r m o p h i l i c t r e a t m e n t and i m p l i e s an i n c r e a s e i n c a p i l l a r y a n d b o u n d w a t e r r e l a t i v e t o f r e e water

(Baserga 1984).

f o r aerobic thermophilic

The d e w a t e r i n g c h a r a c t e r i s t i c s

sludge a f t e r e i t h e r a pretreatment o f a f u l l

b i o d e g r a d a t i o n process has two dominant aspects:

.

t h e i n c r e a s e d p a r t i c l e s i z e has a p o s i t i v e i n f l u e n c e on s l u d g e d e w a t e r i n g e i t h e r by g r a v i t y s e t t l i n g o r c e n t r i -

.

fugation t h e decreased s p e c i f i c s u r f a c e has a n e g a t i v e i n f l u e n c e on t h e s e p a r a t i o n o f b o u n d w a t e r e i t h e r b y f i l t r a t i o n o r drying.

The n e g a t i v e i n f l u e n c e o n l y p l a y s a r o l e when t h e s l u d g e i s dryed a f t e r f u l l biodegradation.

I n a l l o t h e r cases,

the benefit o f

aerobic thermophilic

-

109

s l u d g e t h a t a c c r u e s i s enhanced d e w a t e r i n g cha-

racteristics.

4

CONCLUSIONS The d e v e l o p m e n t o f

an e f f i c i e n t

process f o r the treatment o f

w a s t e sewage s l u d g e i n v o l v e s d e f i n i t i o n a n d q u a n t i f i c a t i o n o f o b j e c tive

functions.

I n t h e c a s e o f a m i c r o b i o l o g i c a l p r o c e s s i n t h e was-

tewater treatment industry, t i o n s between s c i e n t i f i c

t h i s step requires synergistic

authorities,

h o r i t i e s and p l a n t c o n s t r u c t o r s .

interac-

p o l i t i c a l and l e g i s l a t i v e a u t -

R e p o r t e d r e s u l t s and u n c e r t a i n ob-

j e c t i v e s a t t e s t s t h a t t h e s y n e r g i s t i c e f f e c t i s f a r from f i n d i n g a solution.

D i f f e r e n t sources f o r a q u a l i f i e d d e f i n i t o n o f o b j e c t i v e s

f o r o p t i m i z a t i o n were c o n s i d e r e d .

The a p p r o a c h i n v o l v e d i n t e r v i e w s

and d i s c u s s i o n s w i t h p l a n t o p e r a t o r s , suthorities.

c o n s t r u c t o r s and r e g u l a t i n g

The p r o d u c t i o n o f a minimum amount o f t h e r m o p h i l i c b i o -

mass w i t h maximum h e a t p r o d u c t i o n i s t h e m o s t i m p o r t a n t o b j e c t i v e . O t h e r o b j e c t i v e i n c l u d e m a x i m i z a t i o n o f d e g r a d a t i o n p e r f o r m a n c e , enz y m a t i c a c t i v i t i e s and t h e improvement o f d e w a t e r i n g c h a r a c t e r i s t i c s . The i n f l u e n c e o f t h e a e r o b i c t h e r m o p h i l i c

p r o c e s s as a p r e t r e a t m e n t

process w i t h a f o l l o w i n g anaerobic d i g e s t i o n process has a l s o t o be These a d d i t i o n a l o b j e c t i v e s c a n b e i n t e g r a t e d i n t h e i n a c -

considered.

t i v a t i o n o f p o t e n t i a l l y p a t h o g e n i c o r g a n i s m s t h a t a r e p r e s e n t and c o n c e n t r a t e d i n t h e sewage s l u d g e .

The h y g i e n i z a t i o n o f t h e s l u d g e

i s t h e o n l y t a r g e t t h a t i s f o r m u l a t e d by t h e S w i s s l a w . Aerobic

thermophilic

take the greatest

s l u d g e t r e a t m e n t p l a n t s o p e r a t i n g a t 65 O C

a d v a n t a g e o f maximum t h e r m o p h i l i c m i c r o b i a l r e s p i -

r a t o r y a c t i v i t y and a s s o c i a t e d h e a t p r o d u c t i o n . t h a n 60 cy.

OC

a r e u n i n t e r e s t i n g because o f l o w h y g i e n i z a t i o n e f f i c i e n -

Temperatures g r e a t e r t h a n 70

t o m i c r o b i a l reaources. process efficiency sary,

Temperatures l e s s

OC

are too r e s t r i c t e d w i t h respect

The pH v a l u e i s n o t a r e l e v a n t f a c t o r

p r o v i d e d i t exceeds 6.8

a l t h o u g h pH measurement

for

pH r e g u l a t i o n i s u n n e c e s -

i s an i m p o r t a n t p r o c e s s i n d i c a t o r .

Ae-

r o b i c t h e r m o p h i l i c m i c r o o r g a n i s m s a r e i n a c t i v e s t pH v a l u e s b e t w e e n 5 and 6,

b u t s u c h pH v a l u e s o f t e n o c c u r i n u n t r e a t e d w a s t e s l u d g e .

Proteolytic a c t i v i t y i s the only extracellulsr

enzymatic a c t i v i t y

associated with the growth o f thermophilic process microbes, always d e t e c t e d d u r i n g an e f f e c t i v e t r e a t m e n t .

that i s

I t h a s an a c t i v i t y op-

t i m u m i n v i t r o a t 80 OC. P r o t e a s e s p r e s e n t i n t h e r a w s l u d g e a r e in a c t i v e a t t h i s temperature.

Therefore,

i t i s possible t o quantify

- 110

-

t h e p e r f o r m a n c e o f a t r e a t m e n t u n i t by m e a s u r i n g t h e p r o t e o l y t i c a c t i v i t y a t t h i s temperature. E x t r a c e l l u l a r p r o t e a s e s a r e t h e enzymes t h a t a r e a c t i v e d u r i n g normal o p e r a t i n g c o n d i t i o n s , b u t t h e presence o f s t a r c h i n s l u d g e rapidly induces t h e production of amylases. Experiments a t l a b o r a t o r y s c a l e COLOR b i o r e a c t o r s o f 7 and 50 1 i n d i c a t e t h a t d e c r e a s e s i n a c t i v i t y d u r i n g b a t c h and fed b a t c h c u l t i v a t i o n s a r e d u e t o c a r b o n l i m i t a t i o n . The h e a t i n a c t i v a t i o n o f pot e n t i a l l y p a t h o g e n i c m i c r o o r g a n i s m s was a p u r e t e m p e r a t u r e e f f e c t w i t h no s y n e r g i s t i c e f f e c t s from t h e t h e r m o p h i l i c p o p u l a t i o n .

Neit-

h e r a n t i m i c r o b i a l a g e n t s n o r l y s o z y m e w e r e d e t e c t a b l e i n s l u d g e undergoing treatment,

a l t h o u g h one t h e r m o p h i l i c s t r a i n t h a t was i s o -

lated exhibited l y t i c potential.

Pulse experiments with potentially

pathogenic microorganisms demonstrate t h a t t h e i r i n a c t i v a t i o n is s l o w e d down by s l u d g e c o m p o n e n t s t h a t e x e r c i s e a p r o t e c t i v e a c t i o n . A g r e a t advantage r e s u l t i n g from t h e u s e o f a e r o b i c thermophi-

l i c microorganisms is t h e enhancement o f s l u d g e s e t t l i n g c h a r a c t e -

ristics.

T h i s e x t r a b e n e f i t is due t o a d e c r e a s e o f s m a l l p a r t i c l e s

a n d a n i n c r e a s e o f p a r t i c l e s l a r g e r t h a t 3 0 ,urn d u r i n g t h e p r o c e s s . I n t h e f u t u r e , c o n t i n u i n g i n c r e a s e s i n s l u d g e p r o d u c t i o n and i n c r e a s e d a w a r e n e s s o f e n v i r o n m e n t a l p r o b l e m s may s t i m u l a t e t h e r e s e a r c h and development of t e c h n i c a l - s c a l e using thermophilic microorganisms.

sludge treatment processes

Should t h i s occur, t h e present

work w i l l g i v e p r a c t i c a l b a c k g r o u n d knowledge f o r t h e d e s i g n and c o n s t r u c t i o n of sludge treatment processes t h a t is not subjected t o historical prejudice.

REFERENCES 1

U.

Eaier

Zur P h y s i o l o g i e t h e r m o p h i l e r B a c i l l i .

Diss E T H Z G r i c h ,

Nr 8423. i1987). 2

3

4 5 6

U. Baserga,Der

E i n f l u s s der anaeroben Schlammstabilisation auf d i e F a u l s c h l a m m e n t w l s s e r b a r k e i t . Diss E T H N r 7 5 4 2 , ( 1 9 8 4 ) . P.L. E e r g q u i s t , D . R . L o v e , J.R. C r o f t , M.E. D a n i e l a n d W . H . Morgan, G e n e t i c s and p o t e n t i a l a p p l i c a t i o n s o f t h e r m o p h i l i c and e x t r e m e l y t h e r m o p h i l i c m i c r o o r g a n i s m s . B i o t e c h n o l o g y a n d Genet i c E n g i n e e r i n g r e v i e w s 5:199-24, (1987). M . E o m i o , H y g i e n i s i e r u n g v o n K l a r s c h l a m m . 20. V S A - F o r t b i l d u n g s k u r s , Klarschlamm: Behandlung-Verwertung-Entsorgung, Engelberg,

(1968). M. Eomio, E i o p r o c e s s development f o r a e r o b i c t h e r m o p h i l i c s l u d g e t r e a t m e n t . Diss E T H Z i r i c h N r 9 1 5 9 , ( 1 9 9 0 ) . M. Eomio, 8 . S o n n l e i t n e r and A. F i e c h t e r , Growth and b i o c a t a l y t i c a c t i v i t i e s o f a e r o b i c t h e r m o p h i l i c p o p u l a t i o n s i n sewage sludge. Appl Microbiol Eiotechnol 32:356-362, (1989).

7 8

9

10

11 12

13

14 15

16

17

18 19 20

21 22

23

24

25

111 -

K. B r e i t e n b i c h e r , A e r o b - t h e r m o p h i l e S t a b i l i s i e r u n g von Abwasserschlammen H o h e n h e i m e r A r b e i t e n , R e i h e A g r a r t e c h n i k , ( 1 9 8 3 ) . K. B r e i t e n b d c h e r , E n g i n e e r i n g and p r a c t i c a l e x p e r i e n c e s o f a u t o In: I n a c t i v a t i o n o f micheated aerobic-thermophilic d i g e s t i o n r o o r q a n i s m s i n sewage s l u d g e b y s t a b i l i s a t i o n p r o c e s s e s . D . S t r a u c h , A.H. H a v e l a a r a n d P. L ’ H e r m i n e ( e d s ) , E l s e v i e r A p p l i e d S c i e n c e P u b l i s h e r s , L o n d o n New Y o r k 1 9 2 - 2 0 5 , ( 1 9 8 5 ) . BUWAL S w i s s F e d e r a l D e p a r t m e n t o f t h e I n t e r i o r , E n v i r o n m e n t a l P r o t e c t i o n Agency, p e r s o n a l c o m m u n i c a t i o n , ( 1 9 9 0 ) . Camp, D r e s s e r , McKee, E n g i n e e r i n g a n d e c o n o m i c a s s e s s m e n t o f autoheated thermophilic aerobic d i g e s t i o n w i t h a i r aeration. EPA R e p o r t - 6 0 0 / 2 - 8 1 1 7 1 1-39, (1981). Jewell, Thermophilic aerobic digestion o f R.J. Cummings and W . J . d i a r y waste. I n ; f o o d f e r t i l i z e r and a g r i c u l t u r a l r e s i d u e s , L o e h r R C ( e d ) , Ann A r b o r S c i e n c e P r e s s , Ann A r b o r 6 3 7 , ( 1 9 7 7 ) . A . E . E r a l p J.B. F a r r e l , R e c e n t d e v e l o p m e n t s i n t h e r m o p h i l i c s l u d g e d i g e s t i o n i n t h e U n i t e d S t a t e s . US E n v i r o n m e n t a l P r o t e c t i o n Agency EPA/600/D-86/073, (1986). R.G. Feachem, D . J . B r a d l e y , H . G a r e l i k a n d D.D. Mara, S a n i t a t i o n a n d d i s e a s e . H e a l t h a s p e c t s o f w a s t e w a t e r management W o r l d b a n k S t u d i e s i n V a t e r S u p p l y a n d S a n i t a t i o n 3, ( 1 9 8 3 ) . F e d e r a l D e p a r t m e n t o f t h e I n t e r i o r , K l a r s c h l a m m v e r o r d n u n g vom 8. A p r i l (1981)/814.225.23. EDMZ B e r n , 1 1 9 8 1 ) . G. Hamer a n d C . A . Mason, F u n d a m e n t a l a s p e c t s o f w a s t e sewage s l u d g e t r e a t m e n t : M i c r o b i a l s o l i d s b i o d e g r a d a t i o n i n an a e r o b i c t h e r m o p h i l i c semi-continuous system. B i o p r o c e s s E n g i n e e r i n g 2 ~ 6 9 - 7 7 , (1987). H.E. Hammel, H y g i e n i s c h e U n t e r s u c h u n g e n :her d i e W i r k u n g v o n V e r f a h r e n zur Kompostierung von Entwassertem Klarschlamm und zur a e r o b - t h e r m o p h i l e n S t a b i l i s i e r u n g von Fldssigschlamm. D i s s Just u s L i e b i g - U n i v e r s i t a t Giessen, (1983). W.J. J e w e l l , R . M . K a b r i c k a n d J . S . Spada A u t o h e a t e d , a e r o b i c t h e r m o p h i l i c d i g e s t i o n w i t h a i r a e r a t i o n . E P A P r o j e c t Summary, (1982). R M. K a b r i c k a n d W.J. J e w e l l F a t e o f p a t h o g e n s i n t h e r m o p h i l i c a e r o b i c s l u d g e d i g e s t i o n . W a t e r Res 1 6 : 1 0 5 1 - 1 0 6 0 , (1982). T . K e n k e l and J.M. Trela, P r o t e i n turnover o f t h e extreme t h e r (1979). m o p h i l e Th. a q u a t i c u s . J. B a c t e r i o l 1 4 0 : 5 4 3 - 5 4 6 , G . L a n g e l a n d , 6. P a u l s r u d a n d B.E. Haugan, A e r o b i c t h e r m o p h i l i c s t a b i l i s a t i o n . I n : I n a c t i v a t i o n o f m i c r o o r g a n i s m s i n sewage s l u d H a v e l a a r , P. ge b y s t a b i l i s a t i o n p r o c e s s e s . D. S t r a u c h , A.H. L ’ H e r m i t e ( e d s ) , E l s e v i e r A p p l i e d s c i e n c e p u b l i s h e r s , London New Y o r k 3 8 - 4 7 , ( 1 9 8 5 ) . C . L e n t n e r , G e i g y S c i e n t i f i c T a b l e s . V o l 1, u n i t s o f m e a s u r e m e n t , body f l u i d s , c o m p o s i t i o n o f t h e body, n u t r i t i o n , (1981). C.A. Mason, G . Hamer a n d J . D . B r y e r s , The d e a t h a n d l y s i s o f microorganisms i n environmental processes FEMS M i c r o b i o l o g y Rewiews 3 9 : 3 7 3 - 4 0 1 , (1986). U. M o l l e r , S c h l a m m e n t w a s s e r u n g a l s t e c h n i s c h - w i r t s c h a f t l i c h e s Energieproblem. S t u t t g a r t e r B e r i c h t e zur Siedlunggswasserwirts c h a f t 18:265-280, (1966) Gunson, M.H. L i t t l e w o o d a n d R . W i n s t a n l e y , AeS.F. Morgan, H . G . r o b i c t h e r m o p h i l i c d i g e s t i o n o f s l u d g e u s i n g a i r . I n : Sewage Sludge S t a b i l i s a t i o n and D i s i n f e c t i o n , A. B r u c e ( e d ) , E l l i s Horwood L i m i t e d , C h i c h e s t e r 2 7 8 - 2 9 2 , ( 1 9 8 4 ) . H. N e b i k e r , F l b s s i g r o t t e a l a G r u n d l a g e s t a b i l e r S c h l a m m h y g i e n i s i e r u n g d u r c h n a t G r l i c h e n A n t a g o n i s m u s . C h e m i s c h e Rundschau, (19A1).

26

27

28

29

30

31

112

-

H.U. S c h w e i z e r , R e c h t l i c h e G r u n d l a g e n d e r K l a r s c h l s m m e n t s o r g u n g . 20. V S A - F o r t b i l d u n g s k u r s , K l a r s c h l a m m : B e h a n d l u n g - v e r w e r t u n g Entsorgung, Engelberg, (1988). 8. S o n n l e i t n e r , B i o t e c h n o l o g y o f t h e r m o p h i l i c b a c t e r i a growth, p r o d u c t s and a p p l i c a t i o n . Advances i n B i o c h e m i c a l E n g i n e e r i n g / B i o t e c h n o l o g y 28:70-138, (1983). D. S t r a u c h , H.E. Hammel a n d W. P h i l i p p , I n v e s t i g a t i o n s on t h e h y g i e n i c e f f e c t o f s i n g l e s t a g e and t w o - s t a g e a e r o b i c - t h e r m o p h i l i c s t a b i l i s a t i o n o f l i q u i d raw s l u d g e In: Inactivation o f m i c r o o r g a n i s m s i n sewage s l u d g e b y s t a b i l i s a t i o n p r o c e s s e s . D. S t r a u c h , A.H. H a v e l a a r , a n d P. L ’ H e r m i t e ( e d s ) , E l s e v i e r App l i e d s c i e n c e p u b l i s h e r s , L o n d o n New Y o r k 48-63, (1985). H . Wassen, H y g i e n i s c h e U n t e r s u c h u n g e n g b e r d i e V e r w e n d b a r k e i t d e r U m w a l r b e l u f t u n g (System FUCHS) z u r A u f b e r e i t u n g v o n f l u s s i g e n A b f a l l e n a u s dem kommunalen u n d l a n d w i r s c h a f t l i c h e n B e r e i c h . D i s s J u s t u s L i e b i g - U n i v e r s i t a t Giessen, (1975). N.E. W e l k e r a n d L.L. C a m p b e l l , P u r i f i c a t i o n a n d p r o p e r t i e s o f a l y t i c enzyme f r o m i n d u c e d c u l t u r e s o f B a c i l l u s s t e a r o t h e r m o p h i (TP-1). B a c t e r i o l o g i c a l P r o c A m S O C M i c r o b i o l 66:126-126, lus m 6 6 ) . N. W i t t e k i n d , D e t e r m i n a t i o n o f E n t e r o b a c t e r i a c e a e i n sewage s l u d ge. I n p r e p a r a t i o n , ( 1 9 9 0 ) .

-

ELIMINATION OF SPECIAL BACTERIA FROM TREATMENT EFFLUENT BY CILIATES

M.

M A C E K a n d P. H A R T M A N

H y d r o b i o l o g i c a l I n s t i t u t e , C z e c h o s l o v a k Academy o f S c i e n c e s 370 (15 E e s k d B u d G j o v i c e , C z e c h o s l o v a k i a

INTRtIDUCTION Activated-sludge

treatment i s the best technology f o r the e l i -

m i n a t i o n o f o r g a n i c p o l l u t i o n from waste waters. ciency of

H o w e v e ~ , t h ee f f i -

t h e t r e a t m e n t may b e l o w i f t h e y c o n t a i n s p e c i f i c s u b s t r a -

t e s which a r e degradable o n l y by d i s p e r s e d g r o w i n g b a c t e r i a w i t h very low growth rate. t i v e p r e d a t o r s (e.g.

These b a c t e r i a c o u l d b e e l i m i n a t e d b y e f f e c rotifers)

and nondegraded p o l l u t i o n l e a v e s t h e

system o r , t h e s e substances as t h e dominant s u b s t r a t e promote g r o w t h o f d i s p e r s i o n a n d t h e r e f o r e t h e e f f l u e n t i s p o l l u t e d by b a c t e r i a . C i l i a t e s c o u l d s u p p r e s s t h e s e p r o b l e m s c a u s e d b y t h e means o f t w o mechanisms w h i c h a f f e c t t h e b a c t e r i a l c o m m u n i t y as:

1. a l t e r n a t i v e p r e y ,

2. induced f l o c c u l a t i o n . 1. A l t e r n a t i v e p r e y The a s s u m p t i o n o f m e c h a n i c a l s e l e c t i o n o f o p t i m a l f o o d b y c i l i a t e s has n o t been d i s p r o v e d y e t

111.

S e l e c t i o n o f a p a r t i c u l a r volume

or d i a m e t e r range o f p a r t i c l e s m i g h t be caused by t h e i r mechanical p r o p e r t i e s and t h e o r e t i c a l l y c o u l d e l i m i n a t e t h e p o s s i b i l i t y o f d i f f e . r e n t i a t i o n among b a c t e r i a l c e l l s . b a c t e r i a should be ingeated. were s l i g h t l y

Thus,

a r a t h e r wide spectrum o f

T h i s was a l s o p r o v e d w i t h b a c t e r i a w h i c h

t o x i c f o r c i l i a t e s upon monoxenic c u 1 , t i v a t i o n b u t p r o -

v i d e d g r o w t h i f t h e y were i n g e s t e d a l o n g w i t h s u i t a b l e f o o d (Escher i c h i a c o l i [ 2 1 , S a l m o n e l l a t y p h i m u r i u m 131 ).

However,

t h i s problem

c a n b e s t u d i e d more t h o r o u g h l y now o w i n g t o t h e d e v e l o p m e n t o f a d i r e c t m e t h o d f o r e s t i m a t i o n o f p r o t o z o a n f e e d i n g r a t e s 141.

2.

Induced f l o c c u l a t i o n The e f f e c t o f c i l i a t e s on t h e f l o c c u l a t i o n o f b a c t e r i a a n d a t a -

b i l i z a t i o n o f f l o c c u l a t e d b i o c e n o a i s h a s b e e n s t u d i e d f o r many y e a r s

-

114

-

from t h e v i e w p o i n t o f technology 151,

m a t h e m a t i c a l m o d e l l i n g 161,

a n d m i c r o b i a l e c o l o g y [7]. P r e d a t i o n p r e s s u r e o f p r o t o z o a m i g h t i n duce t h e development o f f l o c s or o t h e r m o r p h o l o g i c a l t y p e s o f b a c t e r i a l growth r e s i s t i n g p r e d a t i o n a t t a c k ( f i l a m e n t s ,

zoogleas etc.),

though pure c u l t u r e s o f these b a c t e r i a are dispersed-growing. o t h e r hand,

On t h e

t h e promotion o f a d i s p e r s e d growth o f f a s t growing bac-

t e r i a was t h e o r e t i c a l l y loaded systems,

too.

p r e d i c t e d and p r a c t i c a l l y v e r i f i e d i n o v e r -

The p r o t o z o a n e f f e c t on t h e d e v e l o p m e n t o f

d i f f e r e n t m o r p h o l o g i c a l t y p e s o f b a c t e r i a i s supposed t o be caused b y c i l i a t e c o n t r o l o f t h e b a c t e r i a l community s t r u c t u r e 1 5 1 . Continuous

-

flow c u l t i v a t i o n

T h e s y s t e m s were o r i g i n a l l y i n o c u l a t e d by t h e s t o c k b a c t e r i a l

s t r a i n NTA-1

i s o l a t e d i n o u r l a b o r a t o r y 181.

The s t o c k c u l t u r e o f

c i l i a t e C o l p i d i u m campylum ( S T O K E S ,

t h e free-swimming

1 8 8 6 ) was u s e d

191. Two c o l u m n t a n k s w e r e u s e d f o r t h e e x p e r i m e n t s .

These s y s t e m s

w e r e c o n t i n u o u s l y f e d ( p e r i s t a l t i c pump) b y a n u t r i e n t medium ( T a b . 1 ) c o n t a i n i n g n i t r i l o t r i a c e t i c a c i d aa t h e s o l e o r g a n i c c a r b o n a n d n i t r o g e n source.

The o u t f l o w was p e r i o d i c a l b y s i p h o n a c t i o n r e a -

c h i n g t h e maximum volume flowed a t a time.

(480 m l ) ,

a n d one s i x t h o f i t (80 m l )

out-

A e r a t i o n a n d t h e s t i r r i n g o f t h e s y s t e m s were a-

c h i e v e d by a i r b u b b l i n g t h r o u g h t h e column f o o t

or by a i r l i f t c i r -

c u l a t i o n o f a c u l t i v a t i o n m i x t u r e f r o m t h e f o o t u p t o t h e maximum volume l e v e l .

The p o s i t i v e p r e s s u r e on a l l o u t f l o w s f r o m t h e s y s t e m

kept the c u l t u r e protozoan-free,

or w i t h j u s t a s i n g l e i n o c u l a t e d

protozoan speciea. During the f i r s t p e r i o d o f c u l t i v a t i o n (a)

biomass l e f t t h e

system as mixed l i q u o r o u t l e t ( l i k e a chemostat).

Then d a i l y b i o m a s s

w a s t i n g was a d d e d b e c a u s e m i x i n g c a u s e d b y a i r l i f t waa n o t a u f f i c i e n t t o p r e v e n t biomass accumulation i n t h e systems (b, m i x i n g was u s e d a g a i n a t t h e e n d o f e x p e r i m e n t

( e , f).

TABLE 1 B a s a l n u t r i e n t medium

NTA NaOH

(mg .l-l)

300 90

c).

A i r

-

-

115

a

b

C

Dilution rate (d-l)

0.5

0.5

D a i l y wasting ( V W / y m a x l

0

0

0.06

0.11

Denotation

C

or9

l o a d i n g (9.1-

.

‘1

d

d

e

f

0.5

0.6

0.5

1.0

1/5

1/5

1/5

1/5

0.11

0.20

0.14

0.70

E v a l u a t i o n o f biomass g r o w t h The number o f N T A - 1

s t r a i n colony

f o r m i n g u n i t s (CFU) was e s -

t i m a t e d on a g a r p l a t e s w i t h N T A medium o n l y , number on a g a r w i t h 200 m g . 1 - l

and t h e t o t a l

o f yeast e x t r a c t

(CFU)

(Difco) or 100-fold

d i l u t e n u t r i e n t agar ( D i f c o ) . T o t a l b i o m a s s was d e t e r m i n e d b y means o f t h e d i c h r o m a t e c h e m i c a l o x y g e n demand

COD

[lo]

from which t h e o r g a n i c carbon c o n t e n t

o f t h e b i o m a s s was c a l c u l a t e d u s i n g t h e q u o t i e n t C O D / C = 2 . 8 1 g. or9 -1 g The d i s p e r s e d p o r t i o n o f b i o m a s s was e s t i m a t e d a f t e r r e m o v i n g

.

f l o c c u l a t e d biomass by low-speed c e n t r i f u g a t i o n

(2000 m i n - l ,

S o l u b l e C O D c o n t e n t was d e t e r m i n e d i n a s u p e r n a t a n t

5 min).

a f t e r 5 minutes

o f c e n t r i f u g a t i o n a t 1 2 500 m i n - l .

The number o f c i l i a t e s was d e t e r m i n e d i n p r e s e r v e d s a m p l e s (Uterrnohl’s

-

DaFano s o l u t i o n s ) b y d i r e c t m i c r o s c o p i c c o u n t s u s i n g

1 m l Kolkwitz’s

s e d i m e n t a t i o n chambers.

was assumed t o b e 1 0 0 m g . m l - I

The o r g a n i c c a r b o n c o n t e n t

o f volume biomass 1111,

w h i c h was c a l -

c u l a t e d from c e l l dimensions u s i n g t h e e q u a t i o n f o r t h e volume of ellipsoid.

F o r t h e measurement o f c i l i a t e s u s p e n s i o n f e e d i n g r a t e s ,

the b a c t e r i a c e l l s from the c u l t i v a t i o n u n i t without f l u o r e s c e n t l y l a b e l l e d (41. r i a f o r 10 minutes, enumerated.

fixed,

The ( v o l u m e )

p r o t o z o a were

C i l i a t e s were l e f t w i t h l a b e l l e d b a c t e and t h e number o f i n g e s t e d p a r t i c l e s was

c l e a r i n g r a t e was e s t i m a t e d

111.

The c i l i a -

t e u p t a k e r a t e o f b a c t e r i a was c a l c u l a t e d f r o m c l e a r l p g r a t e s u p p o s i n g f e e d i n g on d i s p e r s e d b a c t e r i a o n l y . n i c carbon content

For t h e budget,

t h e orga-

o f t h e volume biomass o f b a c t e r i a (measured u s i n g

f l u o r e s c e n t m i c r o s c o p e ) was assumed t o b e 258 mg.ml

-1

1121.

R E S U L T S AND D I S C U S S I O N Though t h e c u l t i v a t i o n c o n d i t i o n s were s t a b l e i n t h e f i r s t p e r i o d ( s e e Tab. (Figs l A ,

1B):

2),

t w o t y p e s o f c o m m u n i t y d e v e l o p m e n t were o b s e r v e d

-

116

-

1. d o m i n a n t l y d i s p e r s e d g r o w t h ( 4 1 s t do 5 5 t h day i n s y s t e m A , t o 4 1 s t day i n

a),

s i m u l t a n e o u s d i s p e r s e d and f l o c c u l a t e d g r o w t h ( s y s t e m A

2.

10th

and

-

10th

t o 41st day).

-

? A

c;-

DISPERSED GROWTH

-

7

6

‘2

4 % 2 m

w

12%

log 8 6 4 2

TIME DAYS 1 F i g . 1. C h a r a c t e r i z i n g d a t a o f s y s t e m s A a n d B A : T o t a l i n o r g a n i c n i t r o g e n ( 0 1 and n i t r a t e n i t r o g e n ( 0 1 B: D i s p e r s e d a n d o u t - f l o w i n g b i o m a s s ( 0 1 ; number o f o u t - f l o w i n g ciliates (0) C : I n - s y s t e m t o t a l b i o m a s s l o ) , a n d number o f c i l i a t e s ( 0 ) . T h i s depended o n t h e b a c t e r i a l c o m m u n i t y d i v e r s i t y :

t h e r e was

a l i n e a r c o r r e l a t i o n o f t h e r a t i o between d i s p e r s e d and t o t a l biomass

(COD),

-

117

versus t h e i n v e r s e r a t i o betwen N T A - 1

a n d t h e t o t a l number o f

b a c t e r i a ( d i r e c t r e l a t i o n s h i p between N T A - 1 p o r t i o n and d i s p e r s e d b i o m a s s p o r t i o n on t o t a l v a l u e s i n F i g .

2).

The e q u a t i o n was a l s o

v a l i d f o r b a c t e r i a l (dispersed) culture A w i t h a i r - l i f t (112nd t o 1 4 3 r d day),

circulation

although the accumulation o f f l o c c u l a t e d bio-

mass m i g h t h a v e b e e n p r o m o t e d b y s e d i m e n t a t i o n . system B i n o c u l a t e d w i t h c i l i a t e s (a+;

Similarly,

4 1 s t day)

d a t a from

agreed w i t h t h i s

r e l a t i o n s h i p u n l e s s t h e c i l i a t e c o n c e n t r a t i o n r e a c h e d c r i t i c a l val u e ( a p p r o x i m a t e l y 5x103 m 1 - l ) . significant

=

(N

34;

The l i n e a r r e l a t i o n s h i p was h i g h l y

r = 0.926)

e x c e p t i n g t h r e e p o i n t s ( t w o o f them

were b i a s e d b y an u n c o n t r o l l e d w a l l g r o w t h f r o m 6 3 r d t o 9 6 t h day). This v e r i f i e d the observed dispersed growth o f pure NTA-1

[el. The r e l a t i o n s h i p d i d n o t

without s e l f f l o c c u l a t i o n tendencies

i f t h e c i l i a t e c o n c e n t r a t i o n came c l o s e t o

apply,

culture

lo4

m1-l.

The ob-

s e r v e d e f f e c t o f c i l i a t e s on b a c t e r i a l f l o c c u l a t i o n was i n c o n c o r dance w i t h t h e d e s c r i b e d e f f e c t o f p r e d a t i o n p r e s s u r e 15,

71. P r o -

d u c t i o n o f autochthonous s u b s t r a t e s c o u l d s e r v e as a f e e d source f o r other (NTA nondegrading) b a c t e r i a . The m i x e d c u l t u r e s u n d e r c o n d i t i o n s o f a i r - s t i r r i n g B ) showed s h i g h e r a m m o n i a - n i t r o g e n

(Figs.

content than n i t r a t e ,

lA,

along

w i t h a r a t h e r l o w c o n c e n t r a t i o n o f i n o r g a n i c n i t r o g e n ( u p t o 20 mg. 1-l).

N i t r i f i c a t i o n (an increase o f n i t r a t e concentration)

started after

t h e a i r l i f t c i r c u l a t i o n was s w i t c h e d - o n

only

(Fig l A ,

B).

Linear increase o f n i t r a t e concentration i n the non-protozoan c u l -

=

t u r e A o v e r t i m e was s i g n i f i c a n t ( N

20;

r = 0.966)

as w e l l as t h e

l i n e a r c o r r e l a t i o n between n i t r a t e and biomass c o n c e n t r a t i o n s 19;

(N

=

r = 0.881). T h i s t r e n d c o n t i n u e d a f t e r c i l i a t e i n o c u l a t i o n and d u r i n g t h e i r

growth.

A d e c r e a s e o f n i t r a t e c o n c e n t r a t i o n was o b s e r v e d a f t e r t h e

r e - i n s t a l l a t i o n o f a i r - s t i r r i n g d u r i n g t h e wash-out

o f t h e biomass.

Though t h e n i t r a t e c o n c e n t r a t i o n i n t h e p r o t o z o a n s y s t e m B was a l s o increasing,

e s p e c i a l l y a f t e r i n o c u l a t i o n o f b s c t e r i s l c u l t u r e from

s y s t e m A on t h e 1 4 3 r d day ( F i g .

lB),

g a n i c n i t r o g e n remained r a t h e r low.

the t o t a l concentration o f i n o r T h i s c o u l d be due t o s i m u l t a n e -

o u s n i t r a t e r e d u c t i o n i n t h e b a c t e r i a l f l o c s w h i c h s e d i m e n t a t e d on t h e column b o t t o m

13

. NTA-1

b a c t e r i a t h e m s e l v e s were n o t a b l e t o

r e d u c e n i t r a t e s 181.

A h i g h e r c i l i a t e a b u n d a n c e was a s a o c i a t e d w i t h a h i g h e r p r o p o r t i o n o f b a c t e r i a o t h e r t h a n NTA-1;

under c u l t i v a t i o n condition8 i t

was a l s o a a s o c i a t e d w i t h a h i g h e r p o r t i o n o f f l o c c u l a t e d b i o m a s s .

-

118

-

D a t a f o r an e x a c t e x p l a n a t i o n o f mass g r o w t h i n b o t h s y s t e m s a f t e r t h e 1 5 0 t h day were n o t a v a i l a b l e .

B a c t e r i a c o m m u n i t y s t r u c t u r e appa-

r e n t l y d i d n o t change and t h e c u l t i v a t i o n c o n d i t i o n s remained unchanged.

Probably,

t h e i n o c u l a t i o n o f b a c t e r i a from system A t o B (143rd

day) promoted h i g h e r growth o f c i l i a t e s .

Also,

under extremely h i g h

s u b s t r a t e l o a d i n g o f system B ( f ) t h e c i l a t e s were e b l e t o c o n t r o l dispersed growth,

$1

9

X

and t h e c u l t u r e r e m a i n e d f l o c c u l a t e d .

0.8

0,2 0.1 0.1 0.2

0,4

0.6

BACTERIAL NUMBER RATIO

0,8 NTA X1

F i g . 2. The r e l a t i o n s h i p b e t w e e n d i s p e r s e d b i o m a s s p o r t i o n ( l )o g - l i n e a r (Xl;disg/X1) and N T A - 1 b a c t e r i a p o r t i o n ( X ~ ; N T A / X ~ r e g r e s s i o n except o u t - s t a n d i n g p o i n t s s i g n e d + and d a t a from c u l t i v a t i o n with c i l i a t e s o f 1041nl-~).

The e s t i m a t e d r a t e s o f s u s p e n s i o n f e e d i n g o f c i l i a t e s ( t h e mean v a l u e o f c i l a t e p o p u l a t i o n up t o 20 n 1 . h - l ) ssary

f o r providing growth [ l ] ,

g e s t i o n was supposed.

were l o w e r t h a n necce-

i f only the dispersed bacteria i n -

U n d e r - e s t i m a t i n g m i g h t be caused by s e l e c t i v e

p r e d a t i o n on b a c t e r i a o f p a r t i c u l a r shape,

b u t e s p e c i a l l y by a l t e r -

n a t i v e p r e d a t i o n on b a c t e r i a l f l o c s [ 9 1 . CONCLUSIONS

1. The s e l e c t i v e e f f e c t o f t h e s u b s t r a t e on t h e b a c t e r i a l c o m m u n i t y s t r u c t u r e was v e r i f i e d u s i n g n i t r o l o t r i a c e t i c a c i d a s t h e s o l e o r g a n i c c a r b o n and n i t r o g e n source.

The s i g n i f i c a n t r e l a t i o n s h i p

-

119

-

b e t w e e n t h e N T A - 1 p o r t i o n a n d t h e d i s p e r s e d b a c t e r i a p o r t i o n on t h e t o t a l v a l u e s was o b s e r v e d i n t h e s y s t e m s w i t h o u t c i l i a t e s o r

w i t h a l o w c o n c e n t r a t i o n o f them. 2.

I n t h e b a c t e r i a l c u l t u r e g r o w i n g on s l o w l y - d e g r a d a b l e

substrate,

t h e p o s i t i v e r o l e o f c i l i a t e s on s t a b i l i z i n g t h e f l o c c u l a t e d , well-separable

b i o m a s s was p r o v e d .

Complete f l o c c u l a t i o n o f bac-

t e r i a was .observed a l o n g w i t h c i l i a t e c o n c e n t r a t i o n s a r o u n d

10~~1-l. 3.

The d e g r a d a t i o n o f N T A waa n o t d i s t u r b e d by c i l i a t e p r e d a t i o n . The s t a b i l i z i n g e f f e c t o f c i l i a t e s on b a c t e r i a l f l o c s m i g h t b e caused d i r e c t l y by p r e d a t i o n ( d i s p e r s i o n d i d n o t aerve as t h e sole food source f o r c i l i a t e s )

b u t a l s o by autochthonous s u b s t r a -

t e p r o d u c t i o n d u r i n g d i g e s t i o n w h i c h c o u l d be used by N T A nondegrading f l o c c u l a t i n g b a c t e r i a .

4. P r o d u c t i o n o f n i t r a t e s was o b s e r v e d i n t h e s y s t e m A a n d i t s lin e a r d e p e n d e n c e on b i o m e s s c o n c e n t r a t i o n r e s i d e n c e t i m e ) was v e r i f i e d .

( i n d i r e c t l y on biomass

In well-flocculated

system 8 con-

c e n t r a t i o n o f t o t a l i n o r g a n i c n i t r o g e n was r a t h e r l o w p r o b a b l y due t o p a r a l l e l d e n i t r i f i c a t i o n i n s i d e f l o c s .

REFERENCES

1

2 3

4 5 6 7

8 9 10

11 12 13

T. F e n c h e l , Prog. P r o t i a t o l . , 1 (1986) 65-113. C.R. C u r d s a n d G.J. F e y , W a t e r Res., 3 ( 1 9 6 9 ) 853-867. L.M. M a l l o r y , Ch. Yuk, L . L i a n g a n d M. A l e x a n d e r , A p p l . E n v i r o n . M i c r o b i o l . , 46 (1983) 1073-1079. 13.F. S h e r r , E.8. S h e r r a n d R.D. F a l l o n , Appl. Environ. M i c r o b i o l . , 53 ( 1 9 8 7 ) 9 5 8 - 9 6 5 . R. Sudo a n d S . A i b a , Adv. B i o c h e m . Engn., 29 ( 1 9 8 4 ) 1 1 7 - 1 4 1 . C.R. C u r d s , W a t e r Rea. W. GUde, C u r r . M i c r o b i o l . , 7 ( 1 9 8 2 ) 347-350. J.S. t e c h , P. H a r t m a n a n d J. Chudoba, N e w s l e t t e r Spec. Group A c t i v a t e d S l u d g e P o p u l . D y n a m i c s I A W P R C , 1 ( 1 9 8 9 ) 29 3 0 . M. L e g n e r , P r o c . 7 t h Symp. C o n t i n u o u s C u l t i v a t i o n o f M i c r o o r g a n i s m s P r a g u e ( 1 9 8 0 ) 371-381. J . H e j z l a r , J. Chudoba a n d P. Chudoba, P r o c . H y d r o c h e m i a B r a t i a l a v a ( C z e c h o a l o v a k i s ) (1981) 129-144. K.G. B d r a h e i m a n d G. B r a t b a k , M a r . E c o l . P r o g . S e r . 3 6 ( 1 9 8 7 ) 171-175. M. Simon a n d F. Azam, Mar. E c o l . P r o g . S e r . 5 1 ( 1 9 8 9 ) 201-213. J . Wanner, K. Kucman a n d P. G r a u , W a t e r Res. 22 ( 1 9 8 8 ) 207-211.

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M I C R O B I O L O G I C A L T R E A T M E N T OF M U N I C I P A L S E W A G E SLUDGE AND REFUSE A S MEANS OF D I S I N F E C T I O N P R I O R T O R E C Y C L I N G I N A G R I C U L T U R E

D.

STRAUCH

I n s t i t u t e o f Animal M e d i c i n e and A n i m a l Hygiene, U n i v e r s i t y o f Hohenheim, P.O.B. 70 0 5 6 2 , D-7000 S t u t t g a r t 70, FRG

INTRODUCTION I t i s a well established

as a r u l e ,

f a c t t h a t m u n i c i p a l sewage s l u d g e s ,

do c o n t a i n a b r o a d v a r i e t y o f b a c t e r i a l ,

t i c a l and o t h e r pathogens,

viral,

parasi-

which a r e e x c r e t e d by i n f e c t e d i n d i v i but i n a

d u a l s (Tables 1-4).

The same a p p l i e s t o m u n i c i p a l r e f u s e ,

m o d i f i e d dimension.

These p a t h o g e n s may c o n s t i t u t e a h e a l t h h a z a r d

f o r man a n d a n i m a l when t h e s l u d g e and r e f u s e a r e u s e d a s o r g a n i c f e r t i l i z e r s o r f o r amending s o i l s .

TABLE 1 B a c t e r i a l p a t h o g e n s t o b e e x p e c t e d i n sewage a n d sewage s l u d g e ( C A R R I N G T O N , 1 9 7 8 ; WHO, 1981; LEHMANN a n d WALLIS, 1 9 8 2 ) ~~~

Primary pathoqenic

Secondary p a t h o q e n i c

S a l m o n e l l a spp.

Escherichia

S h i g e l l a spp.

Klebsiella

E s c h e r ic h ia c o 1i

Enterobacter

P s e u d om on a s ae r u g in o s a

Serratia

Yersinia enterocolitica

C itrobacter

Clostridium perfringens

Proteus

Clostridium botulinum

Providencia

Bacillus anthracis L i s t e r i a monocytogenes V i b r i o cholerae M y c o b a c t e r i u m spp. L e p t o s p i r a spp. Campylobacter spp. Staphylococcus '

5 t rep t oc oc c u a

-

122

-

T h e r e f o r e t h e C o u n c i l o f t h e European Communities has i s s u e d a D i r e c t i v e o n t h e u s e o f sewage s l u d g e i n a g r i c u l t u r e on 1 2 J u n e 1986.

W i t h i n 3 y e a r s t h e Member S t a t e s h a v e t o b r i n g i n t o f o r c e t h e

laws,

r e g u l a t i o n s and a d m i n i s t r a t i v e p r o v i s i o n s n e c e s s a r y t o comply

w i t h t h i s Directive (1). TABLE 2 Human e x c r e t e d v i r u s e s w h i c h c a n b e e x p e c t e d i n sewage a n d sewage s l u d q e (FEACHEM e t a l . , 1 9 8 1 ) lirusgroup

D i s e a s e s o r symptoms c a u s e d

Number o f t y p e s

Interovirus

3

Poliovirus Coxsackievirus A

24

Coxsackievirus B

6

Poliomyelitis, Herpangina, meningitis,

New e n t e r o v i r u s e s

r e s p i r a t o r y disease, fever

M e n i g i t i s , r e s p i r a t o r y disease, rash, diarrhoea, fever Meningitis, encephalitis, reapir a t o r y disease, a c u t e haemorrhag i c c o n j u c t i v i t i s , fever

4

30

idenovirus

fever

Myocarditis, congenital heart anomalies, m e n i n g i t i s , r e a p i r a t o r y disease, pleurodynia, rash, fever

34

Echovirus

meningitis,

Respiratory disease, tions

eye i n f e c -

Not c l e a r l y e s t a b l i s h e d

leovirus

3

l e p a t i t i s A-virus

1

Infectious hepatitis

Iotavirus

?

V o m i t i n g and d i a r r h o e a

rstrovirus

?

?

:alicivirus

?

V o m i t i n g and d i a r r h o e a

:oronavirus

?

Common c o l d

l o r w a l k agent and o t ier s m a l l r o u n d v i r u s e s

?

V o m i t i n g and d i a r r h o e a

tdenoaasociated

4

Not c l e a r l y established b u t associated with respiratory d i sease i n c h i l d r e n

virus

I n t h e F e d e r a l R e p u b l i c o f Germany (FRG)

t h e Government h a s i s -

s u e d an " O r d i n a n c e on Sewage S l u d g e " w h i c h came i n t o f o r c e 1 A p r i l 1983.

T h i s o r d i n a n c e p r o v i d e s t h a t t h e u s e o f sewage s l u d g e o n p a s -

t u r e and f o r a g e l a n d i s n o t l o n g e r a l l o w e d i f i t i s n o t " h y g i e n i c a l l y safe"

(sanitized,

disinfected)

not define the term "hygienically

(2).

safe",

Since the l e g i s l a t o r d i d a w o r k i n g g r o u p was e s t a b -

l i s h e d t o e l a b o r a t e s u c h a d e f i n i t i o n and a l s o t o d e f i n e t h e appro-

-

123

-

p r i a t e technologies t o achieve " h y g i e n i c a l l y safe" finitions

sludge.

These de-

a r e a l s o a d o p t e d f o r p r o d u c t s made f r o m m u n i c i p a l r e f u s e .

The t e c h n o l o g y f o r s a n i t a t i o n m u s t r e s u l t i n s p r o d u c t i n w h i c h d i r e c t l y a f t e r i t s t r e a t m e n t i n one gram

-

no s a l m o n e l l a s and n o t more t h a n 1 0 0 0 e n t e r o b a c t e r i e c e a e c a n b e d e t e c t e d f u r t h e r m o r e t h e t e c h n o l o g y must e n s u r e t h a t a s c s r i s ova have l o s t their infectivity.

TABLE 3 P a r a s i t e s t o b e e x p e c t e d i n sewage a n d sewage s l u d g e (HANNAN, WHO, 1981) Protozoa

Entamoeba h i s t o l y t i c a Giardia lamblis Toxoplasma g o n d i i S a r c o c y s t i s spp.

Ce s t o d e s

Taenia saginata Taenia solium Diphyllobothrium latum Enchinococcus granulosus

Nematodes

Ascaris lumbricoides Ancylostoma duodenale Toxocara c a n i s Toxocara c a t i Trichuris trichiura

1981;

TABLE 4 P a t h o g e n i c y e a s t s a n d f u n g i t o be e x p e c t e d i n sewage a n d sewage s l u d g e (WHO, 1981)

Yeasts

Candida a l b i c a n s Candida k r u s e i Candida t r o p i c a l i s Candida g u i l l e r m o n d i i Cryptococcus neoformans Trichosporon

Funqi

A s p e r g i l l u s spp. Aspergillus fumigatus Phialophora r i c h a r d s i i G e o t r i c h u m candidum T r i c h o p h y t o n spp. E p i d e r m o p h y t o n spp.

-

124

-

S a n i t a t i o n m e t h o d s b a s e d on m i c r o b i o l o g i c a l p r o c e s s e s a n d e f f e c t s o f m i c r o b e s a r e 1. a e r o b i c - t h e r m o p h i l i c q u i d sludge

a n d 2.

s t a b i l i z a t i o n o f li-

composting o f dewatered sludge or m i n u c i p a l re-

fuse i n windrows or r e a c t o r s (in-vessel-composting).

I n these techno-

l o g i e s e x o t h e r m i c a n d a n t a g o n i s t i c m i c r o b i a l e f f e c t s c a n a c h i e v e an i n a c t i v s t i o n o f p a t h o g e n s w h i c h may e x i s t i n t h e r a w m a t e r i a l s . INVESTIGATIONS UNDER PRACTICAL C O N D I T I O N S I N SEWAGE T R E A T M E N T PLANTS

(3

-

9)

Aerobic-thermophilic

s t a b i l i z a t i o n o f l i q u i d sludqe (ATS)

T h i s t e c h n o l o g y uses t h e a e r e t i o n o f l i q u i d s l u d g e t o i n i t i a t e the growth o f aerobic thermophilic

microorganisms which are produ-

c i n g exothermic heat j u s t l i k e i t occurs d u r i n g composting o f r e f u se, manure w i t h s t r a w e t c .

t h a t a one-stage

system,

I n v e s t i g a t i o n s i n AT5 p l a n t s h a v e shown

u s i n g o n l y one r e a c t o r c a n n o t p r e v e n t s h o r t

c i r c u i t s w h i c h may r e s u l t i n t h e s u r v i v a l o f p a t h o g e n s i n t h e e f f l u ent (Fig.

1). T h e r e f o r e i t i s n e c e s s a r y t o o p e r a t e an AT5 p l a n t

w i t h a t l e a s t two r e a c t o r s .

I n v e s t i g a t i o n s i n such a p l a n t under

TEMPERATUR “T 1

. TT ee mm ppeerraattuurrev ecrul ar vuef

-.

pH c o u r s e :pH-Verlauf T o t a l germ c o u n t 0 0 : Gesamtkeime Coliforms *-d:Coliforme Keime

-----

0

a ANZAHL KEIME / rnl NUMBER OF GERMSlrnl

1

Salmonellas i s o l a t e d :Salmonellen nachweisbar Salmonellas not i s o l a t e d :Salmonellen n i c h t nachweisbar Coliforms not isolated : C o l i f o r m e n i c h t nachweisbar

F i g . 1. B a c t e r i o l o g i c a l r e s u l t s o f a e r o b i c - t h e r m o p h i l i c s t a b i l i z a t i o n ( A T S ) o f l i q u i d sewage s l u d g e i n a o n e - s t a g e r e a c t o r . p r a c t i c a l c o n d i t i o n s h a v e shown t h a t b y t h i s c o n s t r u c t i o n t h e m i c r o b i o l o g i c a l disadvantages o f h y d r a u l i c s h o r t - c i s c u i t s

c a n be a v o i -

ded i n t h a t t h e aecond r e a c t o r i s a c t i n g a s s e c u r i t y r e s e r v e i n

-

125

-

TABLE 5 R e s u l t s o f m i c r o b i o l o g i c a l s t u d i e s w i t h sewage s l u d g e o f two d i f f e r e n t two-stage aerobic-thermophilic s t a b i l i z a t i o n p l a n t s (Strauch 1988). .ITUS ATS -

ddedova S E suum inactivat.

.

100 100 100

Rs ATS

--

Raw sludge Effluent of aerobic-thermphilic Stabilization

TABLE 6 R e s u l t s o f a Windrow Composting E x p e r i m e n t (Summer). Material: Germ Carrier: Test Germs: ComFOsting Time:

Samples Taken After Weeks 1

2

3

9 m3 S l u d g e + 1.350 kg Straw Silk Gauze Pieces + Ampules Salmonella typhimurium, senftenberg 9 Weeks - Turning 1 x Weekly 12 May - 13 J u l y

Placement

Peak Margin Nucleus

S . typhimurium Silk Piece Ampule

+ +

+

+

Peak

1

Margin Nuc 1e u 5

+

+ + +

Peak Margin

Nucleus

+

4

Peak Margin Nucleus

+

5

Peak Marg." Nucleus

6

s. senftenberg Silk Piece1 Ampule

Peak Margin Nucleus

7

Peak Margin Nucleus

8

Peak Margin Nucleus

-

126

-

w h i c h t h e complete d e s t r u c t i o n o f pathogens i s a c h i e v e d once t h e y h a v e s l i p p e d o u t o f r e a c t o r No. 1 d u e t o a s h o r t - c i r c u i t

(Fig.

2).

M i c r o b i o l o g i c a l c o n t r o l i n v e s t i g a t i o n s i n t w o o t h e r A T S p l a n t s have proved t h a t not only enterobacteriaceae, duced and,salmonellas

f e c a l s t r e p t o c o c c i are re-

are r e l i a b l y i n a c t i v a t e d by t h i s technology

b u t a l s o e n t e r o v i r u s e s a n d A s c a r i s suum o v a ( T a b l e 5 ) .

REAKTOR

I

REAKTOR

1 Temperature curve .Temperaturverlauf pH c o u r s e -_-__. :p H - V e r l a u f T o t a l germ c o u n t 0 0 : Gesamtkeime Coliforms 8 - -e: C o l i f o r m e Keime -*

50 c 40

2

- o..,

30 -

A ........ .......

. ..... .

\.

20 -

\

I lot a

1

1 2

2

3

,

4

lo2

3 4 5 6 7 8 ENVlRKUhGSZElT [TAGE 1

0 1

Salmonellas i s o l a t e d :S a l m o n e l l e n n a c h w e i s b a r Salmonellas not i s o l a t e d :S a l m o n e l l e n n i c h t n a c h weisbar Coliforms not isolated : C o l i f o r m e n i c h t nachweisbar

F i g . 2. B a c t e r i o l o g i c a l r e s u l t s o f a e r o b i c - t h e r m o p h i l i c s t a b i l i z a t i o n (ATS) o f l i q u i d sewage s l u d g e i n a t w o - s t a g e r e a c t o r .

Compostinq o f dewetered s l u d q e w i t h s t r a w i n windrows The i n v e s t i g a t i o n s w e r e made i n t h e s l u d g e c o m p o s t i n g p l a n t o f a small municipality. bales o f straw.

L i q u i d s l u d g e i s s p r a y e d w i t h a t a n k wagon on

W i t h a s p e c i a l t u r n i n g d e v i c e d r a w n by a t r a c t o r

s t r a w and s l u d g e a r e m i x e d a n d t h e c h a r a c t e r i s t i c t r a p e z o i d a l shape o f a windrow i s formed.

The w i n d r o w s t h e n were t u r n e d once a weak,

one w i n d r o w was n e v e r t u r n e d a s a c o n t r o l . The r e s u l t s show t h a t d u r i n g w i n d r o w c o m p o s t i n g o f s l u d g e w i t h s t r a w i n t h e warm s e a s o n s a l m o n e l l a s were d e s t r o y e d w i t h i n s e v e n weeks ( T a b l e 61, w h e r e a s t h e y s u r v i v e d u n t i l t h e e n d o f t h e e x p e r i ment a f t e r n i n e weeks i n t h e w i n d r o w w h i c h was n e v e r t u r n e d ( T a b l e

7).

D u r i n g t h e l o w e r t e m p e r a t u r e s i n w i n t e r t h e k i l l i n g e f f e c t on

-

127

-

salmonellas

and E C B O - v i r u s

the surface

o f t h e windrows w i t h a 1 0 - 1 5

was u n r e l i a b l e ( T a b l e 8 ) .

By covering

cm t h i c k l a y e r o f c u r e d

compost t h e d i s a d v a n t a g e s o f t h e l o w e r t e m p e r a t u r e s d u r i n g t h e w i n t e r p e r i o d can b e compensated.

TABLE 7 R e s u l t s o f a Windrow C o m p o s t i n g E x p e r i m e n t ( S u m m e r ) . 6 m3 S l u d g e + 9W kg S t r a w S i l k Gauze P i e c e r + Ampules Salmonella typhhurium, senftenberg 9 Weeks - N o T u r n i n g 1 2 May - 1 3 J u l y

Material: Germ C a r r i e r : T e s t germs: Cornposting T h e :

jamplea Taken 4 f t e r Weeks

S. typh S i l k Piece

+++

1

+++ +++

2 3

+++ +++

4

5 6

+++ +++ +++ +++

7 8 9

+++

-

+++

S. a e n f r Silk Piece

+++

+++ +++

Ampule

+++

+++ +++ +++

+++ +++ +++ +++ +++ +++ +++ +++

+++ +++

3eTq

+++

+++

+++ +++ +++ +++ +++

irium Ampule

+++ +++

+++ +++

+++ +++

+++

+++ +++

+++

+++ +++ +++ +++ +++ +++

+++

+++

+++ +++ +++ +++ +++ +++

+++

+++

I

S a l m o n e l l a s C u l t u r e d frcm Germ C a r r i e r s i n P e a k , Margin and NUc l e u s of Windrow

TABLE 8 R e s u l t s o f a Windrow C o m p o s t i n g E x p e r i m e n t ( W i n t e r ) . 0 m3 Sludge + 1200 kg S t c a w hterlal: C m p s t i n g T i n e : 6 Weeks-Turning 1 x Weekly 31 October - 1 2 December

Samples Taken A f t e r Weeks

Placement

s.

typhiaurium

5 . senftenbers

ECBO V I r u 6

Nucleus

/

Peak Maigin

Peak Margin N"C1.US

40 20 44

50 40 55

33 20 30

60 16 63

51 18

50

Peak

/

60

nargm

/

NuCleub

/

20 56

Peak Margin Nucleus

++-++ +++*+

. . . .

T e s t Germs On S i l k PleCeE virus Culture ~n O ~ d l y s l sTubes

++-++ +++++ -++-+

40

11 61

57 9 65

53

59

60 14 59

15

12

56

50

65

52 16 56

6 0 51 I 4 I5 59 5 0

59 12 65

52 16

Peak Marg," N"C1.US

I

30 30

15

+

NYCl."S

D =

-

50 7.0 44

Peak Margin

S

Maximum Temperatur Durlng 1 6 Weeks in "C 1 . 2. 3. 4 . 5. 6.

20 5 20 20 5 20

10 4 13

-

128

-

I n s t e a d o f u s i n g s t r a w a l s o o t h e r b u l k i n g a g e n t s may b e u s e d t o i m p r o v e t h e p o r e volume o f t h e s l u d g e and t h u s t h e o x y g e n s u p p l y a n d t h e d e g r a d a t i o n t e m p e r a t u r e w h i c h i s t h e most i m p o r t a n t f a c t o r

for

t h e pathogen k i l l (Table 9 ) .

TABLE 9 B u l k i n g A g e n t s R e p o r t e d As S a t i s f a c t o r y I n C o m p o s t i n g T e s t s P e r f o r m e d A t B e l t s v i l l e , MD, And O t h e r L o c a t i o n s ( E p s t e i n e t a l . , 1981) Wood c h i p s

Cotton g i n trash

F l y ash-wood

chips

Cereal straws

Saw d u s t

Leaves

Tree t r i m m i n g s

Dry,

Bark

U n c r e e n e d compost

Shredded b a r k

Refuse

Lorn stover

A i r c l a s s i f i e d refuse

c o m p o s t e d sewage s l u d g e

Sugarcane bagasse

Refuse d e r i v e d f u e l cubes,

Rice h u l l s

Automobile Tires-woodchips

pellets

Peanut h u l l s

Compostinq o f dewatered sludqe i n b i o r e a c t o r s ( I n - v e s s e l - c o m p o s t i n q ) B i o r e a c t o r s are p l u g - f l o w v e s s e l s f o r composting dewatered sludge.

U s u a l l y saw d u s t ,

ground bark,

r e f l u x c o m p o s t o r o t h e r sub-

s t a n c e s a r e added a s b u l k i n g a g e n t s and t o i n c r e a s e t h e l o w c a r b o n c o n t e n t o f t h e s l u d g e a n d t h u s i m p r o v e t h e C/N

r a t i o which r e s u l t s

i n a more i n t e n s i v e c o m p o s t i n g p r o c e s s a n d i n h i g h e r t e m p e r a t u r e s . The m i x t u r e i s t r a n s p o r t e d w i t h a c o n v e g o r i n t o t h e b i o r e a c t o r ,

rested and e m p t i e d f r o m b e l o w .

a-

The c o m p o s t i n g t i m e i s a b o u t t w o

weeks and t h e s y s t e m i s a c o n t i n u o u s one. E x p e r i m e n t s were made i n a b i o r e a c t o r w i t h s a l m o n e l l a s a n d ECBO-virus.

As shown i n T a b l e 1 0 b o t h t y p e s o f p a t h o g e n s w e r e d e -

s t r o y e d d u r i n g t h e p a s s a g e o f t h e compost

through the reactor.

Another t y p e o f r e a c t o r i s t h e B i o - C e l l r e a c t o r n i c a l l y c l a s s i f i e d as f l o w r e a c t o r w i t h s t o r i e s . watered s l u d g e w i t h a b u l k i n g agent and/or ported t o the top story.

which i s tech-

The m i x t u r e o f de-

carbon c a r r i e r i s trans-

The b o t t o m s o f t h e s t o r i e s a r e a l u m i n u m

f l a p s w h i c h c a n b e t u r n e d down s o t h a t t h e c o m p o s t f a l l s down i n t o the next story.

I n e a c h s t o r y t h e compost l a y e r o f a b o u t 4 0 cm h e -

i g h t i s aerated.

129

-

T h i s c o n t i n u o u s c o m p o s t i n g p r o c e s s t a k e s u s u a l l y 30

days ( 3 days p e r s t o r y ) . TABLE 1 0 R e s u l t s o f Composting E x p e r i m e n t s w i t h S a l m o n e l l a s and E n t e r o v i r u s i n a B i o - R e a c t o r ( S y s t e m KNEER; Fa. W E I S S ) .

Material: Germ C x r i e r s : T e s t Organisms:

Sludge-Compost-Saw D u s t Salmonellas S i l k Gauze P i e c e s + Ampules Virus D i a l y s i s T u b e + Ampules Salmonella typhimirium, e n t e ritidis, senftenberg V i r u s S t r a i n ECBO-LCR-4

-

-~

S. t y p h i m u r i u m S. e n t e r i t i d i s S. s e r i f t e n b e r g Virus Strain ACBO-LCR-4

S

A

/

/

(15)

Exp. 5 1 2 Samples (12)

-

/

Exp. 4 1 5 Samples

Exp. 3 10 Samples (10) S A

EXp. 2 10 Samples (10)

Exp. 1 30 Samples (5) S R

Test Organisms

/

/

1

/

Maximum Temperature

7 0 OC

74

"c

P a s s a g e Time i n Days

14

11

14

Sludge

40 a

40 %

50 %

50 a

50 %

Compost

10 %

10 %

10 a

10 %

25 %

Saw D u s t

50 a

50 %

40 %

40 %

40 %

7

I

-

6

I

= T e s t Organisms n o t Detectable

/ = N o t Done o f Detected Samples ( N ~ & ~= ~Number ) S = T e s t O r g a n i s m s on S i l k G a u z e A = C u l t u r e s i n Ampules D = V i r u s c u l t u r e i n D i a l y s i s Tube

1 5 experiments under p r a c t i c a1 c o n d i t i o n s i n a composting p l a n t showed t h a t s a l m o n e l l a s , stroyed,

ECBO-virus

a n d a s c a r i s o v a w e r e de-

p r o v i d e d t h a t t h e compost m a t e r i a l i s homogeneously mixed

and w e l l and e v e n l y a e r a t e d i n each s t o r y o f t h e r e a c t o r .

One t y p i -

c a l r e s u l t i s shown i n T a b l e 11 w i t h s a l m o n e l l a s a n d B a c .

anthracis

spores.

The l a t t e r w e r e n o t c o m p l e t e l y d e s t r o y e d ( 4 o u t o f 1 5 sam-

p l e s remained p o s i t i v e ) .

Since spore-forming

b a c t e r i a g e n e r a l l y do

n o t p l a y an i m p o r t a n t r o l e f o r h y g i e n e o f c o m p o s t i n g , t h e i r

survival

i s not o f importance. B u t i n any c a s e o f t e c h n i c a l d i s t u r b a n c e s o f t h e b i o r e a c t o r c o m p o s t i n g o p e r a t i o n which r e s u l t s i n a d e c r e a s e o f t e m p e r a t u r e b e -

low 5 0

OC,

t h e b a t c h has t o be c o n s i d e r e d as n o t d i s i n f e c t e d and

-

-

130

f o r a second t i m e o r s h o u l d

m u s t p a s s t h e r e a c t o r as r e f l u x compost

b e c o m p o s t e d i n w i n d r o w s f o r a t l e a s t t h r e e weeks w i t h one t u r n i n g . TABLE 11 R e s u l t s o f Composting E x p e r i m e n t s w i t h S a l m o n e l l a s and A n t h r a x Spores i n a Bio-Cell-Reactor ( S y s t e m DAMBACH-SCHNORR; 1 0 S t o r i e s ) . Mate r ia1 :

Refuse-Sledge (4x) Bark-Composit-Sludge (1x1 Germ Carriers: Silk Gauze Pieces + Ampules Test Germs: Salmonella typhimurium, senftenberg, enteritidis, dublin Spores of Bacillus anthracis 28 - 31 Days Passage Time:

Exp. 2 3 0 Samples S A

Exp. 1 50 Samples S A

Test Germs

S. typhimurium S . senftenberg S . enteritidis S . dublin S . anthracis

Maximum Temperature - = = / =

+

-(7)

-(a)

-(7)

-(7) -(15)

/ /

/

-

80

"c

3 0 Samples

S

S

A

Exp. 4 A

Exp. 5 30 Samples S A

/ /

/ /

-

-

-(7) -(8)

-(15) /

Exp. 3 3 0 Samples

-

/ /

-

79

+(4)

C(4)

-(15)

-(15)

"c

Test Germs not Detectable Growth of Test Germs Not Done

/ /

/ /

/ /

-

70 "C

7 0 "C

/ /

-

75

"c

S = Test Germs on Silk Gauze A = Culture in Ampules (Number) = Number of Detected Samples

As an a i d f o r t h e e v a l u a t i o n o f t h e e f f i c a c y o f c o m p o s t i n g temp e r a t u r e s on t h e i n a c t i v a t i o n o f v a r i o u s p a t h o g e n i c microorganisms t h e g r a p h i n F i g u r e 3 c a n be u s e d . DEFINITIONS OF THE MICROBIOLOGICAL T R E A T M E N T METHODS As a l r e a d y m e n t i o n e d i n t h e i n t r o d u c t i o n l a German w o r k i n g p a r t y h a s b a s e d on i n t e r n a t i o n a l e x p e r i e n c e s e l a b o r a t e d d e f i n i t i o n s f o r s l u d g e d i s i n f e c t i o n t e c h n o l o g i e s under t h e o n l y p r o c e s s a i m o f a secured s s n i t a t i o n .

The d e f i n i t i o n s f o r t h e d i s i n f e c t i o n o f s l u d g e a n d

r e f u s e w i t h m i c r o b i o l o g i c a l methods a r e g i v e n i n t h e f o l l o w i n g . Aerobic-thermophilic

s t a b i l i z a t i o n o f sludqe (ATS)

Process d e s c r i p t i o n I n t h e c o u r s e o f t h e ATS-process

caused by a i r (oxygen) s u p p l y

e x o t h e r m a l m i c r o b i a l d e g r a d a t i o n and m e t a b o l i c p r o c e s s e s r e s u l t i n a r i s e o f t e m p e r a t u r e a n d o f t h e pH up t o v a l u e s a b o u t 8. t h a t t h e r e a c t i o n v e s s e l h a s a good i n s u l a t i o n ,

Provided

the a i r supply i s

c o r r e c t l y c a l c u l a t e d and t h e s l u d g e has a s u f f i c i e n t c o n c e n t r a t i o n o f o r g a n i c d r y m a t t e r i n t h e ATS-process

t e m p e r a t u r e s c a n be r e a c h e d

-

131

-

which besides t h e s t a b i l i z a t i o n a l s o ensure a s a n i t a t i o n o f the sludge. ATS-processes

s h o u l d be o p e r a t e d a t l e a s t as t w o - s t a g e r e a c t o r s

( 2 vessels i n series connection)

( t o avoid the microbiological d i -

sadvantages o f h y d r a u l i c s h o r t - c i r c u i t s .

As d e t e n t i o n t i m e s i n t h e

c o m p l e t e s y s t e m a t l e a s t 5 d a y s a r e t o be c a l c u l a t e d when t h e r e a c t o r s have t h e same volume.

-

Y r n I

8

2 8

65

$ 6 0

+

55 50

45

LO 35

30 25 2c I

F i g . 3. E f f e c t o f t e m p e r a t u r e and t i m e on some e n t e r i c p a t h o g e n s (Feachem e t e l . 1978).

Under c o n s i d e r a t i o n o f t h e b a t c h - t y p e

operation

(e.9.

f e e d i n g p e r day) and 23 h o u r s s t a b i l i z a t i o n ( r e a c t i o n t i m e )

one h o u r and o f

t h e temporary decrease o f temperature i n e v i t a b l y connected w i t h t h i s type o f operation, r e s are necessary:

t h e f o l l o w i n g r e a c t i o n t i m e s and t e m p e r a t u -

-

23 h o u r s a t 5 0

OC

-

10 h o u r s a t 55

OC

-

4 h o u r s a t 60

132

-

or or

OC.

Process c o n t r o l

For supervision o f the process c o n d i t i o n s t h e f o l l o w i n g param e t e r s a r e t o be c o n t r o l l a b l e r e g i s t e r e d b y c o n t i n u o u s p l o t t l n g :

-

t h e t e m p e r a t u r e s and t h e i r r e a c t i o n t i m e s i n t h e r e a c t o r s i n a t l e a s t two p o s i t i o n s w i t h r e c o r d i n g i n s t r u m e n t s

-

t h e pH-values

o f t h e raw s l u d g e and o f t h e e f f l u e n t o f t h e

ATS-process

-

t h e d a i l y s l u d g e volume f l o w ,

b y w h i c h a t known v o l u m e s o f t h e

r e a c t o r s t h e r e a c t i o n t i m e can be c a l c u l a t e d .

Aerobic-thermophilic

s t a b i l i z a t i o n o f sludqe (ATS)

w i t h subsequent

anaerobic diqestion T h i s c o m b i n s t i o n o f an a e r o b i c w i t h an a n a e r o b i c m i c r o b i o l o g i c a l t r e a t m e n t i s d e f i n e d 8s f o l l o w s .

Process d e s c r i p t i o n

I n the aerobic-thermophilic

f i r s t stage t h e sludge i s sanitized.

The s a n i t a t i o n i s e n s u r e d b y s u f f i c i e n t l y h i g h t e m p e r a t u r e s i n t h e s l u d g e which can be produced by a d d i t i o n a l h e a t i n g w i t h e x t r a n e o u s e n e r g y and e x o t h e r m i c m i c r o b i a l p r o c e s s e s d u r i n g t h e p a r t i a l s t a b i lization.

By t h e s u b s e q u e n t a n a e r o b i c m e s o p h i l i c or t h e r m o p h i l i c

s t a b i l i z a t i o n t h e necessary s e c u r i t y o f the s a n i t a t i o n process i s ensured. By t r e a t m e n t i n t h i s t w o - s t a g e

process t h e sludge i s considered

as s a n i t i z e d ifi n t h e f i r s t s t a g e e i t h e r t h e c o n d i t i o n s o f p r e - p a s t e u r i z a t i o n ( s e e No. t u r e of

a t l e a s t 60

11/11 a r e f u l f i l l e d or i f t h e r e a c t i o n t e m p e r a OC

is c o n t i n u o u s l y k e p t f o r a t l e a s t f o u r h o u r s .

D u r i n g t h e s e f o u r h o u r s n o r a w s l u d g e may b e added. (anaerobic)

I n t h e second

s t a g e a p r o c e s s t e m p e r a t u r e o f a t l e a s t 30

OC

must be

kept.

Process c o n t r o l

F o r s u p e r v i s i o n o f t h e p r o c e s s c o n d i t i o n s t h e f o l l o w i n g psramet e r s a r e t o be c o n t r o l l a b l e r e g i s t e r e d by c o n t i n u o u s p l o t t i n g :

-

133

-

I n the f i r s t stage:

-

the temperature i n two p o s i t i o n s the reaction time

i n t h e second stage:

-

t h e temperature.

Compostinq o f s l u d q e i n windrows

Process d e s c r i p t i o n The s a n i t a t i o n o f s l u d g e by c o m p o s t i n g i n w i n d r o w s w i t h b u l k i n g m a t e r i a l (e.g. vings)

municipal refuse,

straw,

saw d u s t ,

wood s h a -

i s caused by t h e h e a t generated d u r i n g composting by m i c r o -

b i a l processes.

B e s i d e s t h i s t e m p e r a t u r e and t h e r e a c t i o n t i m e a l s o

m i c r o b i a l metabolic substsnces w i t h a n t i b i o t i c e f f e c t s a r e o f i m portance. Requirement for t h e s a n i t i z i n g e f f e c t i s a s u f f i c i e n t a e r a t i o n o f t h e m i x t u r e o f s l u d g e a n d b u l k i n g m a t e r i a l b y t e c h n i c a l means a s

for i n s t a n c e t u r n i n g o f t h e windrows or f o r c e d a e r a t i o n o f s t s t i c windrows o r p i l e s ( w h i c h s r e n o t t u r n e d ) . I t must be e n s u r e d t h a t t h e e f f e c t i v e t e m p e r a t u r e s r e a c h e a c h p a r t o f t h e c o m p o s t i n g mater i a l for t h e necessary r e a c t i o n time. The i n i t i a l w a t e r c o n t e n t o f t h e c o m p o s t n g m a t e r i a l m u s t b e

4 0 - 6 0 I and t h e r e a c t i o n t e m p e r a t u r e

n t h e w n d r o w a t l e a s t 55

OC

d u r i n g t h r e e weeks.

Process c o n t r o l F o r s u p e r v i s i o n o f t h e p r o c e s s c o n d i t i o n s f o r each windrow t h e

f o l l o w i n g p a r a m e t e r s a r e t o be c o n t r o l l a b l e r e g i s t e r e d :

-

t h e i n i t i a l water c o n t e n t o f t h e m i x t u r e t h e temperature a t l e a s t d a i l y i n t h r e e p o s i t i o n s snd i n d i f f e r e n t d i s t a n c e s from t h e s u r f a c e o f t h e windrow;

one o f t h e p o -

s i t i o n s i n t h e c e n t r a l p a r t e n d one i n t h e o u t e r z o n e

-

t h e c o m p o s t i n g t i m e and t u r n i n g (number,

date).

Compostinq o f sludqe i n r e a c t o r s (In-vessel-compostinq)

Process d e s c r i p t i o n The s a n i t a t i o n o f s l u d g e b y c o m p o s t i n g i n r e a c t o r s ( i n - v e s s e l composting) with b u l k i n g m a t e r i a l (e.9.)

saw d u s t ,

wood s h a v i n g s ,

bark,

reflux material)

134

-

i s c a u s e d b y t h e h e a t g e n e r a t e d d u r i n g com-

p o s t i n g by m i c r o b i a l p r o c e s s e s .

B e s i d e s t e m p e r a t u r e and r e a c t i o n t i -

me a l s o m i c r o b i a l m e t a b o l i c s u b s t a n c e s w i t h a n t i b i o t i c e f f e c t s a r e o f importance. Requirement f o r t h e s a n i t i z i n g e f f e c t

i s a sufficient aeration

o f t h e m i x t u r e o f s l u d g e and b u l k i n g m a t e r i a l b y t e c h n i c a l means. The s t e a d i n e s s o f d e s i r e d t e m p e r a t u r e p r o f i l e s i n t h e r e a c t o r s c a n b e i n f l u e n c e d and c o n t r o l l e d b y t h e t e c h n i q u e s o f a e r a t i o n , and e m p t y i n g .

filling

I t must b e e n s u r e d t h a t t h e e f f e c t i v e t e m p e r a t u r e s

r e a c h each p a r t o f t h e composting m a t e r i a l f o r t h e necessary react i o n time. The i n i t i a l w a t e r n o t exceed 70

X.

content o f t h e composting m a t e r i a l should

The c o m p l e t e m i x t u r e s h o u l d b e e x p o s e d t o a tempe-

rature o f a t least 55 o f a t l e a s t 10 days.

OC

d u r i n g a passage t i m e t h r o u g h t h e r e a c t o r

Besides t h a t t h e composting m a t e r i a l should

n o t pass t h e " h o t zone"

w i t h a t e m p e r a t u r e o f a t l e a s t 65

OC

faster

than i n 48 hours. The r e a c t o r p a s s a g e s h a l l be f o l l o w e d by a p h a s e o f c u r i n g t h e m a t e r i a l i n w i n d r o w s o r p i l e s o f a t l e a s t t w o weeks w i t h a t l e a s t one t u r n i n g o r i n a s e c o n d r e a c t o r by w h i c h m e a s u r e d t h e n e c e s s a r y s e c u r i t y o f t h e s a n i t a t i o n process i s ensured. The a c h i e v e m e n t o f

s a n i t a t i o n depends t o a l a r g e e x t e n t on an

undisturbed operation o f the composting process.

When d i s t u r b a n c e s

o f o p e r a t i o n s are connected w i t h a decrease o f temperatures t h e b a t c h o f compost m u s t e i t h e r b e u s e d as r e f l u x m a t e r i a l a n d t h u s p a s s t h e r e a c t o r f o r a s e c o n d t i m e o r i t must a g a i n b e c o m p o s t e d i n a w i n d r o w u n d e r t h e c o n d i t i o n s d e s c r i b e d i n No.

II/6

f o r composting o f sludge

i n windrow.

Process c o n t r o l

F o r s u p e r v i s i o n o f t h e p r o c e s s c o n d i t i o n s t h e f o l l o w i n g paramet e r s a r e t o be c o n t r o l l a b l e r e g i s t e r e d :

-

t h e i n i t i a l water c o n t e n t o f t h e m i x t u r e

-

the temperature i n a t l e a s t t h r e e p o s i t i o n s .

One p o s i t i o n h a s

t o b e l o c a t e d b e f o r e and one i n t h e " h o t z o n e "

and a t l e a s t

one i n t h e p e r i p h e r a l z o n e o f t h e r e a c t o r

-

t h e s t o r a g e t i m e and t u r n i n g o f t h e c u r i n g w i n d r o w s ( d a t e , number) and r e a c t i o n t i m e i n t h e second r e a c t o r ,

-

d i s t u r b a n c e s o f o p e r a t i o n s (causes,

duration).

resp.

-

135

-

B e s i d e s t h e s e m i c r o b i o l o g i c s l s a n i t a t i o n p r o c e s s e s a l s o some o t h e r s have been d e f i n e d : w i t h l i m e a s Ca(OH)2,

sludge p a s t e u r i z a t i o n ,

sludge treatment

s l u d g e t r e a t m e n t w i t h l i m e a s CaO.

I n addition,

E x e c u t o r y P r o v i s i o n s f o r each o f t h e s l u d g e s a n i t a t i o n systems have been e l a b o r a t e d .

The r e q u i r e m e n t s a r e c l a s s i f i e d a s f o l l o w s :

ral-Pretreatment

o f sludge

-

Constructional requirements

n a l control - - S t a r t i n g operation system

-

-

-

C l e a n i n g and d i s i n f e c t i o n o f t h e

I n s t r u c t i o n o f the s t o f f - Operational malfunctions

stallations for operational control

Gene-

Operatio-

-

-

In-

Sampling f o r process c o n t r o l .

F u r t h e r t h e b a c t e r i o l o g i c a l a n d p a r a s i t o l o g i c a l m e t h o d s f o r t h e det e r m i n a t i o n o f s a l m o n e l l a s e n t e r o b a c t e r i a c e s e and a s c a r i s ova a r e l a i d down f o r t h e s y s t e m a n d p r o c e s s c o n t r o l

(10,

11).

When a l l t h e s e p r e r e q u i s i t e s a r e met i t i s assumed t h a t t h e s l u d g e can be c o n s i d e r e d as h y g i e n i c a l l y s a f e and t h a t t h e s l u d g e complies w i t h the m i c r o b i o l o g i c a l values mentioned i n t h e introduction. REFERENCES C E C ( 1 9 8 6 1 , C o u n c i l D i r e c t i v e of 12. June 1986 on t h e p r o t e c t i o n o f t h e e n v i r o n m e n t a n d i n p a r t i c u l a r o f t h e s o i l , when O f f . J. E sewage s l u d g e i s u s e d i n a g r i c u l t u r e ( 8 6 / 2 7 8 / E E C ) . u r o p . Comm. No L 1 8 1 / 6 , 4.7.86. B R D - FR G ( 1 9 8 2 ) , K l a r s c h l a m m v e r o r d n u n g ( O r d i n a n c e on sewage s l u d g e ) , BGBl I, 7 3 4 . A . M . B r u c e , A.H. H a v e l a a r a n d P. L ’ H e r m i t e (Eds., 19831, D i s i n f e c t i o n o f sewage s l u d g e : t e c h n i c a l , e c o n o m i c a n d m i c r o b i o l o g i c a l a s p e c t s . D. R e i d e l P u b l . Comp., D o r d r e c h t / N L . D. S t r a u c h , A.H. H a v e l a a r a n d P. L ’ H e r m i t e ( E d s . , 1 9 8 5 1 , I n a c t i v a t i o n o f m i c r o o r g a n i s m s i n sewage s l u d g e b y s t a b i l i z a t i o n p r o c e s s e s . E l s e v i e r A p p l i e d S c i e n c e P u b l . , London/New Y o r k . D. S t r a u c h [ H r s g . , 1 9 8 6 1 , B e r i c h t d e s 1. H o h e n h e i m e r S e m i n a r s “ E n t s e u c h u n g von K l a r s c h l a m m ( P r o c . 1. Hohenheim S e m i n a r “ D i s i n f e c t i o n o f sewage s l u d g e ) . D t . V e t . M e t . G e s e l l s c h a f t , F r a n k f u r t e r S t r . 89, D-6300 G i e s s e n . J. H u b e r , 0 . S i g e l a n d P.H. B r u n n e r ( E d s . , 1 9 8 7 1 , S u r v e y o f s e wage s l u d g e d i s i n f e c t i o n p r o c e s s e s . CEC/SL/l21/87-XII/ENV/l5/87. CEC-DG X I I / E - 1 , Rue de l a L o i 2 0 0 , 8 - 1 0 4 9 , B r u s s e l s . D. S t r a u c h (Ed., 1 9 8 8 ) , B e r i c h t des 2 . H o h e n h e i m e r S e m i n a r s “ E n t s e u c h u n g von K l a r s c h l a m m - E r f a h r u n g s b e r i c h t e a u s d e r P r a x i s “ . ( P r o c . 2. Hohenheim S e m i n a r “ D i s i n f e c t i o n o f sewage s l u d ge R e p o r t s on p r a c t i c a l e x p e r i e n c e s “ ) . D t . V e t . Med. G e s e l l s c h a f t , F r a n k f u r t e r S t r . 89, D-6300 G i e s s e n . J.H. Hannan a n d P. L ’ H e r m i t e ( E d s . , 1 9 8 7 ) , H y g i e n i c a s p e c t s o f t h e t r e a t m e n t and u s e o f o r g a n i c s l u d g e a n d l i q u i d a g r i c u l t u r a l CEC-DG X I I / E - 1 , Rue de w a s t e s ( I ) . CEC/SL/120/87-XII/ENV/l4/87. l a L o i 200, 8-1049 B r u s s e l s . D. S t r a u c h , H . Over and P. L ’ H e r m i t e ( E d s . , 1 9 8 9 ) , H y g i e n i c a s p e c t s o f t h e t r e a t m e n t and u s e o f o r g a n i c s l u d g e a n d l i q u i d aCEC-DG X I I / g r i c u l t u r a l w a s t e s ( 1 1 ) . CEC/SL/29/89-XII/ENV/l/89. E - 1 , Rue de l a L o i 2 0 0 , 8-1049 B r u s s e l s .

-

10

11

136

-

A T V / V K S ( 1 9 8 8 ) : 2. A r b e i t s b e r i c h t d e r A T V / V K S - A r b e i t s g r u p p e 3.2.2 " E n t s e u c h u n g v o n K l a r s c h l a m m " ( 2 n d r e p o r t o f t h e A T V / V K S w o r k i n g p a r t y " D i s i n f e c t i o n o f sewage s l u d g e " ) . K o r r e s p o n d e n z Abwasser 35 ( 1 ) 71-74. A T V / V K S ( 1 9 8 8 ) : 3. A r b e i t s b e r i c h t d e r A T V / V K S - A r b e i t s g r u p p e 3.2.2 " E n t s e u c h u n g v o n K l a r s c h l a m m " ( 3 r d r e p o r t o f t h e A T V / V K S w o r k i n g p a r t y " D i s i n f e c t i o n o f sewage s l u d g e " ) . K o r r e s p o n d e n z Abwasser 3 5 ( 1 2 ) 1 3 2 5 - 1 3 3 3 .

M O D E L I N G OF O R G A N I C MATTER D E S T R U C T I O N BY MICROORGANISMS COMMUNITY

V.A.

VAVILIN

W a t e r P r o b l e m s I n s t i t u t e USSR Academy o f S c i e n c e

The m i c r o o r g a n i s m s w o n d e r f u l a b i l i t y t o u t i l i z e a w i d e s p e c trum o f o r g a n i c substances has been used f o r a l o n g t i m e i n a r t i f i c i a l b i o l o g i c a l treatment plants.

Simulating b i o l o g i c a l treatment

s y s t e m s where t h e d e s t r u c t i o n o f a m u l t i - c o m p o n e n t ce i s caused by microorganisms community,

organic substan

p r e s e n t s a c o n v e n i e n t ob-

j e c t o f p o p u l a t i o n dynamics research. B i o l o q i c a l t r e a t m e n t s y s t e m s a s open s y s t e m s .

The t h e o r y o f

pure microorganisms c u l t u r e growth i s u s u a l l y used f o r t h e d e s c r i p t i o n o f b i o l o g i c a l treatment processes.

Through b i o l o g i c a l t r e a t m e n t

systems t h e c o n t i n u o u s f l o w o f biomass and s u b s t r a t e s i s p a s s i n g . U s i n g Monod f u n c t i o n s t h e s t e a d y s t a t e p r o c e s s o f b i o l o g i c a l t r e a t ment i n a c o m p l e t e - m i x i n g

a e r a t i o n t a n k can be d e s c r i b e d by t h e

f o l l o w i n g system o f e q u a t i o n s

1 - ( L T o

where L

e'

Xe

1

pm ' e L e =

- L ) - Y

K L + L

a r e t h e summary p o l l u t a n t a n d t o t a l b i o m a s s c o n c e n t r s -

t i o n i n aeration tank,

Le i s the input p o l l u t a n t concentration,

T i s the water r e t e n t i o n time, me,

CI

i s t h e mean b i o m a s s r e s i d e n c e t i -

i s t h e maximum s p e c i f i c g r o w t h r a t e o f b i o m a s s ,

yield coefficient, microorganisms,

K

L Traditionally,

Y i s the

k d i s t h e d e a t h and a u t o o x i d a t i o n c o n s t a n t f o r i s the half-saturation coefficient. t h e phenomenological d e s c r i p t i o n o f t h e t r e a t -

ment p l a n t o p e r a t i o n d o e s n o t t a k e i n t o a c c o u n t t h e f a c t t h a t h e t e -

-

138

-

r o t r o p h i c microorganisms are d i f f e r e n t i a t e d .

The m o r e c o m p o n e n t s

t h e more t y p e s o f m i c r o o r g a n i s m s a r e p r e -

t h e waste w a t e r s c o n t a i n ,

A general multi-component m u l t i -

s e n t i n t h e biomass composition.

s p e c i e s m o d e l o f b i o l o g i c a l t r e a t m e n t was s u g g e s t e d b y t h e a u t h o r

(2) d Si

1

-

-

(sOi - Si) T p o l l u t a n ts i n f l o w and outflow

d t

psi ,

i

= 1,2,

...,N,

removal o f pollutants

(2)

- -fB g r o w t h and death o f biomass w h e r e Si,

j

1,2,

j

'

...,M ,

withdrawal o f bi.omass

6 . a r e t h e c o n c e n t r a t i o n s o f t h e i - t h p o l l u t a n t and j - t h

J

qBj

are t h e r a t e s o f removal o f the i - t h group o f biomass, psi, i s the i n f l u e n t s u b s t r a t e and g r o w t h o f j - t h group; Soi = ciLo c o n c e n t r a t i o n o f t h e i - t h p o l l u t a n t , w h e r e Ei is t h e f r a c t i o n o f t h e i - t h p o l l u t a n t i n t h e summary p o l l u t a n t c o n c e n t r a t i o n L o . Two t y p e s o f w i t h d r a w a l o f b i o m a s s c o r r e s p o n d t o t h e t w o t y pes o f reactors:

y =1 /

[ Z, rBj M

d = const

F'=

max

/ j;

,

0

j=1

chemostat

turbidostat where X

7 0 i s a constant,

t o t a l biomass.

equaled t h e s t a t i o n a r y concentration o f

For t h e r a t e s o f

psi

and

PBj

t h e f o l l o w i n g expre-

i

1,2

s s i o n s were used

M;1 ysi

,urn. 6 . ri.

'i

j

= 511

N

Yij

(KLj+

r. .S.)

i=l 1J 1

,

,..., N

M-1 P 'EM

j=1

-

XjkdjBj

where flmj, k d j ,

KLj,

-

139

kdMBM,

Y..

a r e t h e c o r r e s p o n d i n g Monod c o e f f i c i e n t s

1J

f o r j - t h group o f microorganisms;

j = M corresponds t o a non-viable

b i o m a s s ; kdM i s t h e r a t e c o n s t a n t o f d e g r a d a t i o n o f n o n - v i a b l e mass;

X

.i

bio-

r. . i s the substrate pre-

i s the stechiometric c o e f f i c i e n t ,

13

ferability coefficient.

1 i l l u s t r a t e s t h e adequancy o f model ( 2 ) i n t h e case o f

Fig.

two s u b s t r a t e s ( g l u c o s e ,

phenol).

A n a l y s i s o f t h i s a n d some o t h e r m u l t i - c o m p o n e n t m o d e l s i n d i c a tes that the complexity,

stability,

p r o d u c t i v i t y and e f f i c i e n c y o f

t h e community a r e i n t e r r e l a t e d c h a r a c t e r i s t i c s

o f t h e open b i o l o g i -

c a l s y s t e m d e p e n d e n t o n s u c h m a c r o p a r a m m e t e r a s mean b i o m a s s r e s i dence ( r e n e w a l )

t i m e and o r g a n i c l o a d i n g ( t a b l e 1).

Black-box models o f b i o l o g i c a l t r e a t m e n t . I n t r o d u c i n g t h e aggreN M L = . c S1 a n d X-C B o b t a i n i n s t e a d o f t h e m u l t i 1=1 =1 .i component m u l t i - s p e c i e s m o d e l ( 2 3 t h e f o l l o w i n g one gated v a r i a b l e s

d L d t

where

-

yL =

1 (Lo

1

-

N

c fSi i=l

Le)

,

- YL

ex

9

d X -

ex

d t

- yx ,

M

N

ysj

=

and

Lo

j=l

I t is e a s y t o s e e t h a t i n s t e a d y - s t a t e t y p e r e a c t o r and Monod f u n c t i o n s , equivalent t o (1).

qL

and

=

c Soi . i=l

case o f t h e chemostat

f', t h e model ( 5 ) i s

A t t h e r e s u l t o f multi-component model's calcu-

l a t i o n s i t was p r o v e d t h a t t h e b e a t a p p r o x i m a t i o n o f t h e

vL

function

was n o t Monod b u t t h e f o l l o w i n g o n e s : , n

where n, p a r e t h e c o n s t a n t s , u t i l i z a t i o n rate,

y,

i s t h e maximum s p e c i f i c p o l u t a n t

i s t h e non-degradable f r a c t i o n o f organic matter,

X i s t h e biomass c o n c e n t r a t i o n i n a e r a t i o n tank.

-

140

F=

BOD5 ICOD = 0.59

5-

5.8 rng COD Irng MLSS . d

2320

I-

BIOMASS

d

a

OCOD --

I-

x GLUCOSE

z w V z 0

o PHENOL

............ BOD5 ICOD = 0.61

V

-______

L a - - . - -0 -.......

. . . . . . . . . . . . . . . . . . . . . . . . . . . . " n

0

I

' O . . '

X-

2-2

11

F = 0.58 mg CODImg MLSS.d

0

22

11

TIME [ H R S I F i g . 1. K i n e t i c c u r v e s f o r a c t i v a t e d s l u d g e o x i d i z i n g a m i x t u r e o f g l u c o s e and p h e n o l a t d i f f e r e n t o r g a n i c l o a d i n g d u r i n g a s e q u e n c i n g batch expariment.

Simultaneous carbon and n i t r o q e n removal model. tertuary

t r e a t m e n t i s v e r y a c t u a l now. A t p r e s e n t ,

technological

schemes a r e b a s e d o n t e m p o r a l o r s p a t i a l s e p a r a t i o n o f

such proceases as carbonaceous o x i d a t i o n , fication.

The p r o b l e m o f

a l l t h e known t

n i t r i f i c a t i o n and d e n i t r i -

T h r e e m o d i f i c a t i o n s o f a n a c t i v a t e d a l u d g e s y s t e m a r e u-

s u a l l y u s e d f o r m u n i c i p a l sewage w a t e r Scheme

(Fig.

2):

I i s a t w o - s e c t i o n s a e r a t i o n tank. There a r e a e r o b i c

c o n d i t i o n s i n t h e f i r s t s e c t i o n and a n o x i c c o n d i t i o n s i n t h e second s e c t i o n f o r t h e d e n i t r i f i c a t i o n process. Scheme I 1 i s f o u r - s e c t i o n s

a e r s t i o n tank.

I n t h e second and

f o u r t h sections the aerobic conditions are held.

-

141

-

Scheme 111 i s t h e s o c a l l e d B a r d e n p h o p r o c e s s w i t h a d d i t i o n a l r e c i r c u l a t i o n o f s l u d g e m i x t u r e from t h e second s e c t i o n t o t h e i n l e t o f the f i r s t section.

I n s u c h a scheme,

a l l r a w sewage w a t e r

comes t o t h e f i r s t s e c t i o n o f t h e a e r a t i o n t a n k .

REACTOR

qF

SCHEME

REACTOR

I

SCHEME

ANOXIC REACTOR

AEROBIC REACTOR

ANOXIC REACTOR

II

AEROBIC REACTOR

t

1 ANOXIC REACTOR

qF

AEROBIC REACTOR

I Fig.

2.

ANOXIC REACTOR

AEROBIC REACTOR

F ‘

t

M o d i f i c a t i o n s o f a c t i v a t e d sludge system.

T h e r e a r i s e a t h e q u e s t i o n o f how t o d e t e r m i n e t h e o p t i m u m val u e s o f t h e p o r t i o n s o f t h e t o t a l f l u x o f sewage w a t e r Ai,

inflo-

wing i n t o t h e s e p a r a t e s e c t i o n and t h e r e l a t i v e volume o f t h e such s e c t i o n Zi.

A t these optimum values,

t h e f i n e s t l e v e l o f w a s t e wa-

t e r t r e a t m e n t from n i t r o g e n i s achieved.

One o f t h e waya o f s o l v i n g

t h i s p r o b l e m c o n s i s t s o f t h e a p p l i c a t i o n o f t h e m a t h e m a t i c a l model i n g technique.

For such a purpose t h e model w i t h t h r e e main v a r i a b -

l e s (sewage w a t e r o r g a n i c c a r b o n c o n c e n t r a t i o n , gen c o n c e n t r a t i o n ,

SD)

SN,

was s u g g e s t e d ( 7 ) .

Sc,

ammonium n i t r o -

and c o n c e n t r a t i o n o f n i t r a t e s and n i t r a t e s , The v i a b l e s l u d g e b i o m a s s i s c o n s i d e r e d aa

b e i n g composed o f h e t e r o t r o p h i c X,,

and a u t o t r o p h i c XA p a r t s .

In

t u r n t h e h e t e r o t r o p h i c biomass can u t i l i z e for o x i d a t i o n o f organ i c carbon e i t h e r d i s a o l v e d oxygen o r t h a t c o n t a i n e d i n n i t r i t e s and n i t r a t e s .

The m a t e r i a l b a l a n c e e q u a t i o n s f o r t h e i - t h s e c t i o n

o f the n-sectional

a e r a t i o n t a n k may b e w r i t t e n a s f o l l o w a :

- 142

-

TABLE 1 Some p r o p e r t i e s o f o p e n b i o l o g i c a l c o m m u n i t i e s

Typical values o f macroparameters

P r o p e r t i e s of b i o l o g i c a l c o m m u n i t i e s w i t h fixed spectrum of available substrates

Large loading, small biomass t u r n - o v e r time

fast-growing species and organisms s e t t l e d , low s p e c i e s and groups d i v e r s i t y becouse o f displacement o f slow-growing organisms, simple trophic structure, i n s t a b i l i t y t o environmental changes, consumption of easyto-degradate p r o f i t a b l e f r a c t i o n s o f organ i c substance only

Small loading, l a r g e biomass t h r n - o v e r time

slow-growing s p e c i e s and organisms s e t t l e d , l a r g e portion of non-viable biomass, l a r g e s p e c i e s and groups d i v e r s i t y , complicated trophic structure, s t a b i l i t y to environmental changes, u t i l i z a t i o n o f a wide spectrum of s u b s t r a t e s

2. Direction of evolution processes

Chemostat: given values o f subs t r a t e s and biomass f l o w s , s t e a d y - s t a t e reg i m e i s d e t e r m i n e d by deficient substrate

K-strategy: increasing organisms a d a p t a b i l i t y t o biomass growth a t d e f i c i e n t s u b s t r a t e , i . e . of s y s t e m t h r i f t n e s s

Turbidostat: g i v e n s t a t i o n a r y value o f biomass, high r a t e of biomass growth is provided

r-strategy: i n c r e a s i n g maximum b i o m a s s g r o w t h r a t e , i . e . o f system p r o d u c t i v i t y

3 . S p a t i a l settlement of organisms

Type o f o p e n system w i t h s u b s t r a t e flow and biomass exchange

Organisms a b i l i t y t o s p a c e expansion i n continuous flow system

System with suspended biomass

o r g e n i e m s move t o g e t h e r w i t h s u b s t r a t e f l o w and according t o s u b s t r a t e s consumption t h e i r proportion changes

~~~

System with a t t a c h e d biomass

compact s t r u c t u r e o f d i f f e r e n t organisms g r o u p s i s f o r m e d i n s p a c e , when f a s t - g r o w i n c groups, consuming easy-to-degradate s u b s t r a tes s e t t l e n e a r t h e s u b s t r a t e s s o u r c e and slow-growing groups, u t i l i z i n g hard-to-degradate substrates, are displaced t o distant regions

-

where 1 = 1 , 2 , 3

143

-

ammonium n i t r o g e n N , n i t r i t e s

c o r r e s p o n d t o c a r b o n C,

and n i t r a t e s D ,

and t h e s p e c i a l e x p r e s s i o n s u s e d t o d e s c r i b e t h e

r a t e s o f c o n c e n t r a t i o n s c h a n g e s yil,

qi

i s the discharge o f the

sludge mixture s t the o u t l e t o f the i - t h section, Vi

flux,

qF i s t h e t o t a l

i s t h e volume o f t h e i - t h s e c t i o n .

The e x s m p l e o f t h e r e s u l t s o f t h e m o d e l ' s c a l c u l a t i o n s u n d e r t h e s e l e c t e d v a l u e s o f t h e c o n s t a n t s and parameters a r e p r e s e n t e d i n T a b l e 2. Ai

L e t u s n o t e t h a t t h e optimum v a l u e s o f t h e p a r a m e t e r s

s t w h i c h t h e t o t s 1 c o n c e n t r a t i o n o f n i t r o g e n compounds a t t h e

Zi, aeration tank o u t l e t ,

STN

SNn

+

SDn

,

become m i n i m a l d e p e n d i n g

c o n s i d e r s b l y on t h e t o t a l r e t e n t i o n t i m e T ,

concentration of organic

p o l l u t a n t i n raw wastes SCF and t h e r e c y c l e c o e f f i c i e n t R .

TABLE 2 The v a l u e s o f Scn a n d SNn ( m g / l ) a t t h e o p t i m u m v a l u e s o f p s r s m e t e r s Zi a n d Ai f o r t h e d i f f e r e n t schemes.

Scheme CF

h$s

2 75 10

150

-

I

R

Sc2

STN

I Sche I TN c4 --

Sche

rn

sc4

'TN

1

9.90

7.70

9.64

6.82

8.67

6.40

3

9.55

6.46

10.2

5.51

8.72

5.36

1

5.52

6.17

5.77

4.94

4.11

3.92

3

6.67

4.86

4.76

3.82

5.42

3.57

1

14.6

4.36

14.6

4.72

13.4

3.79

3

12.7

2.97

10.2

3.14

10.9

2.67

4.27

2.31

3.62

1.30

4.59

1.58

4.91

1.18

1 lo3

4.78

1.99

5.67

1.53

--

T one-section

12.9

15.7

11.1

15.7

8.39

15.8

8.00

15.9

19.6

10.5

15.7

10.5

9.86

11.0

9.01

11.1

From T a b l e 2 i t i s e a s y t o s e e t h s t n i t r o g e n r e m o v a l e f f i c i e n cy o f the t r a d i t i o n a l one-section F o r carbonaceous removal,

a e r a t i o n t a n k i s bad.

t h e B a r d e n p h o p r o c e s s (scheme 1 1 1 )

i s more e f f e c t i v e i n t h e c s s e o f t h e m a j o r i t y o f r e g i m e s s t u d i e d , a n d i t i s t h e b e s t one f o r t h e n i t r o g e n r e m o v a l u n d e r t h e d i f f e r e n t v a l u e s o f SCF

,

T and R.

But f o r t h i s process,

s d d i t i o n a l energy i s

required f o r the r e c d r c u l a t i o n o f the sludge mixture. t h e complete-mixing (9).

aeration tank the plug-flow

By i n c r e a s i n g

regime i s created

-

-

144

The p h y s i o l o q i c a l a d a p t a t i o n m o d e l o f b a c t e r i a p o p u l a t i o n o f a c t i v a t e d sludqe. essentially

I n t h e u s u a l model o f b a c t e r i a g r o w t h ,

assumed t o be some k i n d o f c a t a l y s t

oxidizing characteristics

a c e l l was

w i t h a fixed set o f

w h i c h were i n t r o d u c e d t h r o u g h t h e Monod

c o n s t a n t s . A t t h e same t i m e ,

t h e b a c t e r i a c e l l i s capable o f i n t e n -

s i f y i n g i t s own r e s o u r c e s s o a s t o p r o v i d e a maximum g r o w t h r a t e a t given conditions.

O n t h e b a s i s o f knewn e v i d e n c e on t h e c e l l ’ s

s t r u c t u r e and f u n c t i o n i n g ,

3 i n d i c a t e s the processes o f i n t e -

Fig.

r a c t i o n s between t h e elementq c o n s t i t u t i n g a c e l l . scheme,

the d i f f e r e n t substrates

S1, S 2 ,

...,

S.

According t o the

are oxidizing w i t h

t h e h e l p o f t h e c o r r e s p o n d i n g f e r m e n t a t i v e s y s t e m s Xfl,

Xfi

thesis. sis.

Xf2,

...,

t o some u n i v e r s a l s u b s t r a t e which was a p r e d e c e s s o r t o b i o s y n This storage substrate X8

i s consumed b y means o f b i o s y n t h e -

For a d e s c r i p t i o n o f t h i s process,

c e n t r a t i o n o f b i o s y n t h e s i s systems.

Xa

i s i n t r o d u c e d a s t h e con-

Biosynthesis production i s u t i -

l i z e d b o t h f o r t h e g r o w t h o f b i o s y n t h e s i s systems and t h e d e v e l o p ment o f f e r m e n t a t i v e systems,

Xfi.

Additionally,

some p o r t i o n o f

r e s o u r c e s i s s p e n t o n t h e c o n s t r u c t i o n o f c e l l u l a r membranes,

PAR AME T E R 5

I II

xs

-

xrn

F i g . 3 . Block-scheme o f a b a c t e r i a c e l l u t i l i z i n g a m i x t u r e o f substrates.

Xm.

-

145

-

Membranes p l a y an i m p o r t a n t r o l e i n t h e mechanism o f t h e c e l l ’ s functioning. The c o r r e s p o n d i n g m a t h e m a t i c a l e x p r e s s i o n s were s u g g e s t e d , d e s c r i b e t h e dynamics o f a balanced g r o w t h o f a c e l l , a polyaubstrate. conditions,

which

w h i l e consuming

I n o r d e r t o p r o v i d e t h e maximum g r o w t h r a t e a t g i v e n

a c e l l m u s t r e d i s t r i b u t e i t s r e s o u r c e s i n a n o p t i m a l way.

F o r t h i s p u r p o s e t h e c o n t r o l l i n g p a r a m e t e r s urn, ua, ufi duced i n t o t h e suggested model.

Thus,

on n o r m a l l y d u r i n g t h e g r o w t h process,

were i n d t r o -

i n order f o r a c e l l t o functi-

t h e r a t i o Xm/B s h o u l d b e m a i n -

t a i n e d a t a c o n s t a n t l e v e l w i t h t h e h e l p o f t h e p a r a m e t e r urn. With the help of

t h e ua parameter,

b o l i c ferments i s adjusted.

t h e o p e r a t i o n o f t h e c a t a b o l i c and sna-

I f there are a l o t o f storage substrates,

p r e f e r e n t i a l development i s g i v e n t o b i o s y n t h e a i s systems.

Otherwise

t h e m a j o r p a r t o f resources i s s p e n t on t h e g r o w t h o f c a t a b o l i c s y s tems,

which a r e t h e main s u p p l i e r s o f r e s e r v e d s u b s t r a t e s .

Finally,

w i t h t h e h e l p o f t h e c o n t r o l l i n g p a r a m e t e r s ufi,

t h e remaining re-

sources a r e s p e n t on t h e g r o w t h o f t h e ferments,

w h i c h t r e a t t h e ea-

a i l y degradate substrate.

A t t h e aame t i m e ,

synthesis o f t h e remai-

n i n g f e r m e n t a t i v e s y s t e m s i s r e p r e s a e d . F u r t h e r on,

a cell starts

t o u t i l i z e t h e s u b s t r a t e s w h i c h a r e more d i f f i c u l t t o u t i l i z e . The c h o i c e o f t h e a p p r o p r i a t e f o o d s t r a t e g y h a s a n e f f e c t u p o n t h e s e t t l e m e n t o f t h i s o r some o t h e r b a c t e r i a i n a c t i v a t e d s l u d g e . The p h y s i o l o g i c a l a d a p t a t i o n m o d e l m e n t i o n e d above,

was i n v o l v e d i n

t h e c y c l e model o f t h e a c t i v a t e d s l u d g e system ( 3 ) .

I t was shown t h a t

t h e b a c t e r i a - g e n e r a l i s t s capable o f u t i l i z i n g a broad substrates range a r e s e t t l e d i n a c t i v a t e d s l u d g e u n d e r a l o w o r g s n i c l o a d i n g o n l y . Under a h i g h o r g a n i c l o a d i n g t h e y a r e d i s p l a c e d by t h e b a c t e r i a - s p e cialists.

I n d e p e n d e n t o f what k i n d o f s p e c i e s ( g e n e r a l i s t o r a p e c i a -

l i s t ) i n h a b i t the a c t i v a t e d sludge,

i t s equilibrium composition in-

cludes a r e l a t i v e l y greater proportion o f the d i f f e r e n t catabolic ferments.

With t h e d e c r e a s e o f t h e l o a d i n g o f t h e s p e c t r u m ,

these

f e r m e n t s become w i d e r . CONCLUSION M o d e l i n g o r g a n i c m a t t e r by d e s t r u c t i o n o f t h e m i c r o o r g a n i s m s community p r o v i d e s t h e u s e f u l knowledge f o r t h e o r y and p r a c t i c e development. REFERENCES 1 A.W. L a w r e n c e a n d P.L. V. 9 6 , N3, 757-700.

McCarty,

J. S a n i t . Eng. D i v . ASCE, 1 9 7 0 ,

-

9

146

-

V.A. V a v i l i n , B i o m a s s R e s i d e n c e Time a n d O r g a n i c M a t t e r D e a t r u c t i o n i n B i o l o g i c a l T r e a t m e n t S y s t e m s . Moscow: Nauka P u b l i s h e r s , 1986. V.A. V a v i l i n a n d V.B. V a s i l i e v , E f f i c i e n c y o f M i c r o o r g a n h m s Community o f O r g a n i c M a t t e r D e s t r u c t i o n ( t o b e p u b l i s h e d ) . V.A. V a v i l i n , V o d n i e R e s u r s i , 1 9 8 8 , N 1 , 91-99. V.A. V a v i l i n , V o d n i e R e s u r s i , 1 9 8 8 , N4, 1 3 7 - 1 4 3 . V.A. V a v i l i n e t a l . A c t a H y d r o c h i m . H y d r o b i o l . , 1987, v.15, N6, 665-668. V.B. V a s i l i e v a n d V . A . V a v i l i n , Vodnie R e s u r s i . , 1990, N1, 119127. A.M. N i e k e r k e t a l . . J.WPCF. 1 9 8 8 . v . 60, N1, 1 0 0 - 1 0 6 . J. Chuddoba e t a l . W a t e r Res., 1 9 7 3 , v . 7., N8, 1 1 6 3 - 1 1 7 1 .

EFFECT OF P R O T O Z O A ON BACTERIAL DEGRADATION O F A R O M A T I C HYDROCARBONS

Yu.

L.

G U R E V I C H and V . P .

LADYGINA

I n s t i t u t e o f B i o p h y s i c s , U S S R Academy o f S c i e n c e s , 6 6 0 0 3 6 K r a s n o y a r s k , USSR

r h e p r o t o z o a sp. wastewaters.

are o f t e n found i n the communities p u r i f y i n g

The m o s t p r o b a b l e and m a i n r o l e o f p r o t o z o a i s t h o u g h t

t o maintain a bacterial population a t the condition o f physiologic a l youth,

and t o promote t h e c e l l s '

a g g r e g a t i o n 11, 2 1 .

The i d e a o f p h y s i o l o g i c a l y o u t h o f a b a c t e r i a l p o p u l a t i o n i s t o o general. rification.

Indeed,

o f water q u a l i t y , shouldn't

cells'

f l o c c u l a t i o n improve t h e wastewater pu-

This f a c t i s displayed by generalized c h a r a c t e r i s t i c s

C O D a n d BOD

e.g.

111.

Nevertheless,

that

fact

be i n t e r p r e t e d as a d i r e c t a f f e c t o f p r o t o z o a on t h e b i o -

c h e m i c a l p r o c e s s o f an o r g a n i c s o x i d a t i o n .

Hence,

t h e i r role i n the

biodegradation o f organic matter i s not obvious yet.

We h a v e s t u d i e d

t h i s p r o b l e m i n e x p e r i m e n t s on p h e n o l a n d n a p h t h a l e n e d e g r a d a t i o n by the simple t r o p h i c chain "substrate + b a c t e r i a " ,

and " s u b s t r a t e +

b a c t e r i a + protozoa".

M A T E R I A L S AND METHODS E : x p e r i m e n t s were c a r r i e d o u t i n a s i n g l e - s t a g e aeration,

mixing,

from 0.06

h - l t o 0.12

We've

pH and t e m p e r a t u r e c o n t r o l .

chemostat w i t h

The d i l u t i o n r a t e was

h-I.

used s e l e c t i v e b a c t e r i a l c u l t u r e s ,

which

1) u t i l i z e d naphthalene,

2) u t i l i z e d phenol. C i l i a t e Colpoda sp.

was u s e d a s a p r e d a t o r ;

t h i s l a t t e r was i s o l a -

t e d from t h e wastes t r e a t m e n t f a c i l i t y o f coke and b y - p r o d u c t

plant.

Colpotla c e l l s a r e a b l e t o g r a z e b o t h suspended b a c t e r i a l c e l l s ,

and

t h e a t t a c h e d ones a t t h e s u r f a c e . The c u l t i v a t i o n medium was a s o l u t i o n o f N H 4 C 1 , MgSO& i n p o t a b l e w a t e r .

KH2P04, Na2HP04,

P h e n o l was s u p p l i e d w i t h t h e medium,

and i t s

-

148

-

i n p u t c o n c e n t r a t i o n was e q u a l t o 0.8 9.1-'. was i n d e p e n d e n t f r o m t h e medium i n p u t .

The n a p h t h a l e n e s u p p l y

The n a p h t h a l e n e f l o w was e -

nough t o m a i n t a i n t h e o u t p u t biomass c o n c e n t r a t i o n i n t h e range o f

0.5

-

1.0

g.1-l

(dry weight).

The r e s i d u a l n a p h t h a l e n e c o n c e n t r a t i o n was d e t e c t e d b y g a s c h r o -

matography;

t h a t o f p h e n o l was d e t e c t e d b y p h o t o m e t e r ,

u s i n g 4 - ami-

n i a n t i p y r i n e as a c o l o r i n d i c a t o r . RESULTS

1. N a p h t h a l e n e + b a c t e r i a s y s t e m s The b a c t e r i a l c o n c e n t r a t i o n was c l o s e t o 1 g . 1 - l

d/w.

n i m a l r e s i d u a l n a p h t h a l e n e c o n c e n t r a t i o n was e q u a l t o 0 . 1

I t s h o u l d be n o t e d , tuated considerably.

t h a t the output naphthalene czncentration Probably,

Fig. 1). t o 1.32 mg.1-' The o u t p u t b i o m a s s c o n c e n t r a t i o n i n c r e a s e d , Hence,

fluc-

The a v e r a g e o v e r t h e

w i t h no c o n s i d e r a b l e d i s t u r b a n c e s o f t h e p r o c e s s ,

input increased

.

-1

the low s o l u b i l i t y o f the substrate

made t h i s i n p u t i n t o t h e c u l t u r e n o n u n i f o r m . period,

The m i mg.1

was e q u a l

as t h e substrate

t h i s form t h e ground t o c o n s i d e r . n a p h t h a l e -

ne the l i m i t i n g factor.

a

R

3

1

5

9

7

11

DAYS

3

DAYS

w

a

b

I

,

u0

I 3 0

1

2

3

DAYS

0

1

2

F i g . 1. D e g r a d a t i o n o f n a p h t h a l e n e i n c h e m o s t a t c u l t u r e o f Pseudomonas ap. a b a c t e r i a l c u l t u r e , 100 L phosphorus; b bacterial + c i l i a t e , 100 L p h o s p h o r u s ; c b a c t e r i a l + c i l i a t e , 1 0 Vn p h o s p h o r u s .

-

-

-

2.

Naphthalene

-

149

+ b a c t e r i a + Colpodae systems

The a v e r a g e o u t p u t n a p h t h a l e n e c o n c e n t r a t i o n i n c r e a s e d u p t o

6.7

rng.l-',

when t h e c i l i a t e was i n t r o d u c e d i n t o t h e c u l t u r e ( u n d e r

t h e same c u l t i v a t i o n c o n d i t i o n s ) . m a i n e d p r a c t i c a l l y t h e same. 3 4.10 cells/rnl.

The mean t o t a l c o u t p u t b i o m a s s r e -

-

number was e q u a l t o 0.5

Ciliate's

P h o s p h o r - l i m i t e d n u t r i t i o n o f t h e c u l t u r e made t h e r e c o v e r y o f t h e q u a l i t y o f b i o p u r i f i c a t i o n o f water.

Moreover,

t h e a v e r a g e nap-

h t a l e n e c o n t e n t was l o w e r d u r i n g t h e p e r i o d s w i t h n o l a r g e f l u c t u a tions.

lo3

30.

The C o l p o d a sp.

p o p u l a t i o n d e n s i t y v a r i e d f r o m 5 . 1 ~ 1t ~o

cells/ml.

The s i g n i f i c a n t p h o s p h o r u s l i m i t a t i o n r e s u l t e d i n a h e a v y i n The C i l i a t e were f o u n d t o b e v e r y

crease o f naphthalene content.

sensible t o increased naphthalene concentration;

e x p e r i m e n t showed

or

e n c y s t a t i o n a n d w a l l g r o w t h a t t h e c o n c e n t r a t i o n o f 30 mg.1-' more.

Our m a i n s t a t e m e n t i s t h a t t h e r e i s an o p t i m a l p h o s p h o r u s c o n centration,

t h a t r e s u l t e d i n t h e most e f f i c i e n t u t i l i z a t i o n o f a t o -

x i c carbon source (here naphthalene).

Both the c e l l s '

aggregation

a n d w a l l g r o w t h were o b s e r v e d d u r i n g t h e e x p e r i m e n t s .

3.

Phenol

+

b a c t e r i a systems

The d i l u t i o n r a t e a t 0.07 medium o f 8 0 0 mg.1-'

4.

mg.1-

Phenol

and a p h e n o l c o n c e n t r a t i o n i n a

r e s u l t e d i n an a p p e a r a n c e o f t h e a v e r a g e d r y

w e i g h t o u t p u t biomass 0.61 0.36

h-l,

.

g.1-l

and o u t p u t p h e n o l c o n c e n t r s t i o n a t

1

+ b a c t e r i a + p r o t o z o a systems

As i n t h e f i r s t e x p e r i m e n t ,

the i n t r o d u c t i o n o f Protozoa bro-

u g h t about c o n s i d e r a b l e d i s t u r b a n c e i n t h e system. t r a t i o n of growth-limiting 150 mg.1-l.

R e s i d u a l concen-

a o u r c e o f c a r b o n s h a r p l y i n c r e a s e s up t o

T h i s i n c r e a s e was a c c o m p a n i e d b y an i n c r e a s e o f t h e t o -

t a l biomass and t h e numbers o f t h e c i l i a t e a t t h e o u t p u t . the values of

t h e observed v a r i a b l e s s t a r t e d t o recover,

reaching the steady state, 0.12

h-'.

t h e d i l u t i o n r a t e waa i n c r e a s e d up t o

A t t h i s d i l u t i o n r a t e 1 3 0 mg.1-'

p h e n o l a n d 128.103

cells/ml

Afterwards, but before

mg.1-'

of

o f c i l i a t e established (Table 1).

biomass,

2,2

The

p o p u l a t i o n o f p r o t o z o a v a r i e d w i t h i n t h e l i m i t s o f 20.103

-

140.10

cells/ml. As e a r l i e r ,

the ten-fold

decrease of

phosphorus c o n c e n t r a t i o n

3

-

-

150

a t t h e i n p u t made d e c r e a s e t h e a v e r a g e v a l u e o f p h e n o l a t t h e o u t p u t t o 0.66

mg.1-l

(minimum v a l u e s a b o u t 0 . 2

c e n t r a t i o n o f b i o m a s s was 1 5 0 m g . l - l , 179,l-1

cells/ml.

150.103

-

So,

600.10

I n t h i s case con-

mg.1-’).

the population o f c i l i a t e

-

The n u m b e r s o f P r o t o z o a v a r i e d w i t h i n t h e l i m i t s 3

cells/ml.

t h e experiments proved t h e p o s i t i v e ( w i t h l i m i t e d nonenergy

substrate)

and n e g a t i v e ( w i t h l i m i t e d s o u r c e o f c a r b o n and e n e r g y )

e f f e c t o f predation o f b a c t e r i a l oxidation o f t o x i c substrates. TABLE 1 D e g r a d a t i o n o f p h e n o l by b a c t e r i a o r b a c t e r i a Input concentration o f phosphorus a t 7;

O il u tion rate

[h-l]

+

c i l i a t e i n chemostat

Dry weight o f phenol [ m q 111

[g/ll

[c e 11s/m 1 1

I

-

I

100

0.07

0.61

100

0.12

0.13 flocs, wall growth

2.2

128.10~

I

10

0.12

0.15 more w a l l growth

0.66

178,6.

L

0.36

lo3

THEORETICAL ANALYSIS The m o d e l o f t h e “ p r e d a t o r - p r e y - s u b s t r a t e ” presumes t h e p r e s e n c e o f t u r n o v e r l i m i t i n g factor. o f t h e system’s

system (nonenergy)

i n a simple trophic chain o f the

Under t h e c o n d i t i o n s o f t h e t u r n o v e r ,

the region

e x i s t e n c e i n t h e p l a n e o f c o n t r o l l i n g parameters So,

D ( l i m i t i n g substrate a t the input,

d i l u t i o n r a t e ) as w e l l as t h e

d i v i s i o n o f t h i s r e g i o n i n t o zones o f s t a b i l i t y and i n s t a b i l i t y , n o t change. ters,

However,

do

a t t h e same v a l u e s o f t h e c o n t r o l l i n g parame-

the population o f Protozoa increases,

o f t h e s y s t e m on t h e w h o l e ,

ensuring the survival

as t h i s is much more t h a n t h e minimum

c r i t i c a l value o f the s i z e o f the population.

T h i s r e s u l t o f nume-

r i c a l s i m u l a t i o n i s i n agreement w i t h t h e e x p e r i m e n t a l d a t a . Taking i n t o account t h a t t h e l e a d i n g r o l e i n t h e *preypredator” s y s t e m is p e r f o r m e d n o t o n l y b y t h e t u r n o v e r a s an a d d i t i o n a l s o u r c e o f biogenous element o f n u t r i t i o n , strate,

b u t a l s o t o t h e a u t o c h t o n e sub-

stimulating b a c t e r i a l reproduction,

the region o f s t a b i l i t y

and t h e r e g i o n o f s u r v i v a l o f t h e system i a c o n s i d e r a b l y e n l a r g e d .

-

151

-

The m o d e l u n d e r c o n s i d e r a t i o n a d m i t s t h a t t h e a u t o c h t o n e s u b s t r a t e i s 11ot t h e p r o d u c t o f l y s i s o f d e a t h o f t h e c e l l s , a s i t i s a s s u med i n t h e w o r k 1 2 1 , b u t t h e r e s u l t o f t h e m e t a b o l i s m o f t h e P r o t o zoa. The e x p e r i m e n t s r e v e a l e d f l o c c u l a t i o n a n d w a l l g r o w t h . T h i s h i n d e r s a d e t a i l e d d e s c r i p t i o n o f t h e p r o c e s s o f naphthalene o r phen o l b i o o x i d a t i o n , may b e t h e o r i g i n o f f l u c t u a t i o n s a n d o t h e r a f f e c t s . On t h e . o t h e r h a n d , w a l l g r o w t h i s t o h e l p s t a b i l i z e t h e p r o c e s s and e n h a n c e t h e q u a l i t y o f s u b s t r a t e b i o o x i d a t i o n . We have an a l y s e d t h i s pnehomenon i n w o r k s 1 3 , 41. We h a v e c o n s i d e r e d t h e mod e l s o f b a c t e r i a l b i o d e g r a d a t i o n o f t o x i c compounds u n d e r c h e m o a t a t c o n d i t i o n s w i t h an a c c o u n t o f f i l m f o r m a t i o n a n d t h e m o d e l o f g r o w t h of

i n f u s o r i a a p t t o s t i c k i n g i n an i n h o m o g e n e o u s c h e m o s t a t c u l t u r e .

DILUTION

RATE

F i g . 2. I n f l u e n c e o f t h e d i l u t i o n r a t e on t h e - g r o w t h o f p r o t o z o a i n c h e m o s t a t ( c a l c u l a t e d c u r v e s ) . An a n a l o g i c a l p i c t u r e f o r b a c t e r i a . X - f r e e - s w i m m i n g c e l l s , X - a t t a c h e d c e l l s , I , 11, 111 d i f f e r g n t r e g i o n s o f s t a b l e and i n s t a b l e s t a t e s and p h y s i o l o g y o f c u l ture.

-

From t h i s a n a l y s i s t h e n e c e s s i t y e n s u e s t o t a k e i n t o a c c o u n t t h e t r a n s i t i o n o f b a c t e r i a and p r o t o z o a from t h e f r e e s t a t e i n t o t h e a g g r e q a t e one a n d b a c k .

T h i s c u l t u r e i s shown t o h a v e a r e g i o n i n t h e p l a n e o f t h e c o n t r o l l i n g p a r a m e t e r s ( S o , D ) , where t h e d e n s i t y o f t h e c u l t u r e i s o b s e r v e d t o depend on n o n - s i n g l e -

valuedly

the d i l u t i o n rate.

D e t e r m i n e d a r e t h e t y p e s o f model s o l u t i o n s , ce o f t h r e e s t a t i o n a r y p o i n t s .

predicting the existen-

The v a r i a n t o f t h e m o d e l w i t h l i m i -

t e d g r o w t h o f aggregate c e l l s i n i t s main f e a t u r e ( a b i l i t y t o recover a f t e r l a r g e disturbances) ses.

corresponda t o d e f i n i t e r e a l proces-

-

152 -

W i t h t h e a c c o u n t o f t h e above m e n t i o n e d p e c u l i a r r e l a t i o n s b e t organisms and t h e s u b s t r a t e i n our t r o p h i c

ween t h e s i n g l e - c e l l chain,

i t can be r e p r e s e n t e d i n t h e f o l l o w i n g f o r m :

attached

free-swimming

bacteria

bacteria

st t ached protozoa

I%-

-a

free-swimming protozoa

C o m p l e t e a n a l y s i s o f s u c h a s y s t e m n e e d s much t i m e .

Besides,

t h i s system c s n h a r d l y be s u f f i c i e n t l y adequate t o a r e a l o b j e c t , under c e r t a i n c o n c e n t r a t i o n s o f naphthalene,

f o r example,

30 mg.1-

as

1

,

t h e c i l i a t e form c y s t s and t h e r e v e r s e excystment p r o c e s s s t a r t s , when t h e c o n c e n t r a t i o n o f n a p h t h a l e n e d e c r e a s e s . n o l wasn't

ss s t r o n g .

S e n s i t i v i t y t o phe-

E n c y s t a t i o n a n d e v e n t h e d e a t h o f c i l i a t e we-

r e o b s e r v e d a t v e r y h i g h p h e n o l c o n c e n t r a t i o n s ( r 500 mg.1

-1

1.

Also

a n a c c o u n t s h o u l d b e made o f t h e p o s s i b i l i t y o f d e p o s i t i o n o f t h e growth-limiting

s u b s t r a t e i n t h e f o r m o f t r a n s f o r m e d compounds i n

the b i o f i l m or i n the c e l l .

R e s u l t s o f n u m e r i c a l c a l c u l a t i o n s by t h e

m o d e l p r o p o s e d do n o t d i s a g r e e w i t h t h e e x p e r i m e n t a l d a t a . CONCLUSION The e x p e r i m e n t a n d t h e o r e t i c a l a n a l y s i b showed t h a t t h e p o s i t i v e e f f e c t o f p r e d a t i o n on b a c t e r i a l d e g r a d a t i o n o f t h e t o x i c agents i s connected w i t h t h e t u r n o v e r o f t h e biogenous elements i n t h e s i m p l e t r o p h i c c h a i n u n d e r i n v e s t i g a t i o n e n d t h e emergence o f autochthonous substrate s t i m u l a t i n g b a c t e r i a l reproduction. REFERENCES

1 2 3

C.R. C u r d s , The e c o l o g y a n d r o l e o f p r o t o z o a i n a e r o b i c sewage t r e a t m e n t p r o c e s s e s . -Ann. Rev. M i c r o b i o l . , 1 9 8 2 , 36, p. 27-46. A . Sambania, S . P a v l o u a n d A . G . Fredrickson, Analysis o f the Chemical dynamics o f c i l i a t e - b a c t e r i a l i n t e r a c t i o n s i n C S T R . Eng. S c i e n c e , v. 41, N6, p. 1 4 5 5 - 1 4 6 9 , 1986. L a d y g i n a , The s t a b i l i t y o f b i o d e g r a d a Yu. L. G u r e v i c h a n d V.P. t i o n o f t o x i c s u b s t r a t e s i n a chemostat with r e s p e c t t o c e l l s Book o f A b s t r a c t s . 9 t h I S C C "Continuous C u l t u r e attachment.

-

-

-

4

153

-

i n B i o t e c h n o l o g y a n d E n v i r o n m e n t C o n s e r v a t i o n " , 19-24 J u l y , 1 9 8 7 , Hradec K r a l o v e , Czechoslovakia. Yu.L. G u r e v i c h and V . P . L a d y g i n a , F o r m a t i o n o f w a l l g r o w t h f i l m s and b i o d e g r a d a t i o n p e r s i s t e n c e o f t o x i c compounds i n c o n t i n u o u s c u l t u r e systems. P r o b l e m o f E c o l o g i c a l M o n i t o r i n g and Ecosystem m o d e l i n g . v . 9 , p . 2 2 - 3 0 , L e n i n g r a d , G i d r o m e t e o i z d a t , 1 9 8 6 .

-

This Page Intentionally Left TBlank

P H E N O L AND NAPHTHALENE DEGRADATION B Y M I X E D CULTURE

OF M I C R O O R G A N I S M S N.S.

MANUKOVSKI,

M.I.

TEREMOVA,

I n s t i t u t e o f Biophysics,

Yu.L.

GUREVICH and I . M .

PAN’KOVA

U S S R Academy o f S c i e n c e s ,

660036, K r a s n o y a r s k , U S S R

Wastewater c o n t a i n s , ces;

as a r u l e ,

a mixture o f organic substan-

i t ’ s b i o d e g r a d a t i o n i s r e a l i z e d by m i c r o b i a l c o m m u n i t i e s .

The

composition o f t h e communities i s e s t a b l i s h e d spontaneously under non-sterile

c o n d i t i o n s o f no e x t e r n a l c o n t r o l

impact.

T h i s communication d w e l l s upon t h e problem o f t h e p u r p o s e f u l f o r m a t i o n o f t h e m i c r o o r g a n i s m community t h a t i s c a p a b l e t o p u r i f y w a t e r more e f f i c i e n t l y .

The o b j e c t u n d e r i n v e s t i g a t i o n was t h e wa-

t e r o f a cokechemical i n d u s t r y .

B i o d e g r a d a t i o n o f p h e n o l a n d na-phtha-

l e n e was m o s t i n t e r e s t i n g t o u s . We h a v e s t u d i e d t w o ways t o i m p r o v e t h e c o m p o s i t i o n o f m i c r o b i a l community, which p u r i f i e d wastewater

-

from aromatic hydrocar-

bons.

1. One c a n i n t r o d u c e a new s t r a i n i n t o t h e m i c r o b i a l c o m m u n i t y . The s t r a i n c o u l d be o b t a i n e d by a r e c o m b i n a n t DNA,

or s p e c i a l l y se-

l e c t e d , or found i n a d i f f e r e n t environment.

2.

The s p e c i e s c o m p o s i t i o n o f a c o m m u n i t y i s n o t a f f e c t e d

(i.e. n o new s p e c i e s o r s t r a i n i s i n t r o d u c e d ) ,

but the r a t i o o f the

densities o f the species present i s appropriately varied. Community c o m p o s i t i o n s h o u l d b e c h a n g e d w i t h r e s p e c t t o t h e feature o f both microorganisms i n t e r a c t i o n ,

and t h e o x i d a t i o n k i n e -

t i c s o f t h e mixed s u b s t r a t e . S p e c i e s and q r o w t h c o n d i t i o n s . monas s p .

and N o c a r d i a sp.

The b a c t e r i a l c u l t u r e o f P s e u d o -

have been i s o l a t e d from t h e wastewater

on t h e s e l e c t i v e media c o n t a i n i n g p h e n o l , g u i a c o l , The g l a s s f e r m e n t o r

o f t h e 0.45-0.6

and

naphthalene

1 e f f e c t i v e v o l u m e was e q u i p p e d

w i t h an a u t o m a t i c d e v i c e t o m a i n t a i n t h e pH a n d t e m p e r a t u r e . was m a i n t a i n e d w i t h i n t h e 6.7-7.5

range,

The pH

a n d t h e c u l t i v a t i o n tempe-

r a t u r e was e q u a l t o 20

156

a n d 30

OC

OC

f o r N o c a r d i a sp.,

b u t 32-34

OC

f o r Pseudomonas s p . Analysis.

The c o n c e n t r a t i o n o f b o t h p h e n o l a n d g u i a c o l was d e -

termined c o l o r i m e t r i c a l l y over 4-aminosntipyrine 2,3-oxygenase

as t h e i n d i c a t o r ;

a c t i v i t y i n c e l l s was d e t e r m i n e d c o l o r i m e t r i c a l l y o v e r

c a t e c h o l ; n a p h t h a l e n e c o n c e n t r a t i o n was d e t e r m i n e d b y t h e c h r o m a t o graph.

I n addition,

s u p e r n a t a n t a b s o r p t i o n maximum was d e t e r m i n e d

-

w i t h i n t h e r e g i o n o f 370

420 n m a t pH 1 1 . 0

111.

1. D e g r a d a t i o n o f a s i n g l e s u b s t r a t e The c u l t u r e s s t u d i e d h a v e shown d i f f e r e n t a b i l i t i e s t o o x i d i z e the substrate

( s e e T a b l e 1). B o t h t h e d e p e n d e n c e o f t h e c e l l s den-

s i t y and t h e s u b s t r a t e c o n c e n t r a t i o n on t h e d i l u t i o n r a t e ,

and t h e

dependence o f t h e s p e c i f i c r a t e o f s u b s t r a t e c o n s u m p t i o n on t h e g r o w t h r a t e f i t i n Monod's and P i r t ' s models ( s e e F i g .

1

-

3).

TABLE 1 Degradation o f aromatic hydrocarbons as s o l e sources o f carbon i n c h e m o s t a t c u l t u r e s o f Pseudomonas a n d N o c s r d i a Dilution rate

Substrate

Culture, substrate

input

output

Gs/ll

[h-']

1000800

< 1

N o c a r d i a spp. guiacol

250

1-5

Pseudomonas sp. + N o c a r d i a sp. p h e n o l

BOO

Pseudomonas sp. phenol

Pseudomonas spp.

Growth yield

0.070.27

0.065-

0.6

0.12

c1

20004000

c 0.5

0.140.20

+

0.0750.12

+

N o t d e t e r m i n e d f o r w a l l g r o w t h and i n s o l u b i l i t y o f n a p h t h a l e n e .

We h a v e m e a s u r e d t h e d e p e n d e n c e o f t h e s p e c i f i c r a t e o f s u b s t r a t e

consumption on t h e d i l u t i o n r a t e . g r o w t h and c e l l s a g g r e g a t i o n ,

The o c c u r r e n c e o f t h e a t t a c h e d

has been measured as w e l l .

o x i d a t i o n d a t a a r e p r e s e n t e d i n T a b l e 2;

I t s h o u l d be n o t e d ,

we'll

d i s c u s s them b e l o w .

t h a t t h e observed v a l u e o f t h e economic

c o e f f i c i e n t i s l e s s tl- n t h e t h e o r e t i c a l one, spent f o r maintenance i s c l o s e t o zero.

a l t h o u g h t h e energy

One c a n e a s i l y see t h a t

d u r i n g t h e p h e n o l d e g r a d a t i o n b y Pseudomonas sp. was a l w a y s c o l o r e d g r e e n ,

The p h e n o l

or b r o w n .

t h e c u l t u r a l medium

-

157

-

0.1

0.2

0.3 DIWTDN RATE [ h-'1

Fig. 1. C h e m o s t a t c u l t u r e o f P s e u d o m o n a s s e . with phenol a s a lim i t i n g substrate. 1 - b i o m a s s , 2 - output c o n c e n t r a t i o n o f phenol

--

- 0.200,

J

0

0.15

-

0.10

.

0.05

-

0

0.05

0.1

0.15

DILUTION RATE

1 0 .h-']

Fig. 2. C h e m o s t a t c u l t u r e o f N o c a r d i a s p . w i t h g u i a c o l a s a limiting s u b s t r a t e ( S o = 0.25 9/11. 1 biomass, 2 autput concentration o f guiacol; a r r o w 8 i n d i c a t e c h a n g e s o f d i l u t i o n r a t e

-

-

-

-

158

1.0

-

'p

c

2

P

n

F i g . 3. S p e c i f i c u p t a k e r a t e o f p h e n o l ( 1 ) a n d g u i a c o l ( 2 ) i n r e l a t i o n t o t h e d i l u t i o n r a t e i n c h e m o s t a t c u l t u r e o f Pseudomonas s p . and N o c a r d i a spp.

N o t e some d i s c r e p a n c i e s b e t w e e n t h e e v i d e n c e a n d t h e m o d e r n theories:

1. N o n - s i n g l e - v a l u e d

r e s u l t o f t h e s u b s t r a t e d e g r a d a t i o n under

c o n s t a n t c u l t i v a t i o n c o n d i t i o n s was o b s e r v e d i n a s e r i e s o f e x p e r i m e n t s (see dash l i n e i n F i g . as w e l l ,

2.

as exp.

7va expa.

Pseudomonaa ap.

2 f o r t h e case o f q u i a c o l o x i d a t i o n ,

1-6 and 13-16 i n Table 2).

c u l t u r e s a l w a y s show m e t e - c l e a v a g e

pathway

f o r catechol oxidation a t the i n i t i a l stage o f c u l t i v a t i o n . (rather long)

2.

t i m e i t disappeared b o t h f o r mixed,

I n some

and m o n o - c u l t u r e s .

Substrate mixture degradation I n s p i t e o f some i n e x p l i c a b l e f e a t u r e s i n s i n g l e s p e c i e s c u l -

t u r e behavior,

t h a t oxidase phenol,

guiacol,

t r i e d t h e m i x e d c u l t u r e o f Pseudomonas aD. them.

+

and naphthalene, N o c a r d i a sp.

we

t o treat

We assumed t h e enzyme k i n e t i c s o f d i f f e r e n t m i c r o o r g a n i s m s t o

d i f f e r f o r v a r i o u s a r o m a t i c h y d r o c a r b o n s . So, complement each o t h e r .

t h e c u l t u r e s should

-

159

-

TABLE 2 Parameters o f phenol d e g r a d a t i o n process i n chemostat ~

NN

Dilution r a p ]

-

Substrate input

hdil

Biomass

output

[mg/l]

1

0.07

1000

0.2

594

2

0.07

1000

0.2

600

Cleavage pathway, absorbtion p e a k [nm]

Economical coef f i c i e n

b/gJ 0.59 0.60

375 meta-

3

0.07

1000

0.3

630

4

0.07

1000

0.2

490

5

0.07

1000

0.2

550

6

0.07

80 0

0.2

510

7

0.07

1000

0.45

440

8

0.08

800

0.2

478

9

0.09

800

0.6

474

10

0.12

1000

0.8

58 2

11

0.14

BOO

0.5

400

12

0.15

1000

0.3

590

13

0.17

880

19.0

454

14

0.17

800

0.5

467

15

0.19

800

21.0

384

0.49

16

0.20

1000

0.5

700

0.70

17

0.23

1000

0.4

510

18

0.26

1000

1.5

672

19

0.27

800

0.9

512

20

0.27

800

1.0

21

0.07

800

0.36

0.63 375

0.55 375 380

0 . 1 h-'.

0.59 0.58 meta-

0.50 0.59 0.50

375

0.58

375

0.51 0.67 0.64

450

410

0.56

610

s m a l l meta

0.76

9/11 and n a p h t h a l e n e because o f t h e e x t r e -

The d i l u t i o n r a t e was e q u a l t o

T h i s i s t h e c r i t i c a l f l o w f o r N o c a r d i a sp.,

i s u t i l i z e d moat e f f i c i e n t l y ;

0.44 0.60

375

t h i s i s the calculated concentration,

mely poor s o l u b i l i t y o f naphthalene).

0.63 ortho-

The m i x e d a u b s t r a t e c o n t a i n e d p h e n o l ( 0 . 5

( 2 g/l);

0.49

that culture didn't

a t which phenol

o x i d i z e t h e naph-

thalene. F o r t h e e x p e r i m e n t we u s e d Pseudomonas ap.

naphthalene.

adapted t o u t i l i z e

The a d d i t i o n o f p h e n o l t o t h e n a p h t h a l e n e u t i l i z i n g

c u l t u r e r e s u l t e d b o t h i n t h e i n t e n s i v e brown c o l o r appearance i n t h e culture,

a n d an i n c r e a s e d r e s i d u a l p h e n o l c o n c e n t r a t i o n .

-

160

-

The m i x e d c u l t u r e was a b s o l u t e l y c o l o r l e s s ,

but the residual

c o n c e n t r a t i o n s o f b o t h p h e n o l and n a p h t h a l e n e were l e s s t h a n 0.2 mg/l.

I t s h o u l d be s t r e s s e d ,

t h a t N o c a r d i a sp.

successfully u t i l i z e d

phenol a t r e l a t i v e l y h i g h i n p u t concentrations o f the former,

with rather great d i l u t i o n rates, The No.

and

mixed c u l t u r e .

2 m e t h o d was i l l u s t r a t e d w i t h t h e f o l l o w i n g e x p e r i m e n t .

The n a p h t h a l e n e o x i d i z i n g c u l t u r e ,

i s o l a t e d from wastewater d i s c h a r -

ged from p h e n o l i c w a t e r b i o l o g i c a l t r e a t m e n t p l a n t , i n t o a n a e r o t a n k i n a s p e c i f i c way.

As s r e s u l t ,

d u a l p h e n o l c o n c e n t r a t i o n d e c r e a s e d a s much a s 30

was i n o c u l a t e d

t h e average r e s i -

X,

b u t the process

s t a b i l i t y o f a sewage b i o l o g i c a l t r e a t m e n t e n h a n c e d ( t h e v a r i a t i o n c o e f f i c i e n t d e c r e a s e d 2.8

4

times).

8

These d a t a s r e shown i n F i g .

12

4.

20

16

MONTMS

F i g . 4. D y n a m i c s o f p h e n o l a t t h e o u t p u t o f b i o l o g i c a l p u r i f i c a t i o n o f wastewater i n coke and b y - p r o d u c t p l a n t . Arrow i n d i c a t e sowing o f nsphth a l e n e - o x i d i z i n g b a c t e r i a

Thus,

t h e e x p e r i m e n t s c a r r i e d o u t show t h a t t h e c h a n g e s i n t h e

q u a l i t y a n d number c o m p o s i t i o n o f a m i c r o b i a l c o m m u n i t y , n a l l y s t a b l e c o n t i n u o u s c u l t u r e have a p o s i t i v e e f f e c t , community r e a r r a n g e .

with functioi.e.

make t h e

-

161

-

Now, w e ’ l l d i s c u s s t h e s e i n e x p l i c a b l e f e a t u r e s m e n t i o n e d a b o v e i n t h e s i n g l e species c u l t u r e behaviour. DlSCUSSION

Both b i o c h e m i s t r y and g e n e t i c s of t h e m i c r o b i a l o x i d a t i o n o f

it is

a r o m a t i c h y d r o c a r b o n s are r a t h e r well s t u d i e d . N e v e r t h e l e s s ,

s t r a n g e , t h a t t h e e v i d e n c e on t h e c a t a b o l i s m m u l t i r o u t i n e s s are n o t i n c l u d e d i n t o w a s t e w a t e r t r e a t m e n t , when c o n s i d e r e d a t t h e p o p u l a t i o n and community l e v e l s .

T h i s e v i d e n c e m a k e s us d i s c u s s t h e p r o -

blem of a m u l t i r o u t i n e s s . Jones e t a l . I 2 1 found t h a t t h e r e g i o n o f m i c r o b i a l growth lim i t a t i o n a s w e l l a s i t s i n h i b i t i o n b y p h e n o l h a d t h e i r s p e c i f i c economic c o e f f i c i e n t s . These r e g i o n s a r e n ’ t o f any i n t e r e s t and have n o g e n e r a l b o u n d a r y , a s i t i s shown i n F i g . 5 [2, 3 1 . Assume t h e g r o w t h r a t e d e p e n d e n c e on s u b s t r a t e c o n c e n t r a t i o n b e a s s h o w n i n F i g . 6 . Q u i t e a few r e s e a r c h e r s s u p p o r t t h i s a s s u m p t i o n , T h e maximum o f regions.

p ( S ) belong t o both t h e l i m i t a t i o n , and i n h i b i t i o n

There is no corresponding p o i n t for i t i n Fig.

5, or a

t r a n s i t i o n a r e a from l i n e 1 t o l i n e 2 . Hence, t h e b a s i c assumption presented a t Figs.

5, 6 should be changed.

The p o s s i b l e c h a n g e f o r

i t i s shown i n F i g s . 5 a n d 6 i n d a s h l i n e s .

0

1

2

5

3

6

7 1l p

f h-’I

F i g . 5. R e c i p r o c a l p l o t o f g r o w t h y i e l d a n d g r o w t h r a t e w i t h p h e n o l oxidizing b a c t e r i a (from /3.2/). 1 limitation, 2 inhibition, 3 h y p o t h e t i c a l l i n e o f p a s s a g e from l i m i t a t i o n t o i n h i b i t i o n

-

-

-

- 162

-

Taking i n t o account k i n e t i c features o f t h e aromatic hydrocarone c a n e a s i l y show t h e e x i s t e n c e o f t h e a r e a o f t h e

bons o x i d a t i o n , bi-stability i n Fig.

i n behaviour.

I t ’ s evident,

i n part,

f r o m o u r d a t a shown

3 a n d T a b l e 2.

P o s i t i o n 7 i n T a b l e 2 shows t h e r e s u l t s t o b e s t a t i s t i c a l l y d i f f e r e n t from t h o s e p r e s e n t e d i n p o s i t i o n s 1-6. meta-cleavage

o f t h e c a t e c h o l wasn’t

s i t i o n 7 v s p o s i t i o n s 1-6

The c o m p a r i s o n o f

shows t h a t m e t a - c l e a v a g e

l e s s output phenol concentration; p o s i t i o n 21,

observed.

I n t h i s case t h e po-

pathway p r o v i d e s

t h e aame shows t h e e v i d e n c e s i n

obtained from another s e r i e s o f long-time

experiments.

W

t-

a

n

SUBSTRATE

F i g . 6. S p e c i f i c growth r a t e vs. s u b s t r a t e c o n c e n t r a t i o n (hypothet i c a l curves; f o r example, 1 meta-cleavage and 2 - o r t h o - c l e a v a g e pathways o f phenol, dash l i n e - o t h e r h y p o t h e s i s )

-

O n t h e o t h e r hand, p o s i t i o n s 1 3 - 1 6 d o n ’ t 2,3-oxygenase way)

(which i s the f i r s t -

activity

o f r e g a r d l e s s s i g n i f i c a n t v a r i a t i o n s i n t h e o u t p u t p h e n o l con-

centration.

Probably,

enzymes ( n a m e l y ,

2,3-

we j u s t o b s e r v e d a v a r i a t i o n o f t h e d i f f e r e n t and 1,Z-oxygenase)

complete e l i m i n a t i o n of ta, Fig.

show t h e d e c a y o f

s t e p enzyme o f m e t a - p a t h -

activities,

t h e c a t a b o l i c pathway.

contrary t o

Our e x p e r i m e n t a l d a -

unfortunately, f a i l s t o prove i t . One c a n e a s i l y d e s c r i b e t h e a r e a s o f m u l t i - s t a b l e

2 ( e a r d i a spe.)

and F i g .

3 (Pseudomonas s p . ) .

behaviour i n

Genetic features

o f m i c r o o r g a n i s m s used t o degrade t h e a r o m a t i c h y d r o c a r b o n s have resulted i n the following. t u r e changed meta-pathway

The p h e n o l o x i d i z i n g Pseudornonas sp. f o r ortho-pathway

shown i n p o s i t i o n 2 1 o f T a b l e 2.

i n some t i m e ;

cul-

that’s

When o x i d i z i n g n a p h t h a l e n e ,

we’ve

-

163

-

i n o c u l a t r e d t h e clones showing a g r e a t a c t i v i t y o f i n t o a culture,

b u t ortho-pathway

2,3-oxygenase

h a s b e g u n t o d o m i n a t e soon.

The d e l a y o f t r a n s i t i o n f r o m m e t e - c l e a v a g e

i n t o the orthoclea-

vage p a t h w a y i s e v i d e n c e o f t h e i n t e r a c t i o n b e t w e e n t h e c e l l s m i c r o b i a l p o p u l a t i o n and s u b s t r a t e b i o d e g r a d a t i o n p l a s m i d s , t h e enzyme a c t i v i t i e s t u r n o u t . e x p e r i m e n t s 1-7,

rather than

T h i s l a t t e r c o u l d be observed i n

T a b l e 2 , t h a t were c a r r i e d o u t as a p i l o t e x p e r i -

ment. Hence,

i t i s evident,

t h a t some f e a t u r e s o f a s u b s t r a t e c a t a -

b o l i s m ( a t i n t r e c e l l u l a r l e v e l ) h a v e a s t r o n g i m p a c t on t h e w a s t e w a t e r p r o c e s s i n g on t h e w h o l e .

REFERENCES

1 2 3

M a n u a l o f M e t h o d s f o r G e n e r a l B a c t e r i o l o g y . P. G e r h a r d t e.a. ed., W a s h i n g t o n 1 9 8 1 . G.L. J o n e s , F. J a n s e n and A . J . McKay, S u b s t r a t e I n h i b i t i o n o f t h e g r o w t h o f b a c t e r i u m NCIB 8250 b y p h e n o l . L . Gen. M i c r o b i o l . , 7 4 ( 1 9 7 3 ) N I , p. 139-148. Yu.L. G u r e v i c h , S t a b i l i t y a n d R e g u l a t i o n o f G r o w t h i n M i c r o b i a l P o p u l a t i o n s . N o v o s i b i r s k , Nauka, 1 9 8 4 , 165 p .

This Page Intentionally Left TBlank

SPECIFIC A D S O R P T I O N O F METAL C A T I O N S ON THE SURFACE OF L I P I D MEMBRANE S Y S T E M S A.M.

OMEL'CHENKO

I n s t i t u t e o f Chemical Technology, Department o f I n o r g a n i c C h e m i s t r y , G a g a r i n a v . , 8 , D n e p r o p e t r o v s k , 320640, USSR INTRODUCTION

S t u d i e s o f t h e m e t a l c a t i o n s b i n d i n g w i t h a r t i f i c i a l l i p i d membrane systems a r e o f s p e c i a l i n t e r e s t i n c o n n e c t i o n w i t h g r e a t phys i o l o g i c a l r o l e o f t h i s cations. life",

Some o f t h e s e s o - c a l l e d " m e t a l s o f

a r e necessary f o r t h e f u n c t i o n o f l i v i n g organisms.

t a l s are t o x i c a l .

O t h e r me-

I n t h e common c a s e t h e r e i s n o t a s t r i c t b o r d e r

between t o x i c a l and u n t o x i c a l m e t a l s because t h e l a r g e c o n c e n t r a t i o n i n o r g a n i s m s some u n t o x i c a l m e t a l s c a n l e a d t o a number o f d i seases.

I n t h i s c o n n e c t i o n t h e e c o l o g i c a l aspect o f t h e m e t a l ca-

t i o n s b i n d i n g w i t h l i p i d membranes i s a t t r a c t i n g s p e c i a l a t t e n t i o n . The b i l s y e r l i p i d membranes (BLMa)

s e p a r a t i n g t w o a q u e o u s so-

l u t i o n s h a v e b e e n m a i n l y u s e d a s a m o d e l o f b i o l o g i c a l membranes. We h a v e c a r r i e d o u t some e x p e r i m e n t a l s t u d i e s o f t h e i n f l u e n c e o f

m e t a l c a t i o n s such as a l k a l i e a r t h c a t i o n s , o f aluminium,

c a t i o n s o f t h e subgroup

f i r s t row t r a n s i t i o n m e t a l c a t i o n s ,

a n d cadmium o n e l e c t r o c o n d u c t a n c e ,

lanthanides,

zink

e l e c t r o c a p a c i t y and d i f f e r e n c e o f

membrane s u r f a c e p o t e n t i a l s o f t h e BLMs. MATERIALS AND METHODS F o r t h e f o r m a t i o n o f t h e BLMs we u s e d t h e c o n v e n t i o n a l m e t h o d worked o u t i n 1961 by M u e l l e r , Rudin, different

T i e n a n d W e a c o t t (1). T h r e e

t y p e s o f ELM s y s t e m a were u s e d :

from bovine b r a i n l i p i d s ,

f r o m egg p h o s p h a t i d y l c h o l i n e a n d f r o m p h o s p h a t i d y l s e r i n e .

I n a l l ca-

s e s t h e B L M s - f o r m i n g s o l u t i o n c o n s i s t e d o f 20 mg/ml c h o l e s t e r o l d i s s o l v e d i n n - h e p t a n e or i n n - o c t a n e . The membrane e l e c t r o c o n d u c t a n c e waa d e t e r m i n e d f r o m Ohm'a

law.

The e l e c t r o c e p a c i t y o f t h e BLMa waa i n v e s t i g a t e d by t h e c y c l i c v o l tammetry method a t l i n e a r v a r i a b l e v o l t a g e with a v e l o c i t y

Of

2

mV/S*

-

166

-

The d i f f e r e n c e o f membrane s u r f a c e p o t e n t i a l s was d e t e r m i n e d b y mea s u r i n g t h e second h a r m o n i c s o f a l t e r i n g c u r r e n t s .

The b a t h i n g e -

l e c t r o l y t e c o n t a i n e d 0 . 0 1 M a q u e o u s K C 1 b u f f e r e d b y 0.002 a c e t a t e o r 0 . 1 M aqueous K C 1 b u f f e r e d by 0.02

4.7-4.9).

M sodium

M s o d i u m a c e t a t e (pH

A l l e x p e r i m e n t s were p e r f o r m e d a t t h e t e m p e r a t u r e 20-22

OC.

RESULTS

I t was shown t h a t i n a d d i t i o n t o b a t h i n g t h e e l e c t r o l y t e i n 10-4-10-2

M M 2 + - s a l t s (M2+

-

mark f o r d i v a l e n t c a t i o n s )

c r e a s e i n t h e b o v i n e b r a i n l i p i d s BLM’s

i n d u c e d de-

X (in

c o n d u c t i v i t y on 1 0 - 2 0

average) b u t e l e c t r o c a p a c i t y i n c r e a s e d from 15-25

X

with small d i f f e -

r e n c e f o r v a r i o u s c a t i o n s . The maximum e f f e c t h a s b e e n o b s e r v e d f o r 2+ Cu T h i s m o d i f i c a t i o n i s t e s t i n g t h e c o n d e n s a t i o n o f t h e membranes

.

s t r u c t u r e and t h e i r t h i c k n e s s d e c r e a s e .

The d i v a l e n t c a t i o n s r e d u c e

the s o l u b i l i t y o f hydrocarbons i n t h e l i p i d r e g i o n o f bovine b r a i n l i p i d membranes.

The a b u n d a n c e o f h y d r o c a r b o n e s o l v e n t i s e x t r u d e d

i n t o t h e t o r u s and m i c r o l e n s e s u p o n a c t i o n o f t h e d i v a l e n t c a t i o n s . C o n s i d e r a b l e i n c r e a s e was shown o f t h e e l e c t r o m e c h a n i c a l s t a b i l i t y o f BLMs i n t h e p r e s e n c e o f t h e d i v a l e n t c a t i o n s .

The e l e c t r o m e c h a n i -

c a l s t a b i l i t y o f BLMs wss d e t e r m i n e d b y m e a s u r i n g t h e b r e a k d o w n v o l -

t age. Consequently, t h e a d d i t i o n o f 10-5-10-3 mon mark f o r t r i v a l e n t m e t a l c a t i o n s )

M M 3 + - s a l t s (M3+

o f c o n d u c t i v i t y o f t h e BLMs b u t t h e n an a b r u p t v i t y was o b s e r v e d .

com-

increase o f conducti-

The minimum c o n d u c t i v i t y o f BLM’s

a t s Concentration o f about

-

induced a t f i r s t a decrease

M M3+-salts.

has been found

The e l e c t r o m e c h a n i c a l

M

s t a b i l i t y o f t h e BLMs h a d a maximum c o n c e n t r a t i o n o f a b o u t M3+-salts also.

A t l o w c o n c e n t r a t i o n s t h e t r i v a l e n t c a t i o n s were

acting s i m i l a r l y t o divalent cations but a t high concentrations the t r i v a l e n t c a t i o n s were

l e a d i n g t o l a t e r a l phase s e p a r a t i o n and t o

t h e f o r m a t i o n o f i o n - c o n d u c t i n g d e f e c t s o f t h e BLM’s

structure.

The b i n d i n g o f t h e m e t a l c a t i o n s c a u s e d a s h i f t o f BLM’s face p o t e n t i a l t o p o s i t i v e r e g i o n .

sur-

T h i s was s u g g e s t e d by t h e c h a n g e

o f d i f f e r e n c e o f membrane s u r f a c e p o t e n t i a l s i n t h e p r e s e n c e o f t h e c a t i o n s w h i c h were m e a s u r e d b y t h e p o t e n t i o d y n a m i c m e t h o d . were a d d e d t o one s i d e o f t h e BLMs.

The s a l t s

The b i n d i n g o f t h e c a t i o n s was

r e v e r s i b l e a n d was r e m o v e d u p o n t h e a d d i t i o n o f E D T A .

E D T A was a d d e d

as t h e disodium s a l t d i s s o l v e d i n c o r r e s p o n d i n g e l e c t r o l y t e . The d i f f e r e n c e

o f surface p o t e n t i a l s o f l i p i d b i l a y e r s from

b o v i n e b r a i n l i p i d s on dependence f r o m m e t a l s a l t s c o n c e n t r a t i o n h a s

-

167

-

5 I

b

E -

-s-

80-

a

201

I

-3

-2

lg CIMI

1

E

E

3-

80

a 80

60

60

LO

40

20

20

-2

-3

-

Lg CIMl

> E

4

lg C[Ml

I

I

a

I.

-

-> s

-2

-3

110

100

80 60 -5

-4

Lg CIMl

-5

-4

-3

ig C[MI

F i g . 1. Dependence o f t h e d i f f e r e n c e o f b o r d e r p o t e n t i a l s o f ELMS f r o m b o v i n e b r a i n l i p i d s on t h e m e t a l l i c cation’s concentration. E l e c t r o l y t e : 0 . 0 1 M KC1+0.002 M a c e t a t e b u f f e r . E a c h p o i n t shows t h e a v e r a g e v a l u e f r o m 4-5 e x p e r i m e n t s . On F i g “ c ” a n d I‘d‘* f o r c u r v e s 2 e l e c t r o l y t e : 0’1 M K C 1 + 0.02 M a c e t a t e b u f f e r . C a t i o n s : Mg2+, .Ca2+, S r 2 + ’ CI Ba2+’ R Co2+, rMn2+, X Ni2+’ re2+’ Q Cu2+’ @Zn2+, Cd2+, fl A l 3 + , ~ p r 3 + ,8 ~ a 3 + , o ~ d 3 + , 0 Cr3+, OGa3+, In3+.

d+,

a)

b e e n shown i n F i g .

168

-

1. S i m i l a r e x p e r i m e n t s h a v e b e e n c a r r i e d o u t

f o r BLMs f r o m egg p h o s p h a t i d y l c h o l i n e a n d f r o m p h o s p h a t i d y l s e r i n e .

I t was d e m o n s t r a t e d t h a t t h e b i n d i n g o f t h e m e t a l c a t i o n s w i t h BLMs decrease i n t h e o r d e r :

-

f o r BLMs f r o m e g g p h o s p h a t i d y l c h o l i n e ( a t any i o n i c s t r e n g h of electrolyte)

-

> Be 2+

(I

Ca2+ > Mg2+

Sr2+

A13+

> La3+ >

P r 3 + > Nd3+ > Tb3+

( 1 group)

re2+

Mn2+

z

Co2+ > N i 2+

(11 g r o u p )

Cd2+ > Cu2+

z

Zn

for

2+

group)

(111 g r o u p )

BLMs f r o m p h o s p h a t i d y l s e r i n e ( a t any i o n i c s t r e n g h o f

electrolyte) Be2+

Ca2+

S r 2+ > Mg2+

A 1 3 + > Tb3+ > P r 3 + > Nd3+ > L a 3 + N i 2 + > Co2+ > F e z + > Mn 2+ Cu2+ > Zn2+ > Cd2+

-

M KC1 buffered

M sodium a c e t a t e )

Ba2+ > S r 2 + > Ca2+ > Mg2+

A13+

( 1 group)

(11 g r o u p ) (111 group)

f o r BLMs f r o m b o v i n e b r a i n l i p i d s ( 0 . 0 1 by 0,002

( 1 group)

z Pr3+ z

Tb3+

5

Nd3+ > L a 3 +

(1 group) (Ig r o u p )

N i 2 + > M n 2 + > Co2+ > Fe 2+

( I 1 group)

Cu2+ > Zn2+ > Cd2+

(111 g r o u p )

F o r BLMs f r o m b o v i n e b r a i n l i p i d s t h e i n c r e a s e o f i o n i c s t r e n g h (0,l

M K C 1 b u f f e r e d b y 0.02

M sodium a c e t a t e )

c a u s e d some a l t e r a -

t i o n s a s c o m p a r e d w i t h t h e above c o n s e q u e n c e s :

2+

Mn2+ > N i 2 + > Co Cu2+ Cd2+ > Zn 2+

( I 1 group) (IIIg r o u p )

I n a l l cases t h e appearance o f t h e above consequences depends

on t h e k i n d o f l i p i d s a n d d o e s n o t d e p e n d o n t h e m e t a l c a t i o n s c o n c e n t r a t i o n i n a s u f f i c i e n t l y wide c o n c e n t r a t i o n r e g i o n .

I n these

c o n s e q u e n c e s t h e m e t a l c a t i o n s were d i s t r i b u t e d i n t o t h r e e , c l a s s i f i c a t i o n groups according t o t h e i r covalent c h a r a c t e r i s t i c s .

The

p r i n c i p l e s o f c a t i o n s c l a s s i f i c a t i o n i a discussed below.

DISCUSSION We c a r r i e d o u t t h e p r o f o u n d a n a l y s i s o f p u b l i s h e d s c i e n t i f i c p a p e r s d e a l i n g w i t h t h e b i n d i n g o f m e t a l c a t i o n s w i t h l i p i d membrane

systems (liposomes,

mono-

169

-

and b i l a y e r s ) .

h e r w i t h o u r own e x p e r i m e n t a l r e s u l t s ,

The r e s u l t i n g d a t a ,

toget-

demonstrated the i n e q u a l i t y

o f adsorption properties o f metal cations.

T h i s i n e q u a l i t y i s usu-

a l y p r e s e n t e d as a c o n s e q u e n c e o f m e t a l c a t i o n s a n d d e a l s w i t h p h y sico-chemical

p r o p e r t i e s o f b o t h m e t a l c a t i o n s and i o n i c g r o u p s o f

l i p i d molecules.

The l i p i d m o l e c u l e s ,

f o r t h e f o r m a t i o n o f BLMs, phosphate group - P o i , ternary -N(CH

3

which have been used m a i n l y

c o n t a i n t h e w e l l known i o n i c g r o u p s :

c a r b o x y l g r o u p -COO-,

+

p r i m a r y -NH3

and q u a r -

) + amine g r o u p s i n d i f f e r e n t c o n f o r m a t i o n s a n d c o m b i -

n a t i o n s f o r each t y p e o f l i p i d .

The a n a l y s i s o f s c i e n t i f i c

litera-

t u r e d e m o n s t r a t e d t h a t t h e c o n s e q u e n c e s o f some t y p e m e t a l c a t i o n s r e p r o d u c e w e l l u n l i k e t h e q u a n t i t i v y d a t a on m e t a l c a t i o n s a d s o r p t i o n a t membrane s u r f a c e .

U s i n g t h e consequences o f m e t a l c a t i o n s

a f f i n i t y t o s u r f a c e o f BLMs we c a n e s t i m a t e t h e p h y s i c o c h e m i c a l nat u r e o f t h e membrane b i n d i n g s i t e s .

F o r t h i s p u r p o s e t h e conaequen-

c e s must i n c l u d e o n l y c a t i o n s h a v i n g c o m p a r a b l e p r o p e r t i e s . I n t h i s connection,

t h e m e t a l c a t i o n s were d i s t r i b u t e d i n t o

t h r e e c l a s s i f i c a t i o n groups a c c o r d i n g t o t h e i r c o v a l e n t c h a r a c t e r i s t i c s (Table 1).

The c o v a l e n t c h a r a c t e r i s t i c s

o f the metal cations

a r e d i f f e r e n t b e t w e e n t h e summary i o n i z a t i o n e n e r g y o f t h e m e t a l

TABLE 1 C l a s s i f i c a t i o n o f t h e m e t a l c a t i o n s on t h e b a s i s o f t h e i r c o v a l e n t characteristics

I Li+,

Na+,

K+,

Cu+, Ag+,

Rb+ Mn2+,

Fe2+,

(Mg2+),

( a b o u t 125 k J / m o l )

Co2+,

(Mg2+)

(300-380 kJ/mol)

y3+,

T1+

( a b o u t 250 k J / m o l )

Cs+ ( a b o u t 85 k J / m o l )

Ca2+, S r 2 + , Be2+

~ n ~ s+c 3, + ,

I11 group

I 1 group

I group

Ni2+

Cu2+, Zn2+, Pb2+,

Cd2+,

5n2+ (more t h a n

560 k J / m o l )

ea3+,

I ~ ~c r+ 3 + ,,

Fe3+, A13+

T13+

(160-590 kJ/mol)

1000 kJ/mol)

(more t h a n

atoms a n d h y d r a t i o n e n e r g y o f c a t i o n s . T h e s e q u a n t i t i e s c h a r a c t e r i z e t h e a b i l i t y o f m e t a l c a t i o n s t o form a c h e m i c a l c o v a l e n t bond.

A l l c o r r e l a t i o n s b e t w e e n c h e m i c a l p r o p e r t i e s a n d membrane a c t i v i t y o f t h e m e t a l c a t i o n s may f u l f i l o n l y one a n d t h e same c l a s s i f i c a t i o n group.

-

170

-

Summary on t h e b a s i s o f a n a l y s i s o f p u b l i s h e d r e s u l t s d e t e r m i ned such consequences:

-

f o r membranes f o r m e d f r o m z w i t t e r i o n i c l i p i d s a n d f r o m a c i -

d i c l i p i d s w h i c h c o n t a i n one a n i o n i c g r o u p p e r m o l e c u l e ( e g g p h o s p h a -

dipalmitoylphosphatidylcholine, phosphatidylglycerol, phosphatidylglycerol + phosphatidylethanolamine): tidylcholine,

phosphatidylcholine +phosphatidic acid,

Na+ > K+

Li+

Ca2+ > Mg2+

Rb+

Sr2+

z

Mn2+ > C o 2 + > N i

Cs

+

Ba 2+

2+

Cd2+ > Cu2+ > Zn

Pb2+

-

5

2+

f o r membranes f o r m e d f r o m a c i d i c l i p i d s w h i c h c o n t a i n t w o

a n i o n i c groups per molecule (phosphatidylserine L i + > Na+ > K+ > Rb+ > Cs Ca2+ > Be2+ > S r 2+ > Mg2+

and c a r d i o l i p i n ) :

+

2+

€la2+ > Ca2+ > S r > Mg2+ €la2+ > S r 2 + > Ca 2+ > Mg2+

N i 2 + > C o 2 + > Mn 2n2+

2+

(only

f o r phosphatidylserine)

(only f o r phosphatidylserine)

Cd2+

>

F o r e s t i m a t i o n o f t h e p h y s i c a l and c h e m i c a l n a t u r e o f b i n d i n g s i t e s on t h e s u r f a c e o f BLMs t h e a l t e r a t i o n o f f r e e e n e r g y

A FiB

f o r i o n exchanged r e a c t i o n

+

A"+

AXn

BXn

+

Bn+

had been c a l c u l a t e d by e q u a t i o n 1

where

AU An+

-

,

AU

X

Bn+

-

are the energies o f e l e c t r o s t a t i c in-

X

t e r a c t i o n o f c a t i o n s A a n d B w i t h a d s o r p t i o n s i t e nX-,

AU An+

-

9

H20

AU B"+

A and 8 , r e s p e c t i v e l y ,

-

respectively,

are the hydration energies o f cations

H20 X i s the anionic centre.

T h i s model i s based

on t h e p u r e l y c o u l o m b i c i n t e r a c t i o n b e t w e e n a n i o n s f o r m i n g t h e b i n d i n g s i t e and t h e m e t a l c a t i o n s .

I t waa shown b y E i a e n m a n ( 2 ) t h a t t h e number o f c o n s e q u e n c e s o f t h e m o n o v a l e n t c a t i o n s a f f i n i t y t o t h e s u r f a c e o f an i o n - e x c h a n g e d

-

171

-

m a t e r i a l as f u n c t i o n o f a n i o n i c r a d i u s r- i s independent f r o m d i s tance d between a n i o n i c c e n t r e s .

However,

i n t h e case o f p o l y v a l e n t

c a t i o n s t h e d i s t a n c e between a n i o n i c c e n t r e s i s e x e r t i n g t h e c r i t i c a l i n f l u e n c e on t h e number o f c a t i o n s c o n s e q u e n c e s .

We s u p p o s e d

that polyvalent cation i s interacting with binding s i t e t h a t i s vieeach w i t h a r a d i u s

wed as c o n s i s t i n g o f t w o a n i o n s , by a distance d (Fig.

r- separated

2).

F i g . 2 . Scheme f o r c a l c u l a t i o n o f e l e c t r o s t a t i c i n t e r a c t i o n o f divalent cation with anionic centres. The e n e r g y o f e l e c t r o s t a t i c i n t e r a c t i o n ( w i t h o u t c a l c u l a t i o n o f t h e i n t e r a c t i o n between a n i o n i c c e n t r e s )

where N A

-

Avogadro's

r i c constant,

-

r+

number,

e

-

cation radius.

a t d > 2r+ i s

e l e c t r o n i c charge,

Lo

-

dielect-

Eq. 2 gives

A t d Q 2r+

n

N~ e L AU

An+ ( B n+) - X

= - -

( + z & ~r

1

+ r-

I n Eqns 2 a n d 3 t h e v a l u e s o f d, and t h e v a l u e s o f Fig.

1

+ 4(d

+ 2r-

1

. lo6

(3)

r + a n d r- a r e s u b s t i t u t i n g i n n m

A U a r e o b t a i n i n g i n kJ/mol.

3 shows t h e i s o t h e r m s o f a l k a l i e a r t h c a t i o n s p e c i f i c i t y

f o r t h e ion-exchanged m a t e r i a l as a f u n c t i o n o f a n i o n i c r a d i u s a t d

=

0-0.160

nm.

V a r i o u s consequences o f a l k a l i e a r t h c a t i o n s c a l c u -

l a t e d f r o m E q n s 1, 2 a n d 3 a r e shown i n T a b l e 2 .

Analogous conse-

q u e n c e s f o r m e t a l c a t i o n s o f a l l c l a s s i f i c a t i o n g r o u p s h a v e b e e n obtained. The a f f i n i t y c o n s e q u e n c e s f o r t h e d i v a l e n t c a t i o n s o f any g r o u p s we o b t a i n e d h e r e f o r t h e membranes f o r m e d f r o m z w i t t e r i o n i c

lipids

a n d f r o m a c i d i c l i p i d s w h i c h c o n t a i n one a n i o n i c g r o u p p e r m o l e c u l e

Table 2 . Consequencee of the cation s p e c i f i c i t y f o r the a l k a l i earth metals (

No

I

I

e2+, Ca2+, Sr2+,

Be2+)

Distances between anionic centres d (Anionic radius), m

Consequencee L

= 0

- 0,1601

d= 0,200

1

d= 0,240

1

d= 0,280

I

1 d= 0 , 3 2 0

- d o go

~~~~

I Ba2% sr2+> Ca2% mg2+ +(more 0,163 +(more 0,163) +(More 0,146) +(more 0,036, +(more 0,043) +(0 160 11 ~ a 2 + ca2+s > Sr2+> +(0 160 0,163) 0,163) +(O 150 +(0 150 0,1609 0,160) +(0,138 +(0,092

-

-

0,150) +(0,134 0,138) +(0,120 -

0,150) +(0,076 0,092) +(0,030 -

a2+

-

+(0,032 0,048) +(0,025

-

0,032) 0,134) 0,076) +(less 0,12O)+(less 0,030) + ( l e s s 0,025)

-

-

-

-

-

-

-

+ ( l e s s 0,026) +(0,036 0,038)

+(more 0,078) +(0,074 0,078) +(0,072 0,074) +(0,071 0,072) +(0,070 0,071) +(0,066 0,070) + ( l e s s 0,066)

-

-

-

+(0,025

-

0,036) +(less 0,025)

-

-

-

-

173

-

[nml

F i g . 3 . Dependence o f a l k a l i e a r t h c a t i o n s p e c i f i c i t y o f i o n e x c h a n g e d m a t e r i a l on a n i o n i c r a d i u s r a t d=O 0 , 1 6 0 nm.

-

a r e e x p l a i n e d on t h e b a s i s o f c a l c u l a t i o n i f t h e b i n d i n g s i t e i s c o n s i d e r e d t o b e f o r m e d b y t w o a n i o n s w i t h c h a r g e -1 a n d s e t t i n g

r-=0.07-0.09

nm w i t h d - 0 . 2 4 0 - 0 . 3 2 0

metal cations belonging t o

1

nm.

For the binding o f t r i v a l e n t

g r o u p t h e b i n d i n g s i t e t o be f o r m e d b y

t w o u n i o n s w i t h c h a r g e -1 a n d r - = 0 . 0 9 5 - 0 . 1 1 0

nm, d = 0 , 2 0 0

nm.

The

decrease o f d p r o b a b l y d e a l t w i t h t h e i n f l u e n c e o f t h e t r i v a l e n t cat i o n s on c o n f o r m a t i o n o f t h e l i p i d p o l a r g r o u p s .

A t the i n t e r a c t i o n o f the p o l y v a l e n t metal c a t i o n s o f group I w i t h membranes f o r m e d f r o m a c i d i c l i p i d s w h i c h a r e c o n t a i n i n g t w o a n i o n i c groups per molecule,

t h e a f f i n i t y consequences can be e x p l a -

i n e d i f d approaches z e r o ( a d i v a l e n t anion)

w i t h r-=0.150

b i n d i n g o f t h e d i v a l e n t c a t i o n s and r-=0.085-0.095

nm f o r

nm f o r b i n d i n g o f

the t r i v a l e n t cations. The a n a l y s i s o f t h e i n t e r a c t i o n o f t h e p o l y v a l e n t m e t a l c a t i o n s b e l o n g i n g t o g r o u p s I 1 a n d I11 w i t h p h o s p h a t i d y l s e r i n e membranes obtained unusually small values of

anionic radius.

T h i s f a c t shows

t h a t t h e i n t e r a c t i o n o f t h e c a t i o n s b e l o n g i n g t o g r o u p s I 1 and I11 w i t h p h o s p h a t i d y l s e r i n e membranes r e a l i z e s a n o t h e r t y p e o f c h e m i c a l bonds t h a n t h a t i n t h e caae o f t h e i n t e r a c t i o n between t h e c a t i o n s belonging t o group

I.

-

174

-

= -11-

0-

0 -10-

0

d

-9-8-7-6-5-4-

-3-2-1 I

1 2 3 4 5 6 7 8

b KO F i g . 4. C o r r e l a t i o n b e t w e e n m e t a l l i c c a t i o n ' s c o n s t a n t s ( K O ) 131 o f t h e b i n d i n g w i t h liposomes f r o m m i x t u r e p h o s p h a t i d y l c h o l i n e + phosp h a t i d i c a c i d a n d s o l u b i l i t y (5) o f m e t a l o r t h o p h o s p a h t e . The l i n e has been drawn u s i n g t h e c o r r e l a t i o n e q u a t i o n l o g S = -1.89-1.22*1og KO ( c o r r e l a t i o n c o e f f i c i e n t 0 . 9 2 ) .

\ Q

'u 10

a

0

d

(

64 -

8

d $9

2-

.2 v

-

175

-

On t h e b a s i s o f t h e c o r r e l a t i o n a n a l y s i s i t was ahown t h a t e x -

p e r i m e n t a l l y o b t a i n e d consequences a r e t y p i c a l i n t h e case o f phosphatidylcholine

+

p h o s p h a t i d i c a c i d membranes f o r t h e b i n d i n g o f t h e

m e t a l c a t i o n s w i t h phosphate groups (Fig.

4) and i n t h e case o f

p h o s p h a t i d y l s e r i n e membranes f o r t h e c h e l a t e b i n d i n g o f t h e m e t a l c a t i o n s w i t h c a r b o x y l a n d amine g r o u p s ( a t a d s o r p t i o n o f t h e t r a n s i t i o n metal cations,

Zn2+, Cd2+ a n d Pb2+ ( F i g .

5) o r f o r t h e e l e c -

t r o s t a t i c b i n d i n g w i t h phosphate and c a r b o x y l groupa ( a t a d s o r p t i o n o f the a l k a l i e a r t h cations,

l a n t h a n i d e s and aluminium).

REFERENCES

1 2

3

P . M u e l l e r , D.O. R u d i n , H.T. T i e n a n d W.C. W e s c o t t , N a t u r e . 1962. V o l . 1 9 4 NO 4832. P . 979-980. G. E i s e n m a n , B i o p h y s i c a l J. 1 9 6 2 . V o l 2. No 2. P a r t 2. p. 259323. P.G. B a r t o n , J. E i o l . Chem. 1 9 6 8 . V o l . 243. No 1 4 . p. 3884-3890

This Page Intentionally Left TBlank

T H E I N V E R S E FLUIDIZATION DESIGN,

L.

-

A NEW APPROACH T O BIOFILM R E A C T O R

T O AEROBIC W A S T E W A T E R T R E A T M E N T

NIKOLOV a n d D.

KARAMANEV

S o f i a U n i v e r s i t y , Center o f Biotechnology Tsankov S t r . , 1421 S o f i a , B u l g a r i a

8 Dr.

The s u b s t a n t i a l a d v a n t a g e s o f f l u i d i z e d s y s t e m s s u c h a s e f f e c t i v e mixing, sons f o r

and i n t e n s i v e mass a n d h e a t t r a n s f e r

are the main rea-

t h e unabated i n t e r e s t i n f l u i d i z a t i o n from s p e c i a l i s t s i n

d i f f e r e n t f i e l d s d u r i n g t h e l a s t 3-4

d e c a d e s [I].

Among t h e v a r i o u s d i f f e r e n t modes o f f l u i d i z a t i o n ,

t h e most

s p r e a d o n e s a r e t h e w e l l known u p f l o w s y s t e m s c o n s i t i n g o f g a s - s o lid,

liquid-solid

or gas-liquid-solid

phases.

I n these systems,

a

b e d o f s o l i d p a r t i c l e s i s s u s p e n d e d i n a f l u i d m e d i a due t o t h e n e t drag force o f the f l u i d s flowing opposite t o the net g r a v i t a t i o n a l f o r c e on t h e p a r t i c l e s [ 1 1 .

The u p f l o w f l u i d i z e d b e d s a r e t h e s u b -

j e c t o f numerous i n v e s t i g a t i o n s and a p p l i c a t i o n s i n t h e f i e l d o f bioreactor design.

Moreover,

the popularity o f t h i s type o f f l u i d i -

z a t i o n i n b i o p r o c e s s e n g i n e e r i n g h a v e l e d t o i n t r o d u c t i o n o f a new term " b i o f l u i d i z a t i o n "

[ 21.

The m a i n a c h i e v e m e n t s o f b i o f l u i d i z a t i o n a r e l i n k e d w i t h t h e development o f u p f l o w b i o p r o c e s s systems w i t h spontaneously

fixed

b i o m a s s on s o l i d p a r t i c l e s o r i n o t h e r w o r d s w i t h b i o f i l m f l u i d i z e d bed r e a c t o r s [ 2 , 31.

A v e r y i m p o r t a n t f e a t u r e o f these systems i s

t h a t t h e d e n s i t y o f t h e b i o p a r t i c l e s i s h i g h e r t h a n t h e media d e n s i ty.

I n t h e s e t y p e s o f b i o r e a c t o r s moving upwards,media

bed o f b i o p a r t i c l e s

-

f l u i d i z e s the

support p a r t i c l e s covered w i t h b i o f i l m , pro-

v i d i n g good c o n d i t i o n s f o r t h e b i o p r o c e s s i n t e n s i f i c a t i o n .

However,

t h e p e c u l i a r i t i e s o f t h e b i o f i l m s I 4 1 make t h e a p p l i c a t i o n o f t h e upflow b i o f l u i d i z a t i o n inconvenient

f o r aerobic processes.

The c o l l i

s i o n s between t h e b i o p a r t i c l e s and t h e s t r o n g s h e a r s t r e s s p r o v o k e d b y t h e i n t e n s i v e h y d r o d y n a m i c s c a u s e some d i f f i c u l t i e s i n t h e s p o n taneous f i x a t i o n o f microorganisms o r c e l l s . t h e b i o f i l m i s formed once,

On t h e o t h e r h a n d ,

if

another very important problem a r i s e s

-

- 178 -

t h a t of biofilm thickness control. A l l t h i s confines the upflow fluidization a p p l i c a b i l i t y t o t h e comparatively narrow f i e l d of liquid-solid anaerobic bioprocess systems.

The s e a r c h f o r n e w o p p o r

t u n i t i e s t o expand t h e p o s s i b l e f i e l d s o f a p p l i c a t i o n o f b i o f l u i d i z a t i o n l e d t o t h e i n t r o d u c t i o n o f i n v e r s e f l u i d i z e d b e d s a s a new a p p r o a c h f o r h i g h p e r f o r m a n c e b i o f i l m r e a c t o r d e s i g n I51 ( F i g .

1).

I n t h i s case the bioparticles with a density lower than t h a t of t h e l i q u i d media a r e suspended i n t h i s media due t o t h e n e t d r a g f o r c e o f t h e f l u i d f l o w i n g d o w n w a r d s e .g .

J

f

o p p o s i t e t o t h e i r buoyancy

force. A t

a glance, one should find no consider a b l e difference between t h e upflow and t h e i n v e r s e (downflow) f l u i d i z e d beds.

However, t h e smaller p a r t i c l e

d e n s i t y c a u s e s a d e c r e a s e o f t h e for-

ce o f t h e c o l l i s i o n s b e t w e e n t h e p a r t i c l e s . I n t h e case o f t h r e e - p h a s e beds,

t h e gas holdup i n t h e inverse

f l u i d i z a t i o n i s much l a r g e r b e c a u s e of t h e countercurrent flow. The s e a r c h f o r a b i o r e a c t o r . F l g . 1. Scheme o f t h e u p f l o w (a) and i n v e r s e ( b ) f l u i d i -

zarqion.

which

could allow t h e control o f t h e biof i l m t h i c k n e s s h a s l e d t o t h e combination of the inverse

fluidization

I t gave

with the a i r l i f t principle.

a s a n o u t c o m e a new t y p e o f b i o f i l m reactor for aerobic processes

-

the

c a l l e d inverse fluidized bed biofilm reactor.

The scheme o f t h e i n v e r s e

f l u i d i z e d bed b i o f i l m r e a c t o r 15, i s shown i n F i g .

2.

61

The a i r i n t r o d u -

ced t o t h e d r a f t tube causes recircul a t i o n f o t h e l i q u i d media

-

upwards

i n t h e d r a f t t u b e and downwards i n t h e annulus.

A bed o f support p a r t i c l e s

w i t h a smaller d e n s i t y t h a n t h a t o f t h e l i q u i d media is placed i n t h e annulus.

*'

The d o w n f l o w i n q l i q u i d e x -

pands t h i s bed t h u s c r e a t i n g a l i q u i d Scheme

Of

the

s o l i d inverse f l u i d i z e d bed.

Initially,

-

179

-

t h e l o w e r l e v e l o f t h e bed i s w e l l above t h e l o w e r o p e n i n g o f t h e d r a f t tube.

The g r o w t h o f t h e b i o f i l m on t h e s u p p o r t p a r t i c l e s c a u -

s e d an i n c r e a s e o f t h e o v e r a l l b i o p a r t i c l e d e n s i t y .

This r e s u l t s i n

t h e e x p a n s i o n o f t h e i n v e r s e f l u i d i z e d b e d and t h e m o v i n g o f i t s l o w e r l e v e l downwards. tube opening,

When t h e b e d l e v e l r e a c h e s t h e l o w e r d r a f t

some o f t h e b i o p a r t i c l e s e n t e r t h e d r a f t t u b e t o g e t h e r The s t r o n g s h e a r s t r e s s i n t h e d r a f t t u b e

w i t h t h e l i q u i d stream.

causes p a r t i a l e r o s i o n o f t h e b i o f i l m and t h e r e f o r e , t h e b i o f i l m t h i c k n e s s and t h e b i o p a r t i c l e d e n s i t y .

bioparticles

a decrease o f

The l i g h t e n e d

move t o t h e t o p o f t h e i n v e r s e f l u i d i z e d b e d ,

t h e process o f b i o f i l m growth continues.

where

I n s u c h a way t h e b i o f i l m

thickness i s controlled. I n i t i a l l y t h i s b i o r e a c t o r was u s e d a s a t o o l f o r b i o f i l m p r o cess research. compared, tor,

The i n v e r s e f l u i d i z e d b e d b i o r e a c t o r

(IFRBR)

on t h e b a s i s o f t h e f e a t u r e s o f t h e " i d e a l "

was

b i o f i l m reac-

w i t h o t h e r apparatuses used f o r e x p e r i m e n t a l s t u d i e s o f b i o -

f i l m processes [ 7 ] .

I t was shown t h a t IFBBR h a s t h r e e i m p o r t a n t ad-

vantages i n comparison w i t h t h e o t h e r b i o f i l m r e a c t o r s , p u b l i s h e d

i n the l i t e r a t u r e

[a].

the b i o f i l m thickness; b i o f i l m formation.

They a r e :

e f f e c t i v e and s i m p l e c o n t r o l o f

l a r g e s p e c i f i c support surface area [91;

fast

A comparison between t h e b i o f i l m f o r m a t i o n i n 4

d i f f e r e n t t y p e s o f b i o r e a c t o r s shows t h a t I F B B R p r o v i d e s b e t t e r c o n d i t i o n s f o r biomass f i x a t i o n .

1.3

The b i o f i l m f o r m a t i o n r a t e i s b e t w e e n

and 2 t i m e s f a s t e r t h a n i n a b i o d i s k r e a c t o r ,

three-phase b i o r e a c t o r s (Fig.

3 ) . The i n v e r s e f l u i d i z e d b e d b i o f i l m

I 3

3 . B i o f i l m formation time. IFBBR; 2 biodisk; 3 fix e d bed; 4 - t h r e e - p h a s e f l u i d i zed bed ( n o t formed). 1

-

-

c

4

TYPE OF BIOREACTOR

Fig.

f i x e d bed and

-

HEIGHT Icm 1

F i g . 4. Oxygen c o n c e n t r a t i o n p r o f i l e s . 1 - experimental data; 2 - theoretical profile, calculat e d by t h e model.

-

180

-

r e a c t o r was u s e d f o r t h e i n v e s t i g a t i o n o f t h e f e r r o u s i r o n o x i d a t i o n k i n e t i c s by b i o f i l m o f T h i o b a c i l l u s

ferrooxidans

-

s i m p l e b i o p r o c e s s w i t h one m a i n p r o d u c t was f o u n d t h a t t h e t e m p e r a t u r e ,

pH,

-

a comparatively

t h e f e r r i c i r o n [lO]:It

t h e p r o d u c t and s u b s t r a t e con-

c e n t r a t i o n s v a r i e d i n w i d e r a n g e s a n d do n o t s i g n i f i c a n t l y a f f e c t the process rate.

The same e f f e c t was o b s e r v e d i n t h e c a s e o f BOD

r e m o v a l by b i o f i l m o f m i x e d a e r o b i c c u l t u r e 1 9 1 . On t h e b a s i s o f t h e r e s u l t s o f t h e l a b o r a t o r y - s c a l e

investiga-

t i o n o f t h e h y d r o d y n a m i c s a n d k i n e t i c s a n d on some l i t e r a t u r e d a t a , a m a t h e m a t i c a l model o f t h e i n v e r s e f l u i d i z e d bed b i o f i l m r e a c t o r was d e v e l o p e d 1111. The m a i n a s s u m p t i o n s o f t h e m o d e l a r e :

plug

f l o w o f t h e l i q u i d i n t h e a i r l i f t s e c t i o n and i n t h e a n n u l u s ;

per-

f e c t m i x i n g o f t h e l i q u i d i n t h e b i o r e a c t o r as whole because o f t h e intensive r e c i r c u l a t i o n ; molecular d i f f u s i o n o f the substrate w i t h i n the biofilm;

Monod k i n e t i c s o f t h e s u b s t r a t e u t i l i z a t i o n ;

f r e e suspended i n t h e l i q u i d c e l l s .

was p e r f o r m e d on t h e b a s i s o f t h e l a b o r a t o r y - s c a l e formance.

no

An i d e n t i f i c a t i o n o f t h e m o d e l bioreactor per-

A comparison between t h e e x p e r i m e n t a l and t h e c a l c u l a t e d

o x y g e n p r o f i l e s b y t h e r e a c t o r h e i g h t a r e shown i n F i g .

4.

I t can

be seen t h a t t h e model p r e d i c t s t h e r e a l s i t u a t i o n w i t h an a c c e p t a b l e accuracy. The m a t h e m a t i c a l m o d e l o f t h e IFBBR waa u s e d f o r d e s i g n o f a pilot-scale

v e r s i o n o f t h e b i o r e a c t o r w i t h 90 m volume.

I t was d e -

v e l o p e d and s u c c e s s f u l l y i m p l e m e n t e d u n d e r i n d u s t r i a l c o n d i t i o n s f o r a e r o b i c wastewater t r e a t m e n t

7 identical sections (Fig.

5),

[12].

The b i o r e a c t o r c o n s i s t e d o f

connected i n series.

-=.

100-

z E ? -

g> 80--

The BOD a n d COD

" BOD

V

z

60n

-

A A

COD

8 40-

n

5 -

3

' F i g . 5 . One s e c t i o n o f t h e p i l o t scale bioreactor.

-

0

2

4

6 8 10 RETENTION TIME I h l

F i g . 6 . The e f f e c t o f t h e r e t e n t i o n t i m e on o u t p u t COD a n d BOD profilea.

-

181

-

p r o f i l e s i n the bioreactor are presented i n Fig.

6.

These d a t a show

t h a t t h e i n v e r s e f l u i d i z e d bed b i o f i l m r e a c t o r has up t o 17 t i m e s h i g h e r e f f i c i e n c y i n comparison w i t h t h e waatewater

with mechanical surface aeration. aame c o n d i t i o n s .

treatment plant

B o t h apparatuaea worked under t h e

The o p e r a t i o n a l c o s t s f o r t h e t r e a t m e n t o f 1 m o f

wastewater d i f f e r e d a l s o about 17 t i m e s . The e x p e r i m e n t s w i t h t h e p i l o t - s c a l e b i o r e a c t o r showed some d i s c r e p a n c i e s between t h e m a t h e m a t i c a l model and t h e r e a l l s r g e scale b i o r e a c t o r performance.

F u r t h e r development o f t h i s model r e -

q u i r e s a m o r e p r e c i s e d e s c r i p t i o n o f t h e phenomena w h i c h t a k e p l a c e i n t h e b i o f i l m r e a c t o r s w i t h i n v e r s e f l u i d i z e d beds.

The h y d r o d y n e -

mic subsystem o f t h e model d e s c r i b e d t h e i n v e r s e f l u i d i z e d bed hydrodynamics by t h e model o f Richardson and Z s k i [131, r i v e d o r i g i n a l l y f o r t h e u p f l o w f l u i d i z e d beds.

w h i c h was de-

The r e c e n t i n v e s t i -

g a t i o n s o f h y d r o d y n a m i c s a n d l i q u i d - s o l i d mass t r a n s f e r o f t h e i n v e r s e f l u i d i z e d b e d showed v e r y i n t e r e s t i n g r e s u l t s .

The e f f e c t o f

d e s c r i b e d w i t h r e l a t i v e l y h i g h accu-

,l.OJ

racy by t h e Richardson-Zski

-m

However,

model.

the terminal velocity

-

one o f t h e m a i n p a r a m e t e r s o f t h i s model

-

can n o t be c a l c u l a t e d by t h e

proposal i n the l i t e r a t u r e equations f o r t h e u p f l o w systems.

0.4+.

. . . . . .

.

. .

Fig. 7' A comparison the expansion c h a r a c t e r i s t i c s o f i n v e r s e end u p f l o w f l u i d i z e d b e d s with the number (Mv 0.8) a n d p a r t i c l e s i z e ( d o = 2.1 m m ) .

The t e r m i n a l

velocity o f l i g h t particles i n in-

f l u i d i z e d b e d s w i t h t h e same a b s o l u t e v a l u e o f t h e d e n s i t y number. We a r e w o r k i n g now o n t h e d e t e r m i n a t i o n o f s new m o d e l f o r p r e d i c t i n g the terminal velocity o f the l i g h t particles.

The l i q u i d - a o l i d maas t r a n s f e r i n t h e t w o - p h a s e d i z e d b e d waa a l a o s t u d i e d .

inverse f l u i -

The e l e c t r o c h e m i c a l m e t h o d o f c a t h o d e

r e d u c t i o n was a p p l i e d . I n the preliminary

experiments

i t waa ahown t h a t i n g e n e r a l

t h e maaa t r a n s f e r r a t e b e t w e e n t h e l i q u i d a n d t h e p a r t i c l e s u r f a c e

X

i s 10

182

-

lower than i n t h e case o f t h e upflow f l u i d i z a t i o n [141.

It

c o u l d be e x p l a i n e d b y t h e s m a l l e r m e c h a n i c a l i n e r t i a o f t h e p a r t i c les.

When t h e i r

i n e r t i a i s small,

t h e p a r t i c l e s move i n t h e t u r b u -

l e n t l i q u i d together w i t h t h e t u r b u l e n t eddies. lative interfacial liquid-solid the mass-transfer

Therefore,

v e l o c i t y decreases,

t h e re-

thus decreasing

coefficient.

The e x p e r i m e n t s w i t h l a b o r a t o r y , f l u i d i z e d bed b i o f i l m r e a c t o r s ,

p i l o t and f u l l - s c a l e

inverse

a n d t h e s i m u l a t i o n b y means o f t h e

mathematical model o f t h e b i o r e a c t o r ,

showed t h a t t h e use o f i n v e r -

se f l u i d i z a t i o n i n b i o p r a c e s s s y s t e m s d e v e l o p m e n t i s v e r y p r o m i s i n g . F u r t h e r i n v e s t i g a t i o n s s h o u l d r e v e a l some new t h e o r e t i c a l a n d p r a c t i c a l opportunities for

implementation o f t h i s approach i n b i o p r o -

cess engineering. REFERENCES 1

2 3 4 5

6 7 8

9

10

11 12 13 14

Muroyama and L . S . Fan, A I C h E J., 311, 1 ( 1 9 8 5 ) . S c h u g e r l , Can. J. Chem. E n g . , 6 7 1 , 1 7 8 ( 1 9 8 9 ) . L . N i k o l o v , B i o t e c h n o l o g y a n d C h e m i s t r y ( b u l g . ) , No 5 , 4 1 (198'9). P.A. W i l d e r e r and W.G. C h a r a c l i s , S t r u c t u r e and F u n c t i o n s o f B i o f i l m s . D . 1 2 , J o h n W i l e v d, S o n s , 1 9 8 9 . L . N i k o l o v ' , D. K a r a r n a n e v a n d D . E l e n k o v , B u l g . P a t e n t No. 32910 ( 1 9 8 1 ) . L . N i k o l o v a n d D . K a r a m a n e v , Can. J. Chem. E n g . , 2 4 , 2 1 4 ( 1 9 8 7 ) . L . N i k o l o v a n d D . K a r a m a n e v , J. F e r m . B i o e n g . ( 1 9 9 0 ) ( i n p r e s s ) . A . Moser, i n : H.-J. Rehm a n d G . R e e d ( e d s . ) , B i o t e c h n o l o g y , WCH P r e s s , W e i n h e i m , 1 9 8 5 . D e B o n t , J. V i s s e r , L . N i k o l o v a n d D. K a r a m a n e v , i n : J . A . M . B. M a t i a s s o n and J . Tramper Eds,), P h y s i o l o g y o f I m m o b i l i z e d 990. C e l l s , E l s e v i e r , Amsterdam, D. Karamanev a n d L . N i k o l o v , B i o t e c h n o l . B i o e n g . , 31, 295 (1988). N i k o l o v a n d J. Champagne, T r a v . C . C h a v a r i e , D. K a r a m a n e v , L de 4 J o u r n e e s E u r o p e e n e s s u r l a F l u d i z a t i o n , 254, T o i l l o u s e (1985). L . N i k o l o v a n d D. K a r a m a n e v . P r o c e e d i n g s o f t h e 2 I n t . C o n f e r e n ce o f Biotechnology, Seoul (1990). J . F . R i c h a r d s o n a n d W.N. Z a k i , T r a n s . I n s t . Chem. E n g . , 3 2 , 3 5 (1954). I . N i k o v , D . K a r a m a n e v a n d D. E l e n k o v , Can. J. Chem. E n g . ( i n press).

K. K.

P R O D U C T I O N OF S U G A R S FROM LIGNOCELLULOSIC W A S T E S BASIC R E S E A R C H AND P I L O T S T U D I E S

H. H.

ESTERBAUER’ STEINMULLER’,

M. H A Y N ~ , w . S A T T L E R ~ , w . STEINER~, Th. S T E I N E R 2 a n d M. S I N N E R ’

I n s t i t u t e o f Biochemistry, A-8010 G r a z , A u s t r i a VOEST-Alpine

AG,

P.O.

U n i v e r s i t y o f Graz,

Box 2 ,

A-4031 L i n z ,

S c h u b e r t s t r a D e 1,

Austria

INTRODUCTION R e s e a r c h and d e v e l o p m e n t i n t h e f i e l d o f e n z y m a t i c s a c c h a r i f i cation of vement.

l i g n o c e l l u l o s e h a s l o s t some o f t h e o r i g i n a l l y l i v e l y mo-

The m a i n r e a s o n f o r t h a t i s c e r t a i n l y t h e l o w p r i c e o f c r u -

de o i l w h i c h p l a c e s r e n e w a b l e r e s o u r c e s i n c l u d i n g l i g n o c e l l u l o s i c s apparently i n a non-competitive f r a n k l y a d m i t now t h a t

position.

Additionally,

one m u s t

t h e o p t i m i s t i c f o r e c a s t s on t h e t e c h n i c a l f e a -

s i b i l i t y made i n t h e e a r l y and m i d t h s e v e n t i e s c o u l d n o t b e v e r i f i e d .

A t t h a t t i m e p r o c e s s e n g i n e e r i n g and economic e v a l u a t i o n were m o s t l y b a s e d on s m a l l s c a l e l a b o r a t o r y e x p e r i m e n t s w h i c h m i g h t s o m e t i m e s e v e n n o t h a v e a l l o w e d t o make a r e a s o n a b l e g u e s s on t e c h n i c a l f e a s i bility.

I n r e t r o s p e c t i v e one m u s t a l s o r e a l i z e t h a t i t t a k e s i t s

t o l l now,

t h a t the basic research i n the f i e l d o f cellulase d i d not

come up t o t h e d e s i r e d l e v e l .

F o r example,

t h e m o l e c u l a r mechanisms

how c e l l u l a s e s c a n h y d r o l y z e c r y s t a l l i n e c e l l u l o s e , t h e s y n e r g i s m o f t h e d i f f e r e n t c e l l u l a s e components, tery.

the basis for i s s t i l l a mys-

The measurement o f enzyme a c t i v i t i e s as FPU g i v e s v e r y d i f f e -

rent results i n different

laboratories,

w h i c h makes i t d i f f i c u l t t o

o b j e c t i v e l y compare enzyme p r o d u c t i o n d a t a .

Briefly,

we n e e d b o t h ,

more b a s i c k n o w l e d g e on c e l l u l a s e s and t h e i r a c t i o n a s w e l l as obj e c t i v e and w e l l p r o v e n d a t a o f p i l o t s t u d i e s . We r e v i e w i n t h i s a r t i c l e some o f o u r r e c e n t r e s u l t s on t h e mo-

I, t h e a d s o r p t i o n o f c e l l u l o -

l e c u l a r structure of

cellobiohydrolase

s e and t h e e f f e c t o f

enzyme dosage on t h e e x t e n t o f h y d r o l y s i s .

We

a l s o r e p o r t some r e s u l t s o b t a i n e d i n p i l o t s t u d i e s p e r f o r m e d w i t h f a c i l i t i e s for

p r o c e s s i n g u p t o 1 t o n l i g n o c e l l u l o s e p e r day.

I n our

long term strategy,

-

184

t h e e n z y m a t i c s a c c h a r i f i c a t i o n i s o n l y one a s -

p e c t o f a much b r o a d e r c o n c e p t f o r t h e d e v e l o p m e n t o f new t e c h n i q u e s f o r t h e p r o d u c t i o n o f base and f i n e c h e m i c a l s ,

fibres,

fuel,

f o o d and f e e d f r o m p l a n t b i o m a s s . MATERIAL AN0 METHODS The d e t a i l e d e x p e r i m e n t a l c o n d i t i o n s f o r t h e s m a l l a n g l e X - r a y s c a t t e r i n g s t u d i e s on c e l l o b i o h y d r o l a s e given i n (ref. VTT-0-80133

I f r o m T.

r e e s e i MCC 77 a r e

l), t h e c o r r e s p o n d i n g s t u d i e s on CEH I f r o m T .

and i t s c o r e p r o t e i n a r e d e s c r i b e d i n ( r e f .

reesei

2).

F o r t h e a d s o r p t i o n s t u d i e s a c e l l u l a s e p r e p a r a t i o n f r o m T. s e i M C G 7 7 g r o w n on a u l f i t e p u l p was u s e d .

p r e c i p i t a t e d from t h e c u l t u r e f i l t r a t e w i t h acetone, r e d e s o l v e d i n w a t e r and f r e e z e d r i e d .

centrifuged,

T h i s enzyme p r e p a r a t i o n h a d

0 . 5 1 FPU/mg p r o t e i n a n d 0 . 2 1 u n i t s O - g l u c o s i d a s e / m g

protein.

s u b s t r a t e f o r t h e a d s o r p t i o n s t u d i e s was A v i c e l f r o m M e r c k . adsorption experiments A v i c e l pH 4.8,

( 0 t o 200 g / L )

200 rpm s h a k i n g w i t h 2.02,

per l i t r e f o r 1 hour.

ree-

The c e l l u l a s e p r o t e i n was

4.05

The For the

was i n c u b a t e d a t 50

OC,

o r 8.1 g c e l l u l a s e p r o t e i n

T h e r e a f t e r t h e s a m p l e s were c e n t r i f u g e d a n d

t h e n o t a d s o r b e d p r o t e i n a n d t h e n o t a d s o r b e d FPU were d e t e r m i n e d . A l s o d e t e r m i n e d were t h e s o l u b i l i z e d s u g a r by HPLC t o c a l c u l a t e t h e r e s i d u a l amount o f A v i c e l .

A l l the other d e t a i l s are given i n (ref.

10). F o r t h e i n v e s t i g a t i o n o f t h e e f f e c t o f enzyme dosage o n t h e e x t e n t o f h y d r o l y s i s C e l l u c l a s t C C N 3000 85/4 ( N o v o i n d u s t r i e s ) , vozym TN 1 8 8 ( 0 - g l u c o s i d a s e ,

p r e t r e a t e d p o p l a r wood r(as u s e d .

Briefly,

FPU/g l i q u i d ) a n d Novozym ( 5 2 0 u n i t s / g c i t a t e b u f f e r pH 4.8, n a l volume o f 100 m l .

0.01

wood)

the l i q u i d Celluclast

(45

l i q u i d ) were a d d e d t o 0.05

M

X Thimerosal (aa b i o c i d e ) t o g i v e a f i -

1 0 0 m l a n d f i n a l enzyme t i t e r s f r o m 10 t o 2 0 0 FPU p e r was a l w a y s 1:l. The 1 0 0 m l b u f f e r

The r a t i o F P U : O - g l u c o s i d a s e

enzyme m i x t u r e was warmed up t o 50 2 g

No-

Novo) a n d S i g m a c e l l 5 0 ( S i g m a ) o r s t e a m

i n a 300 m l E r l e n m e y e r f l a s k ,

OC

Sigmacell (or 2 g water i n s o l u b l e f i b r e s from p r e t r e a t e d poplar were t h e n a d d e d t o e a c h f l a s k .

aluminium f o i l and i n c u b a t e d a t 50

The f l a s k s were s e a l e d w i t h an

OC,

200 rpm s h a k i n g t o p e r i o d s up

t o 9 4 h. S a m p l e s w e r e w i t h d r a w n a t d i f f e r e n t t i m e p o i n t s a n d t h e sol u b i l i z e d g l u c o s e was d e t e r m i n e d b y HPLC.

-

185

-

R E S U L T S AND DISCUSSION

1. M o l e c u l a r s h a p e o f c e l l o b i o h y d r o l a s e

I

C o n s i d e r a b l e p r o g r e s s h a s b e e n made i n t h e r e c e n t y e a r s i n t h e p r e p a r a t i v e s e p a r a t i o n o f t h e i n d i v i d u a l enzyme c o m p o n e n t s c o n t a i ned i n Trichoderma r e e s e i c e l l u l a s e s . c h r o m a t o f o c u s i n g u s i n g a pH g r a d i e n t separated t h e T. proteins.

We ( r e f s .

3 , 4 ) have employed

r a n g i n g f r o m 6.5

t o 3.0

and

r e e s e i M C G 77 enzyme i n t o 1 4 i n d i v i d u a l enzyme

The m a j o r enzyme,

w h i c h c o m p r i s e d a b o u t 60-65

X o f the

t o t a l c e l l u l a s e p r o t e i n was i d e n t i f i e d as c e l l o b i o h y d r o l a s e

I). The CBH I enzyme i s o l a t e d b y us h a d a n i . e . P . a s i n g l e band i n SDS-PAGE c u l a r w e i g h t o f 59000.

o f 3.6

I (CBH

a n d gave

e l e c t r o p h o r e s i s corresponding t o a mole-

O t h e r m e t h o d s g a v e f o r t h e same p r e p a r a t i o n s

somewhat d i f f e r e n t m o l e c u l a r w e i g h t s

(Tab.

s c a t t e r i n g p e r f o r m e d on t h e CBH I f r o m T .

1). S m a l l a n g l e X-ray r e e s e i M C G 77 ( r e f .

1)

gave a v e r y u n u s u a l m o l e c u l a r shape comparable t o a t a d p o l e w i t h a l a r g e h e a d d o m a i n ( l e n g t h 7.0

nm,

d i a m e t e r 4.4

nm) a n d a r a t h e r

l o n g a n d f l e x i b l e t a i l w i t h a l e n g t h o f 11 nm a n d a maximum d i a m e t e r o f 2.2

nm (Fig.

1).

by s m a l l a n g l e X-ray t r a t e o f t h e T.

T h i s f o r m was t h e n f u l l y c o n f i r m e d

r e e s e i s t r a i n VTT-D-80133

n i t y chromatography. and a d i a m e t e r o f 4.4

2)

(ref.

a n a l y s i s o f CBH I p u r i f i e d f o r t h e c u l t u r e f i l (Espoo,

Finland)

by a f f i -

The h e a d o f t h i s enzyme h a d a l e n g t h o f 6 . 7

nm,

diameter o f 3 . 2 nm (Fig.

t h e t a i l i s 12.9

nm

nm l o n g w i t h a m a x i m a l

1).

F i g . 1. M o l e c u l a r shape o f c e l l o b i o h y d r o l a s e I from T . r e e s e i s t r a i n s M C C 77 ( A ) and VTT-D-80133 (8) and t h e c o r e p r o t e i n (C). The s h a p e s were d e t e r m i n e d by s m a l l a n g l e X-ray scattering (refs. 3,4).

18 nm

5nm

C

I

5 nm

-

186

-

TABLE 1 M o l e c u l a r w e i g h t o f CBH I f r o m M C G 7 7 a s e s t i m a t e d by v a r i o u s methods

I method

I

molecular weight 59000

SDS-PAGE e l e c t r o p h o r e s i s Ultracentrifugation

68500

HPLC w i t h LKB G 2 0 0 0 SW c o l u m n

48400

HPLC w i t h L K B G 3 0 0 0 SW c o l u m n

51300

Small angle X-ray

58000

scattering

59400

Amino a c i d a n a l y s i s

I t was known f r o m o t h e r s t u d i e s on t h e s t r u c t u r e o f C B H I t h a t p a r t i a l p r o t e o l y s i s w i t h p a p a i n s p l i t s CBH t h e l a r g e r fragment

(core protein)

I i n t o two fragments,

possesses t h e c a t a l y t i c a c t i v i t y

w h e r e a s t h e s m a l l e r p e p t i d e a p p e a r s t o be r e s p o n s i b l e f o r t h e ad-

5).

sorption t o cellulose (ref.

The s m a l l a n g l e X - r a y

analysis cle-

a r l y r e v e a l e d t h a t t h e core p r o t e i n i s t h e head p a r t o f t h e tadpole.

I t i s o f i n t e r e s t t o n o t e t h a t a s i m i l a r s t r u c t u r e c o u l d be t e n -

t a t i v e l y d e r i v e d f r o m t h e a m i n o a c i d s e q u e n c e o f CBH o f t h e CBH

I gene ( r e f .

I and a n a l y s i s

6 ) . I n t h i s c a s e t h e i n v e s t i g a t o r s came t o

I s h o u l d h a v e a m o l e c u l a r shape l i k e a " d e a d b e a v e r " (J. K n o w l e s , V T T F i n l a n d , p r i v a t e c o m m u n i c a t i o n ) . So f a r i t t h e a s s u m p t i o n t h a t CBH

is n o t known i f a n d how t h e s p e c i f i c p r o p e r t i e s o f CBH I a r e d e t e r m i n e d by t h i s u n u s u a l s h a p e .

2 . A d s o r p t i o n o f c e l l u l a s e on c e l l u l o s e Over many y e a r s we h a v e p e r f o r m e d a d s o r p t i o n s t u d i e s w i t h d i f f e r e n t c e l l u l a s e p r e p a r a t i o n s and v a r i o u s c e l l u l o s e s i n c l u d i n g l i g n o cellulosics.

However,

we e n c o u n t e r e d many p r o b l e m s i n e v a l u a t i n g o u r

e x p e r i m e n t a l l y m e a s u r e d d a t a a c c o r d i n g t o known p h y s i c a l l a w s .

I f

f o r e x a m p l e a d s o r p t i o n s t u d i e s w e r e p e r f o r m e d w i t h a f i x e d amount o f cellulaae

(i.e.

2 , 4 or 8 g p r o t e i n / l )

and v a r i a b l e amounts o f A v i -

c e l (i.e.

1 0 t o 200 g / l )

(ES-/Po*)

was t h e n p l o t t e d a g a i n s t t h e A v i c e l c o n c e n t r a t i o n we ob-

and t h e f r a c t i o n o f p r o t e i n adsorbed

t a i n e d a h y p e r b o l i c c u r v e s u g g e s t i n g t h a t t h e Langmuir t y p e e q u a t i o n

1, w h i c h was a l s o u s e d by o t h e r s ( r e f . sorption studies i s valid.

However,

7) i n cellulase/cellulose

i f we l i n e a r i z e d a n d made t h e

ad-

-

187

-

r e c i p r o c a l p l o t a c c o r d i n g t o e q u a t i o n 2,

t h e c o r r e l a t i o n was v e r y

bad and r a t h e r i n d i c a t e d t h a t t h e e q u a t i o n s 1 or 2 do n o t adequatel y describe the adsorpfion o f cellulaae t o cellulose.

Our f i r s t

s u s p i c i o n was t h a t we made some s y s t e m a t i c e x p e r i m e n t a l e r r o r ( s ) i.e.

t h a t t h e a d s o r p t i o n e q u i l i b r i u m was n o t y e t e s t a b l i s h e d ,

a f r a c t i o n o f c e l l u l o s e was h y d r o l y z e d d u r i n g t h e e x p e r i m e n t ,

that

or

t h a t t h e d i f f e r e n t c o m p o n e n t s o f c e l l u l a s e enzyme i n f l u e n c e d t h e dependancy o f t h e a d s o r p t i o n f r o m t h e c e l l u l o s e c o n c e n t r a t i o n .

W e

then found o u t t h a t t h e a d s o r p t i o n e q u i l i b r i u m i s e s t a b l i s h e d w i t h i n a b o u t one h o u r a n d t h a t t h e f r a c t i o n o f c e l l u l o s e h y d r o l y z e d d u r i n g t h i s t i m e must o f c o u r s e b e t a k e n i n t o a c c o u n t . e v e n i f t h e s e t w o p o i n t s ( a d s o r p t i o n measurement

Nevertheless,

a f t e r one h o u r

measurement o f l i b r a t e d s u g a r s a n d c a l c u l a t i n g r e s i d u a l c e l l u l o s e ) were c o n s i d e r e d n o s t r a i g h t l i n e was o b t a i n e d when t h e d a t a were p l o t t e d according t o equation 2 (Fig.

2).

E S - = adsorbed c e l l u l a a e g/1,

P o = t o t a l i n i t i a l l y added p r o t e i n i n g / l , So-=

c o n c e n t r a t i o n o f c e l l u l o s e i n g / 1 ( i n i t i a l l y added minus f r a c t i o n hydrolyzed during 1 hour),

d

= f r a c t i o n o f Po’which can b i n d t o c e l l u l o s e ,

K-

a d s o r p t i o n e q u i l i b r i u m c o n s t a n t i n gram c e l l u l o s e / l .

Fortunately, Ristroph (ref.

8).

we t h e n r e a d a p a p e r j u s t p u b l i s h e d b y S t u a r t a n d

I n t h i a paper t h e a u t h o r s gave a b a s i c t h e o r e t i -

c a l d e r i v a t i o n f o r t h e d e s c r i p t i o n o f t h e a d s o r p t i o n phenomenom. I n c o n s i d e r i n g t h e v a r i o u s p r o p o s a l s made i n t h i s p a p e r we d e r i v e d

a l l t h e b a s i c e q u a t i o n s w h i c h c o r r e c t l y d e s c r i b e t h e dependancy o f t h e a d s o r p t i o n from b o t h t h e c e l l u l a s e and t h e c e l l u l o s e c o n c e n t r a tion.

A l l t h e d e r i v a t i o n s were b a s e d on t h e a s u m p t i o n t h a t t h e b i n -

ding o f c e l l u l a a e t o c e l l u l o s e f o l l o w s the c l a s s i c a l Michaelia-Ment e n k i n e t i c s described by equation 3 .

-

188

-

A

l2

.

t

0.02 0.04 0,06 0.08 0.10

O,l2 0,U

1 I RESIDUAL AVICET 1I / g 1 F i g . 2 . A d s o r p t i o n o f T . r e e s e i c e l l u l a s e t o A v i c e l e v a l u a t e d by d o u b l e r e c i p r o c a l p l o t . The i n i t i s 1 p r o t e i n c o n c e n t r a t i o n was 2.02 g,'l ( o p e n c i r c l e s ) , 4 . 0 5 g / l ( t r i a n g l e ) a n d 8 . 1 g / l ( c l o s e d c i r c l e s ) . D o t t e d l i n e o b t q i n e d by l i n e a r r e g r e s s i o n a n a l y s i s ( r2 0.900, K * = 4 0 0 g A v i c e l / l , d = - 5 . 1 ) .

Where E i s t h e c o n c e n t r a t i o n o f t h e f r e e enzyme i n m o l / l , t h e concentration o f t h e enzyme-cellulose

complex i n m o l / l ,

the concentration o f the free cellulose i n mol/l,

=

ES

and S

whereby 1 m o l o f

f r e e c e l l u l o s e i s d e f i n e d a s t h a t amount o f c e l l u l o s e i n gram w h i c h has t h e p o t e n t i a l t o

b i n d 1 m o l enzyme.

K i s the Michaelis constant

or adsorption e q u i l i b r i u m constant i n mol/l. I n equation 3 a l l the concentrations are i n mol/l;

t o obtain

t h e corresponding e q u a t i o n s f o r c o n c e n t r a t i o n s i n gram/l, introduce a molecular weight

one m u s t

f o r t h e c e l l u l a s e ME a n d a n a p p a r e n t

m o l e c u l a r weight f o r t h e b i n d i n g c e l l u l o s e MS,

w h e r e MS i s t h a t a-

mount o f c e l l u l o s e i n gram w h i c h c a n b i n d 1 m o l c e l l u l a s e .

With t h e

i n t r o d u c t i o n o f ME a n d M5 t h e f o r m u l a d e s c r i b i n g t h e a d s o r p t i o n f o r v a r i a b l e c e l l u l o s e c o n c e n t r a t i o n s i s g i v e n by e q u a t i o n 4 and t h e formula describing the adsorption for t i o n s i s g i v e n by e q u a t i o n 5 .

variable c e l l u l a a e concentra-

-

[ES

eq.

4

eq.

5

--

189

-

I

Where E S - j s t h e a d s o r b e d c e l l u l a s e i n g / l ,

i n gll,

so*

t o t a l cellulose i n g/l,

t h e f r a c t i o n o f Po*

E*

free

Po*

total protein

cellulase i n g/l,

CL

b g i v e s t h e amount

which can b i n d t o c e l l u l o s e ,

o f c e l l u l a s e i n gram w h i c h c a n m a x i m a l l y b e b o u n d t o 1 g c e l l u l o s e ,

b i s a l s o t h e r a t i o ME/MS, mol/l,

K i s the Michaelis-Menten constant i n

ME i s t h e a v e r a g e m o l e c u l a r w e i g h t o f t h e c e l l u l a s e a n d M S

i s t h e average m o l e c u l a r weight o f t h e c e l l u l o s e . U s i n g t h e t w o e q u a t i o n s a n d an a v e r a g e m o l e c u l a r w e i g h t c e l l u l a s e ME

= 48000

(ref.

91,

we c o u l d o b t a i n some i m p o r t a n t

for the data

d e s c r i b i n g t h e a d s o r p t i o n e q u i l i b r i u m f o r t h e s y s t e m 1. r e e s e i c e l l a ae a n d A v i c e l ( r e f .

10).

The M i c h a e l i s c o n s t a n t i s 2.0

t h e apparent molecular weight

x

Mol/l,

f o r t h e b i n d i n g A v i c e l i s 520.000,

t h e m a x i m a l b i n d i n g c a p a c i t y b i s 9 2 mg c e l l u l a s e p r o t e i n o r 5 5 FPU -6 p e r 1 gram A v i c e l , 1 gram A v i c e l c o n t a i n s 1 . 9 2 x 1 0 mol b i n d i n g sites for cellulase,

t h i s means t h a t o n l y one o u t o f a b o u t 3 0 0 0 g l y -

c o s i d i c bonds i n c e l l u l o s e

f o r m s an e n z y m e - s u b s t r a t e

complex,dthe f o r FPU t h e

f r a c t i o n o f p r o t e i n s w h i c h c a n b i n d t o A v i c e l i s 0.98, d v a l u e i s 0.84.

An a d d i t i o n a l v e r y i m p o r t a n t c o n c l u s i o n r e g a r d s t h e

s u r f a c e c o v e r e d b y t h e enzyme on A v i c e l . m e n t s g a v e f o r t h e 1. r e e s e i c e l l u l a s e

L i g h t s c a t t e r i n g measure-

( = m i x t u r e o f a l l enzymes)

a n a v e r a g e m o l e c u l a r w e i g h t o f 48 0 0 0 ( r e f . l e has a diameter o f

6.58

9),

t h e a v e r a g e molecu-t

n m a n d a v o l u m e o f 1.49

cm3 ( r e f .

x

9). W i t h t h e s e d a t a a n d t h e b i n d i n g c a p a c i t y o f 92 mg c e l l u l a s e / g A v i c e l i t r e s u l t s t h a t o n c o n d i t i o n s o f maximum s u r f a c e c o v e r a g e

( = maximum b i n d i n g , a l l b i n d i n g s i t e s o c c u p i e d ) t h e enzyme c o v e r s 2 3 a s u r f a c e o f 42 m / g A v i c e l e n d a v o l u m e o f 0.186 cm In relative 2 c l o s e a g r e e m e n t w i t h o u r v a l u e s a r e t h e 17.5 t o 35 m / g A v i c e l r e -

.

p o r t e d b y L e e e t a l (11). T h i s l a r g e s u r f a c e c o v e r e d by t h e enzyme s t a n d s i n c o n t r a d i c t i o n t o t h e 2 t o 5 m2 s p e c i f i c s u r f a c e a r e a p e r gram A v i c e l m e a s u r e d by p h y s i c o - c h e m i c a l

methods ( n i t r o g e n adsorp-

tion,

X-ray

diffraction,

190

-

f o r more d e t a i l see r e f .

10).

The f a c t t h a t

c e l l u l a s e o c c u p i e s a b o u t a 10 t i m e s l a r g e r s u r f a c e t h a n a p p a r e n t l y a v a i l a b l e c a n n o t be n e g l e c t e d and d e s e r v e s f u r t h e r

investigation.

P o s s i b l e e x p l a n a t i o n s c o u l d be t h a t c e l l u l o s e i n t h e h y d r a t e d form ( a s used f o r t h e a d s o r p t i o n s t u d i e s )

i n f a c t h a s a much l a r g e r s u r -

f a c e a r e a t h a n d r y c e l l u l o s e as u s e d f o r n i t r o g e n a d s o r p t i o n ,

alter-

n a t i v e l y i t c o u l d b e t h a t t h e enzyme i t s e l f c r e a t e s new s u r f a c e s by forming micropores.

3 . E f f e c t o f enzyme d o s a q e on h y d r o l y s i s o f c e l l u l o s e Many r e s e a r c h e r 3 w o r k i n g on e n z y m a t i c h y d r o l y s i s h a v e p r o b a b l y o b s e r v e d t h a t t h e amount o f c e l l u l o s e h y d r o l y z e d a t a g i v e n t i m e d o e s n o t l i n e a r l y i n c r e a s e w i t h t h e amount o f enzyme added b u t r a t h e r f o l l o w s h y p o b o l i c c u r v e s as shown f o r e x a m p l e i n F i g .

3.

y4n

-

70h 46h 24h

60

c

m

13h 9h

40

r

I

I

I

1

I

20

40

60

80

100

F i g . 3 . E f f e c t o f enzyme d o s a g e o n h y d r o l y s i s o f c e l l u l o s e . A v i c e l ( 2 0 g / l ) was i n c u b a t e d w i t h C e l l u c l a s t C C N s u p p l e m e n t e d w i t h b e t a g l u c o s i d a s e Novozym TN 1 8 8 ( 5 t o 1 0 0 FPU/g A v i c e l ) f o r 2 t o 9 4 h a n d t h e amount o f g l u c o s e f o r m e d was m e a s u r e d .

We f o u n d ( s e e F i g .

3,4) t h a t f o r a l l t i m e p o i n t s ( 2 t o 9 4 h)

t h e enzyme d e p e n d e n c y o f t h e h y d r o l y s i s y i e l d c a n b e d e s c r i b e d by the simple formulas i n equation 6 or 7 ( r e f .

12).

[El eq.

6

Y = Y

max '

1

eq.

7

K

+ [El

K + -- . -1 + 'max

1 Ymax

-

191

-

Where Y i s t h e f r a c t i o n o f a u b a t r a t e ( o r c e l l u l o s e )

hydrolyzed

a t a given time, f r a c t i o n of substrate which c o u l d maximally Ymax i s be h y d r o l y z e d a t i n f i n i t e enzyme c o n c e n t r a t i o n s , E i s t h e enzyme dosage i n FPU/g s u b s t r a t e a n d K i s t h e enzyme d o s a g e i n FPU/g t e yielding

substra-

t h e h a l f maximal (Ymax/Z) h y d r o l y s i s .

0.05 0.04

0.03 0.02 0.01

t

1

4

l

4

I

l

0.04 0.08 0.12 0,16 020 €-'I FPU lg I-' F i g . 4.

D o u b l e r e c i p r o c a l p l o t o f t h e c u r v e s shown i n F i g .

The v a l u e s f o r F i g .

3 a n d 4 w e r e o b t a i n e d by h y d r o l y z i n g on

s t a n d a r d i z e d c o n d i t i o n S i g m a c e l l 50 ( 2 0 9 / 1 1 (Novo)

w i t h C e l l u c l a s t CCN

s u p p l e m e n t e d w i t h Novozym TN 1 8 8 ( b e t a - g l u c o s i d a s e , was a l w a y s 1:l.

t h e r a t i o FPU: O - g l u c o s i d a s e o n l y t h e Novo enzyme-mix,

b u t t h a t a l l t h e o t h e r Trichoderma enzyenzymes p r o d u c e d

b y u s ) f o l l o w e d t h e same c o n f o r m i t y aa ahown i n F i g

7.

Novo),

We a l s o f o u n d t h a t n o t

mes ( v a r i o u s c o m m e r c i a l l y a v a i l a b l e p r e p a r a t i o n s , pressed by e q u a t i o n 6,

3.

3 , 4 and ex-

The e v a l u a t i o n o f enzymes a c c o r d i n g t o

t h i s l a w c o u l d o f f e r a v e r y u s e f u l method t o o b j e c t i v e l y d e f i n e t h e e f f e c t i v e n e s s o f d i f f e r e n t enzymes;

Ymax

good enzymes s h o u l d h a v e a h i g h

v a l u e and a low K v a l u e . Even more i m p o r t a n t c o u l d be t h a t t h e e q u a t i o n s 6 a n d 7 c o u l d

b e u s e d t o d e f i n e a n d compare t h e e f f e c t i v e n e s s o f d i f f e r e n t p r e t r e a t m e n t ~f o r l i g n o c e l l u l o s e .

We h a v e f o u n d t h a t e q u a t i o n s 6 , 7

are not

r e s t r i c t e d t o h y d r o l y a i s o f p u r e c e l l u l o s e b u t o b v i o u s l y c a n b e applied for a l l lignocellulosics.

Wheat a t r a w a n d p o p l a r wood h a v e b e e n

t e s t e d so f a r . p l a r wood.

192

-

5 a s an e x a m p l e shows r e s u t s o b t a i n e d w i t h p o -

Fig.

For that,

t e d s t e a m f o r 10 min.

c h i p s o f p o p l a r wood w e r e t r e a t e d w i t h s a t u r a a t 20 0,

220 a n d 240

OC,

he steamed m a t e r i a l

was washed a n d t h e r e m a i n i n g w a t e r i n s o l u b l e f i b r e s were h y d r o l y z e d

on s t a n d a r d i z e d c o n d i t i o n ( 2 0 g/l, 5 0 same enzyme-mix

s h a k i n g 24 h) w i t h t h e

OC,

a s u s e d f o r t h e e x p e r i m e n t shown i n F i g .

enzyme dosage v a r i e d f r o m 5 t o 1 0 0 FPU/g

substrate.

p r o c a l p l o t a c c o r d i n g t o e q u a t i o n 7 i s shown i n F i g .

3 , 4.

The

A double r e c i 5.

The i m p o r -

t a n t c o n c l u s i o n s w h i c h can be drawn f r o m t h e s e e x p e r i m e n t s a r e as follows: wood,

First,

the equation 7 i s also v a l i d f o r pretreated poplar

the correlation coefficient

t h a n 0.95.

Second,

t e r m i n e t h e v a l u e s o f Ymax perature i . e .

i s for a l l three lines better

t h e t h r e e d i f f e r e n t p r e t r e a t m e n t t e m p e r a t u r e deand K (Tab.

2).

t h e b e s t p r e t r e a t m e n t i s 220

t h e y i e l d i s very c l o s e t o t h e o r e t i c a l (45 and t h e K v a l u e i s l o w e s t (Tab.

Third, OC,

t h e optimum tem-

a t t h i s temperature

I g l u c a n / g p o p l a r wood)

2).

F i g . 5. E f f e c t on enzyme d o s a g e on t h e e x t e n t o f h y d r o l y s i s o f s t e a m p r e t r e a t e d p o p l a r wood. C h i p s w e r e s t e a m e d i n a n a u t o c l a v e a t 200 ( A ) , 220 ( B ) a n d 240 O C ( C ) . The m a t e r i a l was washed a n d t h e w a t e r i n s o l u b l e f i b r e s , WIF ( 2 0 9 / 1 1 were i n c u b a t e d w i t h 5 t o 1 0 0 FPU/g WIF f o r 24 h. The c e l l u l o s e h y d r o l y z e d was c a l c u l a t e d f r o m t h e amount o f g l u c o s e f o r m e d . The enzyme u s e d was a m i x t u r e o f C e l l u c l a s t C C N a n d Novozym T N 1 8 8 ( N o v o ) , t h e r a t i o F P U : f l - g l u c o s i d a s e was a l w a y s 1 : l . The f i g u r e shows t h e d o u b l e r e c i p r o c a l p l o t a c c o r d i n g t o e q u a t i o n 7.

Ordinate:

r e c i p r o c a l v a l u e o f gram c e l l u l o s e h y d r o l y z e d p e r 1 0 0 g WIF,

abscissa:

r e c i p r o c a l v a l u e o f FPU/g WIF u s e d f o r h y d r o l y s i s .

-

193

-

TABLE 2 E v a l u a t i o n o f t h e h y d r o l y s i b i l i t y o f s t e a m e d p o p l a r wood by u s i n g the relationship Y = Y E/K + E (eq. 6 ) . max

.

1. P r e t r e a t m e n t t e m p e r a t u r e 2. Y i e l d o f w a t e r i n s o l u b l e 3.

4.

200

220

OC

240

OC

OC

f i b r e s (WIF) Y m a x , X o f WIF t h e o r e t i c a l l y h y d r o l y s a b l e a t i n f i n i t e en-

86.3 %

69.0

X

6 1 .0

X

zyme d o s a g e ( F i g . 5 , e q . 7) K , FPU/g W I F , e n z y m e l o a d i n g

43.9 %

64.9 X

68.0

x

f o r Ymax/2

9 . 3 9 FPU/g

2 .6 9 FPU/g

4.0

0.999

0.993

0.954

FPU/g

5. Correlation c o e f f i c i e n t f o r

the double reciprocal p l o t

'.

(Fig. 5 ) expressed i n X of t h e

'-max o r i g i n a l p o p l a r wood,

'*max

4.

37.8

= WIF y i e l d x Y m a x

x

4 4 .7

x

41 X

x 100

P i l o t s t u d i e s and economic e v a l u a t i o n A small p i l o t f a c i l i t y was i n o p e r a t i o n from 1982 t o 1986 a t

a p u l p a n d p a p e r company ( S t e y r e r m i h l A C , A u s t r i a , w i t h VOEST-Alpine

AC).

for cellulase production

(one 300 l i t r e s t i r r e d t a n k r e a c t o r , two

250 l i t r e d e e p j e t a e r a t i o n f e r m e n t o r a ) , 50 L,

i n cooperation

The e q u i p m e n t c o n s i s t e d o f t h r e e f e r m e n t o r s f i v e hydrolysis tanks aach

a p u l p e r , a f i l t e r - b e l t p r e s s , a d i s k r e f i n e r a n d s e v e r a l u-

n i t s f o r p r e t r e a t m e n t . A b o u t 1 0 0 k g l i g n o c e l l u l o s i c raw m a t e r i a l s could be processed p e r day. I n l a t e 1 9 8 7 a much l a r g e r p i l o t f a c i l i t y d e s i g n e d f o r p r o c e s s i n g a b o u t 1 t o n raw m a t e r i a l / d a y Alpine AG (Linz, A u s t r i a ) . f o r p r e t r e a t m e n t , 1 5 m3

w e n t i n o p e r a t i o n a t VOEST3 digeator

The m a i n u n i t s h e r e a r e : 3 m

fermentor with prefermentora,

s t i r r e d 15 m

tank for hydrolysis, evaporation u n i t f o r concentration of auger

3

SO-

l u t i o n s and a u n i t f o r f u r f u r a l production , T h e d e s i g n and t h e tests r u n n i n g i n t h i s "Biomass R e f i n e r y " a r e b a a e d on t h e c o o p e r a t i o n i n b a s i c r e a e a r c h ( U n i v e r s i t y o f Graz) and i n d u a t r i a l e n g i n e e r i n g (VOEST-Alpine

I n d u s t r i e a n l a g e n b a u C e s . m . b . H .) .

R e s u l t s o f t h e on-

going tests i n t h e Biomass Refinery w i l l be r e p o r t e d i n d e t a i l elaewhere.

a)

194

-

P i l o t fermentations F e r m e n t a t i o n s were c a r r i e d o u t o n b a t c h ,

n u o u s c o n d i t i o n s w i t h 2-3

7;

i n was i n t h e e a r l y phase Q M 9414 a n d l a t e r T . SVG-17

and V-44,

f e d b a t c h and c o n t i -

s u l f i t e p u l p as c a r b o n s o u r c e .

The s t r a -

r e e s e i mutants termed

w h i c h a r e good c e l l u l a s e p r o d u c e r s w i t h w h e a t s t r a w

as c a r b o n s o u r c e . C o s t a n a l y s i s showed t h a t t h e p r o d u c t i o n c o s t f o r 1 k g enzyme p r o t e i n i n s o l u t i o n ( w i t h 3 FPU/ml a n d 0 . 5 ween 54 A u s t r i a n S c h i l l i n g (AUS)

FPU/mg p r o t e i n )

i s bet-

f o r c o n t i n u o u s f e r m e n t a t i o n and

63 AUS f o r b a t c h f e r m e n t a t i o n ;

35 7;

from t h e s u l f i t e p u l p .

D. t h e r e f o r e a i m e d t o u s e w h e a t s t r a w

Our R . 8

o f t h e p r o d u c t i o n c o s t stem

o r s p e n t h y d r o l y s i s r e s i d u e s f o r enzyme p r o d u c t i o n . W i t h s t e a m t r e a t e d wheat s t r a w and t h e SVC-17

o r V-44 m u t a n t s we now o b t a i n i n

t h e p i l o t f e r m e n t o r enzyme b r o t h s w i t h 6 - 7

3.0-3.7 se/ml.

fPU/ml,

25 u n i t s xylanase/ml

mg c e l l u l a s e p r o t e i n / m l ,

and 2-2.5

units

6-glucosida-

The i n t e g r a t e d p r o d u c t i v i t y i n b a t c h f e r m e n t a t i o n ( f r o m t h e

s t a r t t o t h e end)

v a r i e s b e t w e e n 40-80

FPU/l.h.

b) Pretreatment E n z y m a t i c s a c c h a r i f i c a t i o n o f l i g n o c e l l u l o s i c m a t e r i a l w i l l onl y be f e a s i b l e i n an i n d u s t r i a l p r o c e s s i f a s i m p l e , t i v e pretreatment i s possible. o n l y academic i m p o r t a n c e .

cheap a n d e f f e c -

Many o f t h e p r o p o s e d t e c h n i q u e s h a v e

F o r example,

c h e m i c a l p r e t r e a t m e n t c a n on-

l y be e c o n o m i c i f t h e r e a g e n t s a r e cheap a n d u s e d i n s m a l l q u a n t i t i e s w i t h no w a s t e o r c a n be r e c o v e r e d w i t h h i g h e f f i c i e n c y .

We c o n -

c e n t r a t e d o u r p i l o t s t u d i e s m a i n l y on t h r e e p r e t r e a t m e n t m e t h o d s , w h i c h seemed t o b e m o s t p r o m i s i n g t o u s .

I n t h e following p a r t these

methods a r e b r i e f l y d e s c r i b e d f o r wheat s t r a w ,

t h e y a r e h o w e v e r ap-

p l i c a b l e w i t h some s l i g h t m o d i f i c a t i o n s t o o t h e r l i g n o c e l l u l o s i c s . CaO-pretreatment The w h e a t s t r a w was s o a k e d w i t h C a O - s o l u t i o n straw)

(5

X per dry matter

f o r one h o u r a t a m b i e n t t e m p e r a t u r e a n d t h e n m i l l e d i n a s i n -

gle disk refiner,

d e w a t e r e d on a b e l t p r e s s a n d n e u t r a l i z e d w i t h

H2S04. NaOH-pretreatment Cooking w i t h 12-20

X NaOH p e r d r y m a t t e r i s a w e l l k n o w n d e l i g n i -

f i c a t i o n process i n the pulp industry.

Due t o t h e h i g h c o s t s o f t h e

c h e m i c a l and t h e n e c e s s i t y o f i t s r e c o v e r y , for

the enzymatic s a c c h a r i f i c a t i o n

an N a O H - p r e t r e a t m e n t

c a n a f f o r d o n l y a l o w NaOH c h a r g e .

-

195

-

Our e x p e r i e n c e s showed t h a t t h e m o s t p r o m i s i n g p a r a m e t e r s a r e t h e following:

2

X NaOH p e r d r y m a t t e r , 180

OC cooking temperature,

10

minutes r e t e n t i o n time a t t h e cooking temperature. The e x p e r i m e n t s a l s o showed t h a t t h e h e m i c e l l u l o s e w i l l n o t b e solubized w i t h this pretreatment.

T h e r e f o r e no f u r t h e r

reaction o f

t h e pentoses can t a k e p l a c e as i n t h e a u t o h y d r o l y s i a p r o c e s s w i t h o u t NaOH. N e g a t i v e a s p e c t s o f t h e NaOH p r e t r e a t m e n t a r e t h e l i g n i n d e g r a d a t i o n which n e c e s s i t a t e s t h e c l e a n i n g o f l i q u o r s i n a wastewater treatment plant, production,

l e s s q u a n t i t y o f t h e h y d r o l y s i s r e s i d u e f o r energy

a n d a more d i f f i c u l t r e c o v e r y o f a c e t i c a n d f o r m i c a c i d

f r o m t h e a l k a l i n e c o o k i n g l i q u o r c o m p a r e d t o an a c i d l i q u o r .

Steam p r e t r e a t m e n t

F o r many y e a r s s t e a m p r e t r e a t m e n t h a s b e e n w e l l known i n t h e p u l p and p a p e r i n d u s t r y e s p e c i a l l y

for

the production o f viscose

p u l p by t h e k r a f t p r o c e s s where t h e p e n t o a e a a r e s o l u b i z e d b y a wat e r and/or are:

steam p r e h y d r o l y s i s .

Typical p r e h y d r o l y s i s parameters

t e m p e r a t u r e b e t w e e n 3 0 a n d 60 m i n u t e s a t s maximum t e m p e r a t u r e .

The d i g e s t e r s u s e d a r e e i t h e r b a t c h a u t o c l a v e s o r c o n t i n u o u s t u b e digesters.

A p e n t o s e r e c o v e r y o f up t o 80 % c a n be a c h i e v e d .

We a l s o u s e d t h e s e p a r a m e t e r s f o r t h e p r e t r e a t m e n t o f s t r a w and f o u n d o u t t h a t between 160-170 high extent,

OC

t h e x y l o s e c a n be r e c o v e r e d t o a

b u t t h a t the glucose y i e l d i n the f o l l o w i n g enzymatic

h y d r o l y s i s was l o w ( u p t o 60

X

o f the theoretical).

T h e r e f o r e we r e -

duced t h e r e t e n t i o n t i m e and i n c r e a s e d t h e t e m p e r a t u r e . m a x i m a l g l u c o s e y i e l d a t 190-200 batch cooking.

OC and 10 min.

We f o u n d s

retention time i n

F o r an i n d u s t r i a l p r o c e s a t h e d i g e s t e r s h o u l d be de-

s i g n e d f o r a t r e a t m e n t w i t h s a t u r a t e d s t e a m up t o 220 OC. The x y l o s e y i e l d waa r e d u c e d a t h i g h e r t e m p e r a t u r e s ,

due t o

t h e d e g r a d a t i o n o f t h e x y l o s e t o f u r f u r a l and p o l y m e r i z a t i o n o f f u r f u r a l i n the presence o f l i g n i n .

T h e r e f o r e we d e c i d e d t o a p p l y a

t w o s t e p p r o c e s s where t h e p e n t o a e s a r e s o l u b i z e d i n t h e f i r s t s t e p w i t h t h e known p a r a m e t e r s o f t h e p u l p i n d u s t r y a n d a s e c o n d s t e p a t a t e m p e r a t u r e o p t i m a l f o r t h e g l u c o s e p r o d u c t i o n i n enzymatic saccharification. The p e n t o s e y i e l d i n s a c c h a r i f i c a t i o n o f s t e a m t r e a t e d m a t e r i a l i s l o w because pentoaanea a r e e x t r a c t e d a f t e r t h e f i r s t s t e p o f t h e steam t r e s t m e n t .

c)

-

196

S a c c h a r i f i c a t i o n and c o s t s n a l y s i s U s i n g t h e t h r e e p r e t r e a t m e n t 8 (CaO,

NaOH

o r s t e a m o n l y ) we o b -

tained i n l a b scsle or small p i l o t scale hydrolysis yielda reported i n lab.

3.

TABLE 3 Y i e l d s o f g l u c o s e o b t a i n e d f r o m some s e l e c t e d l i g n o c e l l u l o s i c s b y e n z y m a t i c h y d r o l y s i s on l a b o r a t o r y o r s m a l l p i l o t s c a l e . C o n d i t i o n s f o r h y d r o l y s i s were a l w a y s 1 0 F P U / g , 50 O C , 48 t o 1 2 h.

cellulose content % o f oven d r y w e i g h t

raw m a t e r i a l

glucose y i e l d ?A o f t h e o r y

wheat s t r a w

37

75

s w e a t sorghum b a g a s s e

42

65

palm press f i b r e s

22

60

palm p r e s s s t a l k s

22

h a r d woods birch,

(poplar,

beech,

fruit-tree)

s o f t woods ( s p r u c e ,

31

pine)

38

-

-

42

60

42

50

36

65

reed gras

42

70

s u g a r cane b a g a s s e ,

pith

Pulpe

b a r k from beech

m u n i c i p a l s o l i d waste

(BRAM)

-

37

80

53

100

23

80

cellulose r i c h fraction o f 23

90

70 65

100

corn stover

potato residue,

-

I

-

213

80 65 75

00

about 50

The g r o s s m a t e r i e l b a l a n c e f o r t h e p r o d u c t i o n o f s u g a r a f r o m wheat s t r a w

(450 t o n s / d a y )

g i v e n i n Tab.

f o r the three pretreatment options i s

4.

The l o w e s t c o n v e r s i o n c o s t s o f t h e steam p r e t r e a t m e n t ,

1.49

sugar a r i s e w i t h

AUS/kg

i f c r e d i t f u r f u r a l as a b y p r o d u c t i s t a k e n

i n t o a c c o u n t t h e p r i c e w o u l d b e a t 1.34

AUS/kg

sugar.

s i o n c o s t s a r e baaed on z e r o raw m a t e r i a l c o s t a , a day dream. a procesa,

I n A u s t r i a wheat s t r a w would c o a t ,

a b o u t 0 . 8 AUS/kg

These c o n v e r -

which a r e o f course

i f needed f o r s u c h

a n d b a s e d on t h i s p r i c e t h e p r o d u c t i o n

c o s t s f o r a u g a r a a r e b e t w e e n 3.2 sent t h i s p r i c e i s f a r t o o high

a n d 4.5

AUS/kg

(Tab.

4).

A t pre-

to be c o n s i d e r e d t o b e a b a s i s f o r

t h e production o f b i o t e c h n o l o q i c s l products from sugars derived from

lignocellulosics.

Nevertheless,

197

-

s e n s i t i v i t y a n a l y s e s c l e a r l y show

t h a t c o n v e r s i o n c o s t s c o u l d be c o n s i d e r a b l y r e d u c e d ( i n t o t a l about

5 0 X ) by an i m p r o v e m e n t o f enzyme r e c y c l i n g and r e d u c e d enzyme l o a ding.

Moreover,

t h e p r o c e s s e c o n o m i c s l o o k s much mor'e f a v o r a b l e i f

some o f t h e l i g n i n c o u l d b e s o l d aa b y p r o d u c t s a n d i f a l l t h e p e n t o ses a r e c o n v e r t e d t o f u r f u r a l and i f a c e t i c a c i d and f o r m i c a c i d

are r e c o v e r e d . TABLE 4 P r o d u c t i o n o f s u g a r s f r o m wheat s t r a w . Cross m a t e r i a l b a l a n c e b a s e d on d r y m a t t e r , c o n v e r s i o n and p r o d u c t i o n c o s t s f o r 3 p r e t r e a t m e n t o p t i o n s ( s e e t e x t ) . P l a n t s i z e 450 MT/day, o t h e r c o n d i t i o n s f o r c o s t e v a l u a t i o n see ( r e f . 1 3 ) . MT = m e t r i c tons. ~ _ _ _ _ _

steam

N G 7 - l

CEO

190-200 OC

refiner

milling

steam 180

enzyme p r o t e i n n e e d e d , MT/day

5.30

6.58

6.24

hexoses produced,

121

90

135

50

77

MT/day

pentoses produced,

MT/day

f u r f u r a l produced,

MT/day

fuel,

81

hydrolysis residue,

MT/day

I

'

OC/

__

~~~

4

0

0

164

250

120

conversion c o s t s f o r sugar-mix (hexoses without

+

pentoses)

f u r f u r a l byproduct,

w i t h f u r f u r a l byproduct,

AUS/kg

AUS/kg

1.49

1.99

2.01

1.34

-

-

3.12

4.56

p r o d u c t i o n c o s t s f o r sugar-mix

w i t h a p r i c e o f 0 . 8 AUS/kg straw,

wheat

with f u r f u r a l byproduct,

AUS/kg s u g a r - m i x 12 A u s t r i a n S c h i l l i n g (AUS)

= 1 US

3 71

D o l l a r (June 1988)

I n c o n s i d e r i n g f u t u r e s c e n a r i o s one must a l s o b e aware t h a t p l a n t end f o r e s t g e n e t i c s i s v e r y r a p i d l y d e v e l o p i n g and w i l l w i t h o u t any d o u b t l e a d t o l i g n o c e l l u l o s i c p l a n t s ( t r e e s , s p e c i f i c a l l y d e s i g n e d f o r i n d u s t r i a l needs.

g r a s s e s a.o.1

Short r o t a t i o n forestry

w i t h p o p l a r a n d w i l l o w a l r e a d y g i v e h e c t a r y i e l d s u p t o 20 t o n s d r y matter/year

a s c o m p a r e d t o 2-3 t o n s / y e a r

with conventional forestry

(ref.

14).

198

-

With t h e m o d e r n t e c h n i q u e s o f g e n e t i c e n g i n e e r i n g i t

s h o u l d a l s o be p o s s i b l e t o d e v e l o p p l a n t s w i t h a h i g h and a l o w l i g n i n c o n t e n t .

cellulose

T h i s t o g e t h e r w i t h many o t h e r a s p e c t s

w i l l c o n t i n u o u s l y i n c r e a s e t h e i m p o r t a n c e o f l i g n o c e l l u l o s i c s as a

raw m a t e r i a l source. ACKNOWLEDGEMENT The w o r k o f t h e I n s t i t u t e o f B i o c h e m i s t r y was i n p a r t s u p p o r t e d by t h e B u n d e s m i n i s t e r i u m f;'r

Wissenschaft und Forschung,

Wien.

REFERENCES M . Schmuck, I. P i l z , M. Hayn e n d H. E s t e r b s u e r , I n v e s t i g a t i o n o f c e l l o b i o h y d r o l a s e from Trichoderms r e e s e i by s m a l l angle Xr a y s c a t t e r i n g , B i o t e c h n o l . L e t t . 8 ( 6 ) ( 1 9 8 6 ) 397-402. 2 P . M . A b u j a , M. Schmuck, I. P i l z , P. Tomme, M. C l s e y s s e n s and H. E s t e r b a u e r , S t r u c t u r a l a n d f u n c t i o n a l d o m a i n s o f c e l l o b i o hydrolase I from Trichoderma r e e s e i , Eur. Biophys. J. 1 5 (1988) 339-342. 3 M. Hayn and H. E s t e r b a u e r , S e p a r a t i o n a n d p a r t i a l c h a r a c t e r i z a t i o n o f Trichoderma r e e s e i c e l l u l s s e by f a s t chromatofocusing, J . Chrom. 329 ( 1 9 8 5 ) 379-387. 4 H. E s t e r b a u e r , M. Hsyn, G. J u n g s c h a f f e r , E . T a u f r s t z h o f e r a n d J . Schurz, Enzymatic c o n v e r s i o n o f l i g n o c e l l u l o s i c m a t e r i a l s t o s u g a r s , J. Wood Chem. T e c h n o l . 3 ( 3 ) ( 1 9 8 3 ) 261-287. 5 H. v a n T i l b e u r g h , P. Tomme, M. C l a e y s s e n s , R . B h i k h a b h a i a n d G. P e t t e r s o n , L i m i t e d p r o t e o l y s i a o f t h e c e l l o b i o h y d r o l a s e I f r o m T r i c h o d e r m a r e e s e i , FEBS L e t t . 2 0 4 ( 2 ) ( 1 9 8 6 ) 223-227. 6 T . T . T e e r i , P . L e h t o v s s r a , S. K a u p p i n e n , I. S a l o v u o r i a n d J . K n o w l e s , Homologous d o m a i n s i n T r i c h o d e r m a r e e s e i c e l l u l o l y t i c enzymes: gene s e q u e n c e a n d e x p r e s s i o n o f c e l l o b i o h y d r o l a s e 11, Gene 5 1 ( 1 9 8 7 ) 4 3 - 5 2 . 7 H. Ooshima, M. S a k a t a a n d Y . H a r a n o , A d s o r p t i o n o f c e l l u l a s e f r o m T r i c h o d e r m a v i r i d e on c e l l u l o s e , B i o t e c h n o l . B i o e n g . 25 ( 1 9 8 3 ) 3103-3114. 8 J.Y. S t u a r t and D.L. R i s t r o p h , A n a l y s i s o f c e l l u l s s e - c e l l u l o s e a d s o r p t i o n d s t a : s f u n d a m e n t a l a p p r o a c h , B i o t e c h n o l . B i o e n g . 27 ( 1 9 8 5 ) 1056-1059. 9 J. S c h u r z , J . B i l l i a n i , A . H E n e l , W.D. E i g n e r , A. J a n o s i , M. H s y n a n d H. E s t e r b a u e r , R e a c t i o n m e c h a n i s m e n d s t r u c t u r a l c h a n g e s a t enzymatic d e g r a d a t i o n o f c e l l u l o s e by Trichoderma r e e s e i c e l l u l a s e , A c t a P o l y m e r i c a 36(2) ( 1 9 8 5 ) 76-80. 1 0 W . S t e i n e r , W . S a t t l e r a n d H. E s t e r b a u e r , A d s o r p t i o n o f T r i c h o derma r e e s e i c e l l u l a s e on c e l l u l o s e : e x p e r i m e n t a l d a t a a n d t h e i r a n a l y s i s by d i f f e r e n t e q u a t i o n s , B i o t e c h n o l . B i o e n g . ( 1 9 8 8 i n press), 11 S.B. L e e , H.S. Shin, D.D. Ryu, A d s o r p t i o n o f c e l l u l a s e : E f f e c t o f p h y s i c o c h e m i c a l p r o p e r t i e s o f c e l l u l o s e on a d s o r p t i o n a n d r s t e o f h y d r o l y s i s , B i o t e c h n o l . B i o e n g . 27 ( 1 9 8 2 ) 2137-2153. 1 2 W . S a t t l e r , H. E s t e r b s u e r , 0. G l e t t e r a n d W . S t e i n a r , The e f f e c t o f enzyme c o n c e n t r a t i o n o n t h e r a t e o f t h e h y d r o l y s i s o f c e l l u l o s e , B i o t e c h n o l . Bioeng. (1988 accepted for p u b l i c a t i o n ) . 1

13 14

199

-

H . S t e i n m i l l e r , T h e s i s , Graz U n i v e r s i t y o f Technology, A u s t r i a (1988). C . F . M i t c h e l l , Advances i n h a r v e s t i n g t e c h n o l o g y f o r s h o r t r o t a t i o n p o p l a r and w i l l o w s , i n : D. S c h l i e p h a k e and P . Kramer ( E d . ) A g r i c u l t u r a l S u p l u s e s , DECHEMA, F r a n k f u r t , 1 9 8 5 , 1 2 1 - 1 4 3 .

This Page Intentionally Left TBlank

THE FUTURE OF THE LIGNOCELLULOSIC W A S T E S BIOCONVERSION I. SPILDA,

A.

B L A t E J a n d M.

KOSIK

The S l o v a k T e c h n i c a l U n i v e r s i t y ,

Lignocellulose

r e f o r m ca.

95

X

B r a t i s l a v a 81237,

Czechoslovakia

i s t h e most a b u n d a n t p o l y m e r on t h i s p l a n e t whe-

o f t h e d r y w e i g h t o f p l a n t o r i g i n a t e d wastes.

c o n v e r s i o n o f l i g n o c e l l u l o s e w a s t e s (LCW)

The

i n t o valuable products i s

u s u a l l y t r e a t e d i n t h e b r o a d e r c o n t e x t o f renewable r e s o u r c e s and i s studied from technical,

s o c i a l and economical aspects.

I n recent

y e a r s e c o l o g i c a l a s p e c t s became i m p o r t a n t a l s o a n d o n l y s i g n i f i c a n t p r e s s u r e f r o m t h e p u b l i c s e c t o r f o r e n v i r o n m e n t a l c o n s i d e r a t i o n and r e c y c l i n g dominate t h e s t i m u l a t i o n o f t h e search f o r a b e t t e r u t i l i z a t i o n o f LCW. The b a s i c p r o b l e m w h i c h f a c e d t h e r e s e a r c h e r s i n LCW c o n v e r s i o n have changed l i t t l e . simple

-

Technically speaking the process i s r e l a t i v e l y

t h e h y d r o l y s i s o f c e l l u l o s e polymer i n t o s a c c h a r i d e s and

t h e subsequent b i o c o n v e r s i o n i n t o e t h a n o l o r o t h e r p r o d u c t s . p r o c e s s e s were i m p l e m e n t e d c a . y e a r s ago,

Such

80 y e a r s ago and were i m p r o v e d 5 0

b u t i n l a s t 20 y e a r s t h e y became e c o n o m i c a l l y n o n - c o m p e t i

t i v e w i t h crude o i l bssed products.

I n the future,

a commercially

f e a s i b l e c o n v e r s i o n p r o c e s s would s o l v e a t l e a s t two problems. tly,

Firs-

t h e problem o f waste d i s p o s a l and secondly t h e need f o r a con-

tinuous supply o f low-cost l o s e s a r e renewable,

chemical intermediates.

Since l i g n o c e l l u -

b o t h p r o b l e m s can be s o l v e d .

THE FUTURE OF RESOURCES The p r i n c i p s l p r o b l e m s e m e r g i n g f r o m t h e a n a l y s i s e x t e n s i v e l y discussed i n t e c h n i c a l l i t e r a t u r e ,

are associated with fundamentally

d i f f e r e n t c h a r a c t e r i s t i c s o f LCW r e s o u r c e s . sed and b u l k y .

Also,

when compared w i t h c r u d e o i l r e s o u r c e s . resource tent.

of

They a r e w i d e l y d i s p e r -

t h e d e n s i t y o f energy i s ca. Thus,

one o r d e r l o w e r

LCW r e p r e s e n t s t h e

a d e c e n t r a l i z e d c h a r a c t e r and o f a l o w e r e n e r g y con-

Table 1 l i s t s t h e p o t e n t i a l s o u r c e s and t h e i r d i v e r s e n a t u r e .

-

202

-

TABLE 1 P o t e n t i a l s o u r c e s o f LCW Source

Description

Crop r e s i d u e s

S t r a w , weeds, c o r n c o b s a n d s t a l k s , sugarcane waste, wasted f o d d e r

Forest residues

Bark,

sawdust,

A g r i c u l t u r a l by-products

Bagasse,

S o l i d urban wastes

Waste p a p e r ,

The q u a n t i t i e s ( a n d a l s o q u a l i t i e s ) areas o f t h e world.

branches,

g r a i n bran,

twigs

seeds,

hulls

vegetable wastes

o f LCW a r e d i f f e r e n t i n v a r i o u s

E s t i m a t e s o f a v a i a l i i l i t y o f LCW i n t h e USA i n -

d i c a t e t h a t c o l l e c t a b l e LCW f r o m a g r i c u l t u r e i s i n t h e r a n g e o f 500

400

-

800 m i l l i o n t o n s o f d r y m a t t e r p e r y e a r and from f o r e s t r y ,

600 m i l l i o n t o n s p e r y e a r .

source c o u l d s u p p l y ca.

5

-

When c o n v e r t e d i n t o e n e r g y ,

ca.

this

7 X o f t h e t o t a l consumed e n e r g y .

Ligno-

c e l l u l o s i c s p r o d u c t i o n i n E u r o p e i s g i v e n i n T a b l e 2 a n d shows t h a t ca.

300 m i l l i o n t o n s i s a v a i l a b l e a t p r e s e n t .

TABLE 2 L i g n o c e l l u l o s i c s p o t e n t i a l i n Europe [ i n m i l l i o n s o f t / y ] Source

Now

Future

Wood short r o t a t i o n forestry

1 85

conventional forestry s c r u b woodland Sweet s o r g h u m A g r i c u l t u r a l wastes

1e.g.

straw1

M u n i c i p a l s o l i d wastes

I n the l o n g term perspective, changed d r a m a t i c a l l y , a v a i l a b l e wastes,

100

150

5

10

2

250

250

250

60

75

t h e amount o f LCW i n E u r o p e w i l l n o t b e

although t h e r e i s a wider range o f l o c a l l y

o n l y two c a t e g o r i e s a r e a v a i l a b l e i n l a r g e scale.

T h e r e i s c e r e a l s t r a w a n d a n i m a l manure.

A t present, about a h a l f

o f t h e s t r a w p r o d u c e d i n West E u r o p e i s b u r n t

i n the fields.

The

-

203

-

amount o f manure i s n o t p r o p e r l y r e c o r d e d a n d i s c o n s i d e r e d f o r r e c y c l i n g as f e r t i l i z e r

[ a f t e r p a s s i n g b i o m e t h a n i z a t i o n c y c l e i n some

cases]. The w h o l e p r o b l e m o f LCW h a s b e e n t h e s u b j e c t o f many r e v i e w studies covering regional,

n a t i o n a l o r g l o b a l c o n s i d e r a t i o n and t h e y

i l l u s t r a t e t h e e x t e n t o f i n t e r e s t w h i c h LCW h a s g e n e r a t e d .

On t h e

b a s i s o f the consideration o f the cspacity o f p l a n t s t o produce l i g n o c e l l u l o s e a s s o c i a t e d w i t h t h e a n t i c i p a t e d i m p a c t o f new b i o t e c h n o logies,

i t can be p r e d i c t e d t h a t t h e i n c r e a s e i n p l a n t p r o d u c t i v i t y

w i l l continue.

I f no c h a n g e s o c c u r i n w e s t E u r o p e t h e p r e d i c t e d s u r -

p l u s i n y e a r 2 0 0 0 w i l l be so l a r g e t h a t i t w i l l r e q u i r e t h e u t i l i z a t i o n o f technologies other than the conventional burning applied a t the present time. n o t be used,

I f t h e s u r p l u s o f p r o d u c t i o n a s w e l l a s LCW w i l l

t h i s may f o r m many e c o n o m i c a l a n d p o l i t i c a l p r o b l e m s .

T h i s w i l l a l s o form a major impetus for t h e f u r t h e r development o f conversion technologies. ached t h e i r maximum,

As c h e m i c a l c o n v e r s i o n t e c h n o l o g i e s r e -

the future l i e s i n bioconversion technologies.

CCoommbbuusst it oi onn oor r oot thheer r t h t heer rmmoocchheemmi ci caal l t teecchhnnool o l oggi e i ess aar ree ppr reeddoommi -i nnaannt tl y u s e d i n LCW c o n v e r s i o n p r e s e n t l y . S i n c e t h e l y u s e d i n LCW c o n v e r s i o n p r e s e n t l y . S i n c e t h eyy aar ree nnoot t new, new, i inn hhi isst toor ryy t thheeyy hhaavvee ppaasssseedd ppeer ri o i oddss oof f i innt teennssi ivvee r reesseeaar rcchh aanndd ddeevvee- l ol oppmmeennt .t . They They aar ree eer ri inngg ppr ri inncci ippl leess, , ssuucchh ppr roocceesssseess i iss

bbaasseedd on on t thhee aappppl li iccaat ti ioonn oof f cchheemmi ci caal l aanndd eennggi n i nee- aanndd t thhee ppoot teennt ti iaal l f foorr f fuur rt thheer r i m p r o v e m e n t s o i m p r o v e m e n t s of f

ccoonnssi d i deer reedd aass l o l oww. . PPr roommi si si n i ngg t teecchhnnool o l oggi e i ess oof f t thhee f fuut tuur ree wwoouul dl d bbee bbaasseedd on on bbi o i occoonnvveer rssi o i onn oor r t thhee aappppl li iccaat ti ioonn oof f ppr ri inn- cci ippl leess oof f bbi o t e c h n o l o g y . I n T a b l e 3 i o t e c h n o l o g y . I n T a b l e 3 l li isst tss t thhee mmaai n i n ppr roocceesssseess uut ti il li izzcc c o n v e n s i o n o t h e r t h a n t h e d i r e c t c o n s u m p t i o i inn LCW LCW c o n v e n s i o n o t h e r t h a n t h e d i r e c t c o n s u m p t i onn as as aanni m i maal l f e f eeedd

oor r f of ooodd. .

oe c rhm eo mci hc e am l itceacl h pn rool coegsi e ssa r e predomit icoen s so er s o t h e r t h e r m Th se B i o l o gC i coaml b u p sr o n a n t l y u s e d i n LCW c o n v e r s i o n p r e s e n t l y . S i n c e t h e y a r e n o t new, ) n s i v e r e s e a r c h and deveI n de i ns hi ti u sto r yc a yt h e( Ny ) h a v e p a s s e d p C eo r imo bduss toi of n i n( Ct e T ha e rpmp ol icchae tmi o i cna l o cf o cn hv ee m r si icoanl a (D) C loom ( C ) a r e b a s e d on t h e pp moesnt ti n . g They nd engineC a t a l y t i c l i q u e f a c t i o n ( R ) Le a rni d i tsi o, n a n (0 n fgi l pl r di necpiopsl e d , t hC)e p o t e n t i a l f o r f u r t h e r i m p r o v e m e n t s o f h .y dPr o i si n g(0) ( Cs, i D Fs e ur m c he nptraotci oe n sse s) considered A a sc i dl o w r ol ymsi s technologies o f the A fnuateur roeb i c d i g e s t i o n ( C ) P u l p i n g (C) w o u l d b e b a s e d on b i o c o n v e r s i o n o r t h e a p p l i c a t i o n o f p r i n t i ot hn e ( Cm) a i n p r o c e s s e s u t i l i z c Scaicpcl h e as r iof if c be itoi ot enc h( Rn ,o l oD g) y . I n TDa e b ll ei g 3n i lf ii cs st s i n LCW c o n v e n s i o n o t h e r t h a n t h e d i r e c t c o n s u m p t i o n as a n i m a l f e e d

o r food.

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204

-

The f e a s i b i l i t y o f t h e b i o c o n v e r s i o n o f LCW h a s b e e n s t u d i e d intensively

i n recent years.

I n general,

lignocellulose materials

a r e n o t i n a s u i t a b l e s t a t e f o r immediate b i o c o n v e r s i o n .

The c r y s -

t a l l i n i t y o f the c e l l u l o s e serves t o r e t a r d the r a t e o f production o f monomeric sugars.

Also,

t h e a c c e s s i b i l i t y o f enzyme s y s t e m s t o

p o l y s a c c h a r i d e s i s r e s t r i c t e d by t h e p r e s e n c e o f l i g n i n .

Pretreatment

( p h y s i c a l o r c h e m i c a l ) o f LCW i s t h e r e f o r e n e c e s s a r y t o e v e r c o m e these problems.

A t t h e p r e s e n t t i m e we h a v e a r e l a t i v e l y g o o d f u n d a -

m e n t a l k n o w l e d g e on enzymes d e g r a d i n g l i g n o c e l l u l o s e o r i t s compon e n t s e.g.

cellulase,

xylanases,

l i g n i n a s e s a n d some p i l o t p l a n t s

or s m a l l s c a l e p r o d u c t i o n o f c e l l u l a s e s , m a i n l y o p e r a t i n g f r o m T r i choderma f u n g i .

P o t e n t i a l p o s s i b i l i t i e s f o r the f u t u r e promise the

g e n e t i c a l l y manipulated microorganisms which could increase the p r o d u c t i o n a n d a c t i v i t y o f l i g n o c e l l u l o s e d e g r a d i n g enzymes.

Enzyme

systems d e g r a d i n g c e l l u l o s e e x h i b i t an u n u s u a l c o m p l e x i c i t y

i n the

t e r m o f t h e number o f p r o d u c e d enzymes a n d t h e i r m e c h a n i s m o f a c t i o n . From f u n g i ,

T r i c h o d e r m a h a s b e e n i s o l a t e d a n d c h a r a c t e r i z i d e d t o mo-

r e t h a n 1 8 d i f f e r e n t t y p e s d e g r a d i n g c e l l u l o s e a n d x y l s n . The u n d e r s t a n d i n g o f t h e n a t u r e o f v a r i o u s a c t i v i t i e s o f enzymes on d i f f e r e n t components o f l i g n o c e l l u l o s e i s v e r y i m p o r t a n t f o r p r a c t i c a l reasons. As t h e r e a r e l a r g e p o s s i b i l i t i e s o f i m p l e m e n t i n g x y l a n a s e s i n p u l p production,

there i s s growing i n t e r e s t i n the production o f xyla-

n o l y t i c enzyme s y s t e m s r a t h e r t h a n t h e s e a r c h f o r c e l l u l o l y t i c s y s tems w h i c h a r e a s s o c i a t e d w i t h e t h a n o l p r o d u c t i o n on a l a r g e s c a l e . The q u e s t i o n o f enzyme s p e c i f i c i t y h a s n o t y e t b e e n s o l v e d . I n t h e f i e l d o f c e l l u l a s e s t h e gene f o r enzyme p r o d u c t i o n was a n a l y z e d a n d an a t t e m p t h e r o r g a n i s m s 1e.g.

e x i s t s t o introduce proper vectors i n t o o t -

yeast] f o r the production o f cellulases.

C e l l u l a s e s a r e p r o d u c e d n o t o n l y b y f u n g i b u t a l s o b y many m i c r o o r g a n i s m s s u c h as b a c t e r i a , animals.

Therefore,

a c t i m o m y c e t e s and b y some i n v e r t e b r a t e

the search i s s t i l l continuing f o r the optimal

m i c r o o r g a n i s m p r o d u c i n g c e l l u l o l y t i c enzymes. i s the u t i l i z a t i o n o f thermophilic cellum,

C.

Considered as important

b a c t e r i a e.g.

C l o s t r i d i u m thermo-

thermosaccharolyticum,as h a v i n g advantageous h i g h r e a c t i o n

r a t e s and a wide range o f m e t a b o l i z i n g s u b s t r a t e a . E n z y m a t i c h y d r o l y s i s o f LCW h a s v e r y h i g h y i e l d s ,

because en-

zymes o n l y c a t a l y z e h y d r o l y s i s r e a c t i o n s a n d n o t s u g a r d e g r a d a t i o n r e a c t i o n s as i n t h e case o f chemical h y d r o l y s i s .

But s t i l l a consi-

d e r a b l e i m p r o v e m e n t i s n e c e s s a r y b e f o r e t h e p r o c e s s w i l l be e c o n o m i c a l l y competitive.

The m o a t i m p o r t a n t r e a e a r c h p r o b l e m s a r e :

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205

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a) performance o f pretrentment b ) p r o d u c i n g more e f f e c t i v e a n d l e s s e x p e n s i v e enzymes c) developing a hydrolysis process with greater y i e l d s ,

pro-

d u c t s c o n c e n t r a t i o n and r a t e s .

With t h e c u r r e n t s t a t e o f t h e a r t ,

t h e most c o s t l y p a r t o f t h e p r o -

ceas i s t h e enzyme p r o d u c t i o n w h i c h t a k e s ca.

1 4 days i n a fedup t o

I n the hydrolysis section,

o f enzyme c a n b e o b t a i n e d .

t h e most i m p o r t a n t p a r a m e t e r s as y i e l d , etc.

-

A t these conditons,

b a t c h aystern a t 1 5 0 g o f b i o m a s s / l i t e r . 100 I U / L . h r

10

concentrations,

duration

a r e s t r i c t l y i n t e r r e l a t e d a n d depend o n t h e p r e t r e a t m e n t a n d Y i e l d s a r e h i g h e r i n d i l u t e d systems ( l o w

t h e t y p e o f enzyme u s e d .

i n h i b i t i o n ) a n d l o n g e r r e a c t i o n t i m e s make h i g h e r y i e l d . i s s e l e c t e d as t h e f i n a l p r o d u c t ,

b i o c o n v e r s i o n (SSF p r o c e s s ) c a n b e u s e d . s i b l e products from glucose,

I f ethanol

s i m u l t a n e o u s s a c c h a r i f i c a t i o n and The g e n e r a l scheme o f p o s -

r e s u l t i n g from enzymatic h y d r o l y s i s i s

given i n Fig.

1. The f o u r m a i n g r o u p s o f p r o d u c t s a r e c o n s i d e r e d

(biopolymers,

alcohols,

c a r b o x y l i c a c i d s and b i o c h e m i c a l s ) .

Ethanol Isopropanol Butanol 2,3 b u t a n d i o l Glycerol Carboxylic acids Alcohols Acetic acid Propanoic a c i d Butanoic acid Lactic acid Gluconic a c i d Fumaric a c i d Itaconic acid Malic acid C i t r i c acid

Biopolymers Enzymes Polysaccharides

P HB Biochemicals

! Aminoacids Antibiotics Hormones e t c

Fig.

1. F e r m e n t a t i o n p r o d u c t s o f g l u c o s e As t h e r e a r e p r o b l e m s w i t h t h e n o n h o m o g e n e i t y o f t h e s u b s t r a t e

and l o w y i e l d s o f g l u c o s e ,

t h e i n t e r e s t i n b i o c o n v e r s i o n moved more

t o hemmicellulose p a r t o f l i g n o c e l l u l o s i c s .

Hemmicelluloses are rea-

d i l y s o l u b l e i n a l k a l i a n d a p p r e c i a b l e q u a n t i t i e s o f them a r e c u r r e n t l y n o t u t i l i z e d aa w o u l d b e d e s i r e d . p i n g and p u l p p r o c e s s i n g ,

R e l e a s e d f r o m wood d u r i n g p u l -

xylans are dissolved i n apentliquor.

Some

-

206

-

wood p r e h y d r o l y s a t e s c o n t a i n s s i g n i f i c a n t q u a n t i t i e s o f x y l o o l i q o s a ccharides. z e d Form.

There are a l s o wastes which c o n t a i n x v l a n s i n non-hydroly-

A t steam e x p l o s i o n p r o c e s s water s o l u b l e f r a c t i o n o f x y l a n

i s also released.

S t e a d y i n c r e a s e s o f non-wood p u l p i n g ,

especially

i n wood d e f i c i e n t c o u n t r i e s w i l l b e a c c o m p a n i e d b y an a d d i t i o n a l stream o f xylan,

t h e u s e o f w h i c h must a w a i t t h e d e v e l o p m e n t o f e c o -

n o m i c a l l y and t e c h n o l o g i c a l l y b e n e f i c i a l p r o c e s s .

Direct bioconver-

s i o n o f x y l a n b y m i c r o o r g a n i s m s seems t o b e t h e m o s t p r o m i s i n g approach t o the developing process.

The x y l a n b i o c o n v e r s i o n r o u t e

2.

i s s i m i l a r t o g l u c o s e and i s g i v e n i n F i g .

r

Ethanol Butanol Aceton

I

Acetic acid Lactic acid Butyric acid

X Y L O S E

Polysaccharides

I

Enzymes

sc P Fig.

2 . F e r m e n t a t i o n p r o d u c t s from D-xylose

Xylan can be h y d r o l y z e d t o x y l o s e

(enzymatically or chemically)

and s u b s e q u e n t l y x y l o s e can be c o n v e r t e d t o a s i n g l e c e l l p r o t e i n

or t o low molecular weight products (alcohols).

I n chemical hydro-

l y s i s p a r t o f xylan i s converted i n t o f u r f u r a l ,

w h i c h may i n h i b i t

further microbial fermentation. i n m i l d (enzymic)

F o r t h i s reason t h e r e i s i n t e r e s t

h y d r o l y s i s o f xylans.

The i n d u s t r i a l a p p l i c a t i o n

o f enzymes u t i l i z a t i o n f o r x y l a n c o n v e r s i o n i s h i n d e r e d b y t h e complex structure of

t h e s u b s t r a t e and r e q u i r e m e n t

for specific

com-

p o s i t i o n o f u s e d enzymes.

A t p r e s e n t , m o s t o f LCW b i o c o n v e r s i o n o c c u r s a t a n a e r o b i c d i g e s t i o n o r b i o m e t h a n i z a t i o n l e a d i n g t o b i o g a s and i t s e n e r g y uses. The amount a n d q u a l i t y o f b i o g a s p r o d u c e d d e p e n d s o n t h e LCW u s e d

-

207

-

and e n g i n e e r i n g t e c h n o l o g y o f t h e w h o l e s y s t e m .

The b i o g a s s y s -

tem r e p r e s e n t s t e c h n o l o g i c a l l y a d v a n c e d s m a l l s c a l e LCW u t i l i z a t i o n , however, connected w i t h v a r i o u s e c o l o g i c a l problems. S i n c e b i o c o n v e r s i o n o f c e l l u l o s e and advanced,

hemicelluloses

are w e l l

t h e l i g n i n a l s o a v a i l a b l e i n LCW r e p r e s e n t s an

u n d e r u-

sed r e s o u r c e .

Because o f i t s u n i q u e p o l y p h e n o l i c s t r u c t u r e ,

g r a d a t i o n o f l i g n i n by m i c r o o r g a n i s m s i s v e r y d i f f i c u l t .

biode-

L i g n i n and

i t s degradation products are then major sources o f p o l l u t i o n i n the wood p u l p i n g i n d u s t r y .

I n nature,

the degradation o f l i g n i n i s the

r e s u l t o f t h e c o o p e r a t i v e a c t i o n o f f u n g i and b a c t e r i a ,

but the

a b i l i t y t o d e g r a d e o r m o d i f y l i g n i n i s l i m i t e d t o r e l a t i v e l y few microorganisms.

S t u d i e d t h e most,

f u n g i Phanerochaete

chrysosDorium

s e c r e t s a c o m p l e x o f enzymes who a r e c a t a l y z i n g l i g n i n d e g r a d a t i o n t o l o w m o l e c u l a r compounds.

A l s o i n t h i s area,

t h e usage o f g e n e t i c

m a n i p u l a t i o n i s e x p e c t e d t o e n h a n c e t h e p r o d u c t i o n o f enzymes as w e l l as i t s a c t i v i t i e s . FUTURE P R O S P E C T S FOR LCW B I O C b N V E R S I O N

I t i s v e r y d i f f i c u l t t o p r e d i c t the p r o b a b i l i t y o f success o r failure

o f some f u t u r e b i o c o n v e r s i o n p r o j e c t .

We m u s t r e a l i z e

t h a t f u t u r e m a r k e t s w i l l b e more u n c e r t a i n a n d c a p i t a l r e q u i r e m e n t s l a r g e r than i n the past.

These f a c t o r s c o n t r i b u t e t o m i s t a k e s t h a t

a r e made i n d e v e l o p i n g new t e c h n o l o g i e s .

Despite this,

there i s

common i n t e r e s t i n d e v e l o p i n g LCW ( o r b i o m a s s ) c o n v e r s i o n t e c h n o logies.

I n West E u r o p e u n d e r t h e name o f LEBEN ( L a r g e E u r o p e a n B i o -

mass E n e r g y N e t w o r k )

a s e r i e a o f p r o j e c t s was s t a r t e d s e r v i n g a s an

i n s t r u m e n t f o r r e g i o n a l r e d e v e l o p m e n t a n d f o r m i n g an o p p o r t u n i t y f o r l a r g e s c a l e e x p l o i t a t i o n o f renewable resources.

Such p r o j e c t s

o f f e r a t l e a s t a p a r t i a l s o l u t i o n t o t h e problems associated w i t h a g r i c u l t u r a l surpluses, g l o b a l issues: quality,

etc.

l a n d use,

greenhouse e f f e c t ,

r u r a l e m p l o y m e n t a s w e l l a s some q u a l i t y o f t h e environment,

water

The o b j e c t i v e s o f LEBEN i n c l u d e t h e d e m o n s t r a t i o n o f

i n t e g r a t e d biomass based systems f o r m a t e r i a l and energy p r o d u c t i o n , a n d t h e d e v e l o p m e n t and e x p a n s i o n o f a g r i c u l t u r e and f o r e s t r y

and

t h e improvement o f socioeconomic c o n d i t i o n s w h i l e p r o v i d i n g

b-

?I

s t a n t i a l environmental benefits. T h i s t y p e o f development w i l l r e q u i r e a t f i r s t , changes i n b i o c o n v e r s i o n p r o c e s s e s . aches t o t e c h n o l o g i c a l ,,push"

changes.

theory o f technological

I n theory

t h e r e a r e two appro-

The f i r s t a p p r o a c h , change,

technological

c a l l e d the

c o n s i d e r s i n v e n t i o n and

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208

-

i n n o v a t i o n as s e l f g e n e r a t i n g a c t i v i t i e s . process responding t o technological s i d e r a t i o n o f the f u t u r e market. "pull"

theory

I t i s t h e s c i e n c e pushed

o p p o r t u n i t i e s w i t h o u t t h e con-

The s e c o n d a p p r o a c h c a l l e d t h e

o f t e c h n o l o g i c a l change i s based on i n v e n t i o n s and

i n n o v a t i o n d e t e r m i n e d b y t h e e x i s t i n g n e e d s o r b y e c o n o m i c a l demand

(or m a r k e t p l a c e ) .

I n t h e f i e l d o f b i o c o n v e r s i o n o f LCW,

b o t h appro

aches a r e a c c e p t a b l e because f u t u r e t e c h n o l o g i c a l change o c c u r s as a r e s u l t o f both forces.

However,

t y p e s o f c h a n g e s is d i f f e r e n t .

t h e p r e d i c t a b i l i t y o f t h e s e two

The s c i e n c e p u s h e d i n n o v a t i o n c a n

n o t be p l a n n e d f o r t h e o b v i o u s reasons. o n s a r e more e a s i l y f o r e c a s t e d ,

The demand p u l l e d i n n o v a t i -

b u t i t m u s t be r e a l i z e d t h a t some

new p r o c e s s c a n b e c o m m e r c i a l i z e d o n l y i f i t p r e s e n t s some a d v a n t a ges t o w a r d s t h e e x i s t i n g ones.

B i o c o n v e r s i o n o f l i g n o c e l l u l o s i c can

be d e f i n e d as a demand p u l l e d p r o b l e m , ventions o f biotechnology.

a p p l y i n g new s c i e n t i f i c

F o r the near future,

in-

i t i s expected t h a t

w i t h b e t t e r k n o w l e d g e o f s u b s t r a t e p r o p e r t i e s new a r e a s w i l l b e op e n e d f o r p r o d u c t d e v e l o p m e n t b a s e d on b i o c o n v e r s i o n o f l i g n o c e l l u l o s e and/or

i t s components.

REFERENCES

1

2

3

4 5 6 7

8 9

10

D.O. H a l l and J. Coombs, The A q r o - E n e r g y F i l i e r e : E x p e r i e n c e s a n d P e r s p e c t i v e s , I n : The A g r o - T e c h n o l o g i c a l S y s t e m s t o w a r d s 200 0 (G. A n t o n e l l i , A . Q u a d r i o - C u r z i o e d s . ) , E l s e v i e r , A m s t e r dam, 1 9 8 8 . J. Coombs, B i o t e c h n o l o g y a n d b i o m a s s e n e r g y , I n : Biomass-Regen e r a b l e Energy (D.O. H a l l a n d R . P . O v e r e n d , e d s . ) , W i l e y , New York 1987. M. A r e s t a a n d G. F o r t i , C a r b o n d i o x i d e aa a s o u r c e o f c a r b o n , D . R e i d e l P u b l . Co., 1 9 8 7 . F. P a r i s i , Advances i n l i g n o c e l l u l o s i c s h y d r o l y s i s and i n u t i l i z a t i o n o f hydrolyzates, I n : L i g n o c e l l u l o s i c M a t e r i a l s (A. F i e c h t e r e d . ) , A k a d e m i e V e r l a g , B e r l i n 1 9 8 9 , p . 53. L.R. L y n d l , P r o d u c t i o n o f e t h a n o l f r o m l i g n o c e l l u l o s i c s m a t e r i a l u s i n g t h e r m o p h i l i c b a c t e r i a , In: L i g n o c e l l u l o s i c M a t e r i a l s ( A . F i e c h t e r ed.), Akademie V e r l s g , B e r l i n 1 9 8 9 . A.G. L o b a n o k , B.G. B a b i t s k a y a a n d Zh.H. B o g d a n o v s k a y a , M i k r o b n i j s i n t e s n a osnove t s e l j u l o z y ( C e l l u l o s e based m i c r o b i s l s y n t h e s i s ) , Nauka i t e c h n i k a , M i n s k 1 9 8 8 . A.A. Klesov (eds.), T s e l j u l o l y t i t s c h i j e m i k r o o r g a n i z m y i ferment y ( C e l l u l o l y t i c m i c r o o r g a n i s m and enzymes), I t o g i n a u k i i t e c h n i k y , V o l . 10, V N I I T I Moscow, 1 9 8 8 . M.N. Monakhov, B i o k o n v e r s i y a t s e l j u l o s y : m i k r o b i o l o g i y a i b i o chemia ( C e l l u l o s e b i o c o n v e r s i o n : m i c r o b i o l o g y and b i o c h e m i s t r y ) , I t o g i n a u k i i t e c h n i k y , V a l . 11, V N I I T I M O S C O W 1 9 8 8 . A . B l a z e j and P . B i e l y , B i o c o n v e r s i o n o f p l a n t x y l a n s , I n : Wood and C e l l u l o s i c s ( J . F . Kennedy, G . O . P h i l l i p s , P.A. Williams, e d s . ) , E . Horwood L t d . , C h i c h e s t e r 1987. L.H. S e e n a t h , I n d u s t r i a l B i o t e c h n o l o g y I n t e r n a t i o n a l 1 9 8 8 / 8 9 , Longman, 1988.

DETOXIFICATION OF PHENOL POLLUTED SOIL BY SOME N O C A R D I A

AND BASIDIOMYCETES E.

MALARCZYK,

R.

APALOVIC,

2.

M.

LEWICKA-KRbL,

3.

STASZCZAK and A.

KOCHMAQSKA-RDEST, LEONOWICZ

Department o f B i o c h e m i s t r y , M a r i a Curie-Sklodowska Sklodowska P l a c e 3 , 20-031 L u b l i n , P o l a n d

University,

SUMMARY I t has been s t a t e d t h a t i n t r o d u c i n g t h e m i c r o b i o l o g i c a l mater i a l i n t o t h e s o i l c o n t a m i n a t e d b y p h e n o l and heavy m e t a l s ( m a i n l y i r o n ) improves t h e chances o f d e c r e a s i n g t h e d i s a s t r o u s e f f e c t s o f a d e v a s t a t e d n a t u r a l e n v i r o n m e n t . N o c a r d i a sp. b e l o n g i n g t o A c t i n o m y c e t e s a n d B a e i d i o m y c e t e s f u n g i known a s l i g n i n a n d p h e n o l i c subs t a n c e s t r a n s f o r m e r a . When g r o w n i n t h e medium e n r i c h e d w i t h wood d u s t w h i c h comes f r o m f u r n i t u r e m a n u f a c t u r i n g , were added i n t o t h e c o n t a m i n a t e d s o i l s . As a r e s u l t o f t h e r e l a t i v e l y s h o r t i n c u b a t i o n p e r i o d , a c o n s i d e r a b l e d e c r e a s e o f t h e p h e n o l c o n t e n t and g r o w t h a c c e l e r a t i o n were o b s e r v e d .

INTRODUCTION The p r o b l e m o f r e c u l t i v a t i o n o f f o u n d r y i n d u s t r y d e s t r u c t e d s o i l and t h e u t i l i z a t i o n o f i n d u s t r i a l wastes, l i o n s o f tons every year,

w h i c h amounts t o m i l -

i s u r g e n t because o f c o n t i n u o u s devasta-

t i o n t o t h e human e n v i r o n m e n t [ r e f .

11. The m o s t a r d u o u s c o n t a m i n a n t s

a r e p h e n o l i c s u b s t a n c e s and t h e wastes t h e y i n c l u d e e.g. o f phenol-formaldehyde r e s i n ,

t u t e a source o f about 1 k q o f pure phenol o u t d i n g mass.

t h e remains

which a f t e r depolirnerization,

consti-

o f 1000 t o n s o f mol-

I t i s u s u a l l y s t o r e d i n dumps a n d f r o m t h e r e i t i s washed

directly i n t o the soil, minates t h e environment.

r i v e r s or w a t e r r e s e r v o i r s w h e r e i t c o n t a Therefore,

there i s a very urgent problem

w i t h t h e e f f i c i e n t d e t o x i f i c a t i o n o f s u c h w a s t e m o l d i n g dump mass and i f p o s s i b l e i t s r a t i o n a l u t i l i z a t i o n .

U s i n g i t as a c o n s t i t u e n t

i n t h e b i t u m e n maaa f o r r o a d s u r f a c e s o r t o p r e v e n t s o i l e r o s i o n (what happens from t i m e t o time,

ref.

2 and 3 ) d o e s n o n p a y .

I t re-

q u i r e s huge c o s t s a n d d o e s n o t s o l v e t h e p r o b l e m a t a l l . This r e p o r t deals w i t h t h e a d v i s a b i l i t y o f enrichment o f s o i l s contaminated w i t h p h e n o l i c substances,

w i t h m i c r o o r g a n i s m s decompo-

s i n g them,

-

210

s t a b i l i z e d on t h e w o o d - d u s t

as a n a t u r a l c a r r i e r .

Such

a procedure a c c e l e r a t e s t h e r e g e n e r a t i o n process o f t h e contaminat e d s o i l and makes i t e a s i e r i n i t through environmental

for

o t h e r m i c r o o r g a n i s m s t o s e t t l e down

enrichment

w i t h a v a i l a b l e carbon sour-

ces. METHODS Microbioloqical material preparation

10 s t r a i n s o f N o c a r d i a w e r e g r o w n [ r e f .

4 and 5 1 i n t h e H o r v a t h

4 1 w i t h s u c c i n i c a c i d as a c a r b o n s c u r c e .

and A l e x a n d e r

medium [ r e f .

T h e wood d u s t

f r o m t h e b e e c h t r e e i n 4 g p o r t i o n s was p l a c e d i n a

f l a t b o t t o m e d f l a s k o f 20 m l v o l u m e a n d w e t t e d w i t h 2 5 m l o f m i n e r a l

or

c u l t u r e medium-according t o H o r v a t h and A l e x a n d e r

f o r Nocardia,

a c c o r d i n g t o Czapek-Lindeberq

The m i x t u r e o f

[ref.

Nocardia autotrophica ( 4 s t r a i n s ) ,

61 f o r

fungi.

N o c a r d i a opaca ( 2 s t r a i n s ) ,

&-

c a r d i a q l o b e r u l a ( 3 s t r a i n s ) and s e p a r a t e m y c e l i a o f I n o n o t u s o b l i quus, P l e u r o t u s o s t r e a t u s and P o l y s t i c t u s v e r s i c o l o r ( c u l t i v a t e d according t o r e f .

6 ) was s p r e a d i n d i v i d u a l l y on t h e s t e r i l e medium.

M a c e r a t i o n o f t h e m i c r o b i o l o g i c a l m a t e r i a l w i t h wood d u s t was c a r r i w e t t i n g t h e medium as r e -

ed o u t a t room t e m p e r a t u r e f o r 3 m o n t h s ,

q u i r e d w i t h t h e s t e r i l e c a r b o n w h i c h was f r e e o f m i n e r a l c u l t u r e medium. P r e p a r a t i o n o f s o i l samples contaminated w i t h p h e n o l E x p e r i m e n t s w e r e c a r r i e d o u t on v a r i o u s s o i l s s a t u r a t e d e i t h e r w i t h p h e n o l o r w i t h p h e n o l m i x e d w i t h m o l d i n g mass o b t a i n e d f r o m the Lorry Factory i n Lublin.

S a w d u s t f r o m t h e c o n i f e r s was a l s o i n -

t r o d u c e d t o some p o r t i o n s o f b o t h t y p e s o f s o i l . cluded i n the detailed description.

The RESULTS a r e i n -

D e t e r m i n a t i o n s o f changes i n t h e

p h e n o l c o n c e n t r a t i o n w e r e made u s i n g t h e c o l o r i m e t r i c m e t h o d i n t h e r e a c t i o n w i t h DASA I r e f .

41.

To t h i s a i m ,

and p r e c i p i t a t e d from 5 m l o f w a t e r ,

1 g o f s o i l was a c i d i f i e d

c e n t r i f u g e d and t h e r e a c t i o n

w i t h D A S A was c a r r i e d o u t i n t h e f l u i d .

The a b s o r b a n c e was r e a d a t

4 8 0 nm. RESULTS Recultivation o f the s o i l c u l t i v a t e d w i t h phenol Eight

Portions o f 250 g o f the f e r t i l e ,

non-sterile

s o i l prepa-

r e d a c c o r d i n g t o t h e p r o c e d u r e p r e s e n t e d i n T a b l e 1, were p l a c e d on g l a s s f u n n e l s and w e t t e d w i t h 150 m l o f 0 . 5

X p h e n o l s o l u t i o n added

-

211

-

i n t o t h e s o i l . i n t h r e e p o r t i o n s o f 5 0 m l e v e r y t w o weeks. h r s from t h e l a s t phenol a d d i t i o n i n t o each sample, o a t Avena s a t i v a were sowed i n . t h r e e days.

24

The s e e d s b e g a n t o g e r m i n a t e a f t e r

After

The e x p e r i m e n t l a s t e d t w o weeks.

germination,

After

32 seeds o f t h e seven days from

t h e l e n g t h o f t h e r o o t s and g r e e n o v e r g r o u n d p o r t i o n s

w e r e measured.

After

1 4 d a y s t h e g r e e n mass o f l e a v e s was s e p a r a t e d

f r o m t h e r o o t mass and t h e i r w e i g h t was d e t e r m i n e d .

The p h e n o l c o n -

t e n t i n t h e s o i l was d e t e r m i n e d on t h e f i r s t a n d l a s t d a y s o f t h e experiment.

The r e s u l t s a r e g i v e n i n T a b l e 1 a n d F i g .

1.

TABLE 1 The p h e n o l l e v e l on t h e f i r s t a n d t h e l a s t day o f t h e Avena s a t i v a g r o w t h m e a s u r e d i n 8 t y p e s o f s o i l . P h e n o l c o n c e n t r a t i o n was d e t e r m i n e d a s A400nm i n O A S A r e a c t i o n ( d e t a i l s i n t h e t e x t ) .

;oil dos

Phenol c o n c e n t r a t i o n l t h day 1 0 t h day

Characteristic

1.

Soil (control)

2.

Soil

+

3.

Soil

4.

3.

5.

3.

+ +

0.02

0.05

0.75

1.70

+ p h e n o l + sawdust ( c o n t r o l )

0.55

0.86

Nocardia (10 s t r a i n s )

0.05

0.75

f u n g i ( 3 species)

0.04

0.60

0.05

0.20

phenol ( c o n t r o l )

6.

2. + fungi ( 3 species)

7.

2.

8.

3 . + Nocardia + Fungi

+ N o c a r d i a (10 s t r a i n s )

0.03

0.25

0.04

0.45

R e c u l t i v a t i o n o f s o i l c o n t a m i n a t e d w i t h m o l d i n q mass Soil,

l:l:l,

m o l d i n g mass a n d s a w d u s t were m i x e d a t t h e v o l u m e r a t i o

o b t a i n i n g t h e t o t a l volume o f t h r e e l i t e r s .

The f u n g i and

N o c a r d i a were added t o i n d i v i d u a l s a m p l e s i n g l a s s j a r s as shown i n T a b l e 1. The o b s e r v a t i o n s o f i n d i v i d u a l v a r i a n t s were c a r r i e d o u t f o r 22 m o n t h s w i t h p e r i o d i c a l d e t e r m i n a t i o n s o f t h e p h e n o l c o n t e n t

i n t h e s o i l samples.

E v e r y week t h e s a m p l e s w e r e s t i r r e d f o r b e t t e r

o x i d a t i o n a n d t h e w a t e r was

refilled.

After

a year,

b i o l o g i c a l use-

f u l n e s s o f t h e i n d i v i d u a l v a r i a n t s was e x a m i n e d b y s o w i n g s e e d s o f r e d f e s c u e g r a s s o f Leo s p e c i e s .

A f t e r t h r e e weeks,

d r y masses o f

o v e r g r o u n d and r o o t p a r t s o f a l l t h e v a r i a n t s were compared. p e r i m e n t a l d a t a i s shown i n T a b l e 2 and F i g .

2.

The e x -

-

212

A

I 3

6

7

8

NUMBER OF SOIL

B

3

5

7

8

NUMBER OF SOIL

F i g . 1. The d i f f e r e n c e b e t w e e n w e i g h t ( A ) a n d l e n g h t (8) o f l e a v e s and r o o t s o f Avena s a t i v a a f t e r t h e c u l t i v a t i o n o n 8 t y p e s o f s o i l (according t o Table1)ntaminated with ohenol: leaves, r o o t s I( d e t a i l s i n t h e t e x t ) . '

-

-

- 3 0, I

I-

I

P Y

2

n

1

1

2

3

4

5

6

7

L

8 NUMBER OF SOIL

F i g . 2. The d i f f e r e n c e b e t w e e n w e i g h t o f l e a v e s a n d r o o t s o f r e d fascue g r a s s (Leo s p e c i e s ) a f t e r t h e c u l t i v a t i o n on 8 t y p e s o f s o i l ( a c c o r d i n g t o T a b l e 2 ) e n r i c h e d i n m o l d i n g masses a f t e r a y e a r c u l t i v a t i o n w i t h f u n g i and N o c a r d i a ; 0 leaves, rn - r o o t s ( d e t a i l s i n t h e t e x t ) .

-

DISCUSSION The r e c u l t i v a t i o n o f c o n t a m i n a t e d s o i l b y p h e n o l was r e a c h e d b y i t s e n r i c h m e n t w i t h m i c r o o r g a n i s m a c a p a b l e o f p h e n o l a n d l i g n i n decomposition.

R e c u l t i v a t i o n o f s o i l c o n t a m i n a t e d w i t h dump masses

-

213

-

c a r r i e d o u t i n t h e same way showed t h e a p p l i c a b i l i t y o f t h i s p r o c e dure i n decreasing the degradation o f the n a t u r a l environment.

I t was s t a t e d t h a t t h e p r e s e n c e o f p h e n o l i n c r e a s e s t h e r o o t mass g r o w t h ,

i n h i b i t i n g t h e development o f a p l a n t s green p a r t s .

The

a d d i t i o n o f wood m a t e r i e l seems t o d i m i n i s h t h e s e p r o p o r t i o n s .

TABLE 2 The p h e n o l i c s u b s t a n c e s l e v e l m e a s u r e d i n 8 t y p e s o f s o i l e n r i c h e d i n m o l d i n g mass. The c o n c e n t r a t i o n was d e t e r m i n e d as A4OOnm i n O A S A r e a c t i o n ( d e t a i l s i n t h e t e x t ) a t t h e b e g i n n i n g and end o f a y e a r cultivation.

'Oil

Nos

P h e n o l i c s u b s t a n c e s (A4001

Characteristic

s t a r t 4 t h month 2 4 t h month ~~

1.

0.05

s o i l (control)

0.03

0.04

2.

S o i l - m o l d mass-sawdust

0.41

0.22

0.12

3.

S o i l - m o l d mass ( c o n t r o l )

0.40

0.35

0.30

4.

S o i l - m o l d mass-Nocardia

0.42

0.05

0

5.

S o i 1 - m o l d mass- f u n g i

0.31

0.05

0.21

6.

S o i l - m o l d mass-sawdust-fungi

0.38

0.03

0.35

7.

S o i l - m o l d mass-sawdust-Nocardis

0.40

S o i l - m o l d mass-sawdust-Nocardis-fungi

0.38

0 0

0.33

18.

Enrichment of

(control)

0

t h e c o n t r o l v a r i a n t s i n t h e fungus m a t e r i a l o r

i n Nocardis c e l l s a c c e l e r a t e s s i g n i f i c a n t l y t h e phenol substance d e c o m p o s i t i o n w h i c h i s o b s e r v e d i n more e x u b e r a n t p l a n t g r o w t h . I n b o t h t y p e s o f c o n t a m i n a t i o n t h e most adequate p r o p o r t i o n s between t h e p l a n t s o v e r g r o u n d and u n d e r g r o u n d p a r t s were o b t a i n e d i n t h e variants,

w i t h o u t t h e a d d i t i o n o f sawdust.

g r o w t h p r o v e s t o b e more p r o f i t a b l e ,

The e f f e c t o f f u n g i on p l a n t

w h i c h c s n be s e e n i n t h e b y -

products wich are substances o f a n t i b i o t i c types w i t h Nocardia c e l l s which can a f f e c t

the plants secondarily.

The m o s t u s e f u l v a r i a n t s

seem t o b e t h o s e w h i c h i n c l u d e b o t h f u n g u s m a t e r i a l a n d A c t i n o m y c e -

t e s whose

m u t u a l p r o p o r t i o n s c a n be c h a n g e d .

The p o s i t i v e e f f e c t

b o t h k i n d a o f microorganisms i s o u t o f q u e s t i o n and c r e a t e s t h e

Of PO-

s s i b i l i t y o f a d d i n g m o l d i n g mass i n t o weak s o i l s ( p o o r f e r t i l i t y , b u i l d side-spaces, a t u r f layer,

embankmenta e . t . c . ,

to

where a f t e r t h e f o r m a t i o n o f

t h e y can p r o t e c t t h e ground f r o m l o w e r i n g .

The b i o l o -

g i c a l r e g e n e r a t i o n o f h e a v y i n d u s t r y w a s t e s c r e a t e s a g r e a t e r chance

-

214

-

for c o n t a m i n a t i o n t o d e c r e a s e t h a n i f u s i n g t h e w a s t e s w i t h o u t b e i n g processed.

The s o u r c e o f f u n g u s m a t e r i a l a n d A c t i n o m y c e t e s

can be

i n t e n t i o n a l y c u l t i v a t e d o n w a s t e m a t e r i a l s f r o m t h e f o o d a n d wood industry [refs.

7,

8 a n d 91.

REFERENCES

1

2 3

4 5

6

7 8 9

A . L e o n o w i c z , M . W o j t a s - W a s i l e w s k a , J . R o g a l s k i a n d J. L u t e r e k B i o l o g i c a l decomposition o f l i g n o c e l l u l o s e , I n t e r b i o t e c h '87, P r o c . I n t e r n . Symp. o n B i o t e c h n o l o g y , B r a t i s l a v a , C z e c h o s l o v a k i a , June 25-26, 1987, p p . 416-451. Z . Kin, S t a t e and knowledge on l i g n i n s and t h e i r a p p l i c a t i o n , Chem. T e c h n o l . C ' 'il., 7 ( 1 9 8 5 ) 1 1 - 2 0 . K.-E. Eriksson, Swedish developments i n biotechnology r e l a t e d t o t h e p u l p and p a p e r i n d u s t r y , T a p p i , 68 ( 1 9 8 5 ) 46-55. E . Malarczyk, Transformation o f phenolic a c i d s by Nocardia, Acta M i c r o b i o l . P o l o n . , 38(1) ( 1 9 8 9 ) 4 5 - 5 3 . E . M a l a r c z y k , I. K o r s z e h - P i l e c k a , J. R o g a l s k i a n d A . L e o n o w i c z , G u a i a c o l and i s o v a n i l l i c a c i d a s m e t a b o l i t e s i n t h e t r a n s f o r m a t i o n o f methoxyphenolic a c i d by Nocardia a u t o t r o p h i c a , Phytochem i s t r y , 26(3) (1987) 1321-1324. A . L e o n o w i c z , R.U. E d g e h i l l a n d J-M. B o l l a g , The e f f e c t o f pH o n t h e t r a n s f o r m a t i o n o f s v r i n q i c and v a n i l l i c a c i d s by t h e l a c c a s e o f R h i z o c t o n i a p r a t i c o l a and Trametes v e r s i c o l o r , Arch. Microb i o l . . 137 (1984) 89-96. A . L e o n o w i c z , A . P a s z c z y h s k i a n d J. T r o j a n o w s k i , The way o f h i g h p r o t e i n biomass p r o d u c t i o n f r o m d i s t o l l e r y brew, P o l i s h P a t e n t No 9 3 0 5 4 , 1 9 7 4 . A . L e o n o w i c z a n d J . T r o j a n o w s k i , The way o f h i g h p r o t e i n b i o m a s s p r o d u c t i o n f r o m w h e y , P o l i s h P a t e n t No 9 5 2 2 5 , 1 9 7 5 . J . T r o j a n o w s k i , A . L e o n o w i c z a n d S . L a b e d z i h s k i , The way o f h i g h p r o t e i n biomass p r o d u c t i o n from l i g n o c e l l u l o s i c waste m a t e r i a l s , P o l i s h P a t e n t No 1 1 9 8 9 2 , 1 9 7 6 .

THE DETOXIFYING ROLE OF FERRO-PHENOLIC COMPLEXES PRODUCED B Y N D C A R D I A

E.

MALARCZYK,

A.

LEONOWICZ

J.

KOCHMANSKA-RDEST,

M.

WOJTAS-WASILEWSKA

Department o f B i o c h e m i s t r y , M a r i a Curie-Sklodowska Sklodowska P l a c e 3, 2 0 - 0 3 1 L u b l i n , P o l a n d

and

University,

'

SUMMARY The p o s s i b i l i t y o f r e c u l t i v a t i o n o f s o i l c o n t a m i n a t e d w i t h t h e e x c e s s o f p h e n o l s u b s t a n c e s o r i r o n b y some s p e c i e s o f N o c a r d i a was s t u d i e d . A complex o f t h e p r o p e r t i e s c l o s e t o n a t u r a l s i d e r o p h o r e was f o r m e d u n d e r t h e m o d e l c o n d i t i o n s i n t h e b a r r e n s o i l c o n t a m i n a ted w i t h t h e a d d i t i o n o f p-hydroxybenzoic a c i d i n t h e presence o f c e l l s . I t was s t a t e d t h a t t h e p r o c e s s p r o t e c t e d v e g e t a t i o n , a s c o u l d be seen i n t h e c a s e o f , t h e b e t t e r o a t g r o w t h i n t h e s o i l .

I N T f
and s e l f - p u r i f y i n g

However,

system i n

i n t h e case o f s i g n i f i c a n t

c o n t a m i n a t i o n by i n d u s t r i a l s u b s t a n c e s i t becomes s t e r i l e and r e quires recultivation

( r t t . 1).

One o f t h e f a c t o r s d i s t u r b i n g t h e s o i l s e c o l o g i c a l b a l a n c e i s contamination o f the environment by substrates o f phenol character o f excesses o f heavy m e t a l s i n t h e s o i l . czyk e t a l . ,

ref.

ferro-phenolic

21,

As s t a t e d e a r l i e r ( M a l a r -

some s p e c i e s o f N o c a r d i a c a n f o r m a s p e c i f i c

complex i n t h e c o n d i t i o n o f excess p - h y d r o x y b e n z o i c

and p r o t o c a t e c h u i c a c i d s i n a s l i g h t l y a l k a l i n e medium.

The p r e s e n -

ce o f t h e complex p r o t e c t s N o c a r d i a c e l l s a g a i n s t t h e d i r e c t e f f e c t o f n o n o and d i p h e n o l s a s w e l l a s i r o n e x c e s s . T h i s p a p e r c o n s i d e r s t h e p o s s i b i l i t y o f u s i n g N.

opaca complex

f o r m i n g p r o p e r t i e s t o i n c r e a s e t h e f e r t i l i t y o f s o i l s d e s t r o y e d by c o n l i n u o u s e n v i r o n m e n t a l c o n t a m i n a t i o n on t h e b a s i s o f m o d e l i n v e s tigations. MATERIALS AND METHODS The c o m p l e x f o r m i n g s t r a i n s o f N o c a r d i a o p a c a DSM 43202,PCM

2136

-

216

2).

were u s e d i n t h e i n v e s t i g a t i o n s ( r e f . t i n g i t a t 160 O C f o r 6 h r s . calcium carbonate. zoic acid,

The o b j e c t o f t h e s t u d i e s

i n the experiments,

was f e r t i l e s o i l w h i c h ,

was s t e r i l i z e d b y h e a -

The s o i l was a l k a l i z e d t o p h 7 . 4

using

The c o n t a m i n a t i n g a g e n t u s e d was p - h y d r o x y b e n -

w h i c h was a d d e d i n t o t h e s o i l i n t h e f o r m o f s o d i u m s a l t ,

i n t h e amount o f 0.3

%,w i t h t h e a d d i t i o n o f 0.06

X ferrous sulfa-

te. T h e r e were 3 v a r i a n t s i n t h e e x p e r i m e n t : soil,

non-sterile

non-sterile

acidic

a l k a l i z e d s o i l and s t e r i l e a l k a l i z e d s o i l .

c o n t r o l s a m p l e s were c o n t a m i n a t e d w i t h p - h y d r o x y b e n z o i c ferrous sulfate solution.

The

a c i d and

The b a s i c s a m p l e s i n c l u d e d t h e s u s p e n s i o n

o f N o c a r d i a c e l l s i n t h e amount c o r r e s p o n d i n g t o 3 0 mg o f d r y mass. S o i l p o r t i o n s were p l a c e d i n g l a s s c r y s t a l l i z e r s p r o t e c t e d a g a i n s t drying.

E v e r y day 3 g s o i l p r o b e s w e r e a c i d i e f i e d ,

w i t h 2 5 m l o f w s t e r and c e n t r i f u g e d . content

( b y means o f t h e t e s t w i t h M e r c k r e a g e n t ) ,

acid content

precipitated

Then i n t h e s u p e r n a t a n t i r o n p-hydroxybenzoic

( c o l o r e d r e a c t i o n w i t h DASA w i t h maximum a t 440nm)

and

p r e s e n c e o f t h e c o l o r e d p h e n o l - i r o n camplex (absorbance measurements a t 5 7 0 nm) were d e t e r m i n e d .

RESULTS The e f f e c t o f p - h y d r o x y b e n z o i c

a c i d o n t h e s o i l t h a t was me-

dium r i c h i n i r o n i o n s on t h e f o r m s t i o n o f t h e c o l o r e d p h e n o l - i r o n complex i n t h r e e k i n d s o f s o i l : r i l e was i n v e s t i g a t e d .

acid,

a l k a l i n e and a l k a l i n e b u t a t e -

The r e s u l t s a r e p r e s e n t e d i n F i g .

The r e s u l t s show t h a t t h e f e r r o - p h e n o l i c

1.

c o m p l e x was f o r m e d

only i n the s t e r i l e s o i l with the alkaline reaction.

The amount o f

c o m p l e x was h i g h e s t j u s t a f t e r t h e f i r s t day o f i n c u b a t i o n , c o r r e s p o n d e d t o s b o u t a 50 a c i d content.

which

l o w e r i n g o f primary p-hydroxybenzoic

D e c o m p o s i t i o n o f t h e c o m p l e x l a s t e d a b o u t one week.

The i r o n c o n t e n t i n t h e s t u d i e d v a r i a n t o f s o i l was e q u a l t o t h e complex c o n t e n t , form,

b u t t h e i r o n o c c u r r e d i n t h e medium i n t h e s o l u b l e

contrary t o the o t h e r v a r i a n t s o f the experiment.

s t e r i l e s o i l samples,

p-hydroxybenzoic

t h e same r a t e a s b e f o r e , soil.

I n t h e non-

a c i d decomposed a l m o s t a t

w i t h o u t s t a r t i n g i r o n i o n s adsorbed b y t h e

T h i s i n d i c a t e s a d i f f e r e n t mechanism o f p - h y d r o x y b e n z o i c

decomposition i n n o n - s t e r i l e After

t h e seven day i n c u b a t i o n ,

s o i l o f variant

acid

(but with Nocardia) s o i l s . when t h e c o m p l e x c o n t e n t i n t h e

I 1 1 was c l o s e t o z e r o ( T a b l e 11, t h e o a t s e e d was

sown b o t h i n t h e c o n t r o l a n d p r o p e r s o i l s o f t h i s v a r i a n t .

A t first

-

217

-

-

0

1

2

5;

B

A

3 4 5 TIME [DAYS]

1

2

C

U

3 4 5 TIME [DAYS]

1

2

3 4 5 TIME [DAYS1

F i g . 1. The c o m p s r i s o n o f t h e l e v e l o f i r o n ( 3 + ) , p - h y d r o x y b e n z o i c a c i d and f e r r o - p h e n o l complex i n t h r e e t y p e s o f s o i l . Acid s o i l ( 0 1; a l k a l i n e s o i l ( I 1; ~ l k a l i n es t e r i l e s o i l (A); A p-hydroxybenzoic a c i d l e v e l ; B - i r o n ( 3 + ) l e v e l ; C ferro-phenols complex l e v e l .

-

-

1 2

-

-

1 2 R A N T MATERIAL

F i g . 2 . The d i f f e r e n c e b e t w e e n w e i g h t e n d l e n g t h o f g r e e n p a r t s o f A v e n s s a t i v e p l a n t s a f t e r g r o w t h on a l k a l i n e s t e r i l e s o i l (A) a n d leae n r i c h e d i n Nocardia c u l t u r e s (8) ( d e t a i l s i n t h e t e x t ) ; 1 ves; 2 roots.

-

-

t h e p l a n t s f r o m t h e c o n t r o l were g r o w i n g f e s t e r e n d t h e g r e e n mass increase

WES

greater.

After t h r e e days t h e p l a n t s of both samples

were t h e same. A f t e r t e n d a y s t h e d i f f e r e n c e b e t w e e n t h e p l a n t s was s t a t i s t i c a l l y s i g n i f i c a n t , i n f a v o r o f t h e s o i l e n r i c h e d w i t h Nocerd i e c e l l s . The g r o w t h d i f f e r e n c e s a r e shown i n F i g . 2.

-

-

218

-

TABLE 1 The e f f e c t o f pH a n d s t e r i l e c o n d i t i o n s on t h e l e v e l o f i r o n ( ) + ) , p - h y d r o x y b e n z o i c a c i d and f e r r o - p h e n o l complex i n s o i l s e n r i c h e d i n Nocardia c u l t u r e s ( d e t a i l s i n the t e x t ) . Acid s o i l Ce(3+)

(+)

Alkaline s o i l

pOHB

cpx F e ( 3 + )

Alkaline s t e r i l e s o i l

pOHB

cpx Fe(3+)

pOH8+ c p x + +

I

C - t h e c o n t r o l s o i l , + N - t h e same s o i l w i t h N o c a r d i a c u l t u r e s p-OHB p-hydroxybenzoic acid, cpx - t h e f e r r o - p h e n o l complex

-

+

++

DISCUSSION The o b t a i n e d r e s u l t s a l l o w u t t o d r a w a f e w i m p o r t a n t c o n c l u sions:

1. S o i l i s a b l e t o r e s i s t c o n t a m i n a t i o n b y b o t h p h e n o l s u b s t a n ces and heavy m e t a l s because s t r o n g s o r p t i o n p r o p e r t i e s and a c t i v i t y o f m i c r o o r g a n i s m s make i t i m p o s s i b l e f o r s o i l t o f u l f i l l b a s i c detoxification functions 2.

i n t h e medium.

The mechanism o f t h e f e r r o - p h e n o l i c

complex f o r m a t i o n i n t h e

s o i l seems t o b e q u i t e a n a l o g o u s t o t h a t d e s c r i b e d p r e v i o u s l y ,

re-

f e r r i n g t o c h a n g e s i n t h e s y n t h e t i c N o c a r d i a o p a c a medium e n r i c h e d

with p-hydroxybenzoic a c i d (ref. 2 ) . des t h e p r e s e n c e o f N o c a r d i a opaca, and t h e p r e s e n c e o f p - h y d r o x y b e n z o i c

The n e c e s s a r y c o n d i t i o n ,

or p - a n i s i c a c i d i n excess,

needed f o r p r o t o c a t e c h u i c a c i d f o r m a t i o n . molecules o f p-hydroxybenzoic

besi-

i s a s l i g h t l y a l k a l i n e medium The c o m p l e x is made o f t w o

a c i d and two m o l e c u l e s o f p r o t o c a t e -

-

219

-

c h u i c a c i d w h i c h p r o b a b l y c o m b i n e t h r o u g h a common i r o n i o n .

Du-

r i n g t h e c o m p l e x d e c o m p o s i t i o n ( a l s o w i t h t h e p a r t i c i p a t i o n of>c a r d i a opaca c e l l s ) , takes place,

gradual dearomatization o f protocatechiuc acid

which i s tantamount t o t h e appearance o f e a s i l y a v a i -

l a b l e a l i p h a t i c compounds, H20

-

m e t a b o l i t e s o f K r e b s c y c l e a n d C02 a n d

t h e p r o d u c t s o f p h e n o l compounds c o m p l e t e c o m b u s t i o n .

3 . Formation o f a p e c u l i a r ferro-phenolic

complex i n t h e p r e -

sence o f N o c a r d i a i n s o i l causes r e p e a t e d t r a n s f o r m a t i o n o f i r o n ions i n t o a soluble state,

w h i c h p r o m o t e s t h e s e t t i n g o f new m i c r o -

organisms f r o m a i r and w a t e r .

Gradual d e g r a d a t i o n o f p-hydroxyben-

z o i c and p r o t o c a t e c h i u c a c i d s coming from t h e decomposing complex becomes a new,

a v a i l a b l e source o f carbon.

The p h e n o l s u b s t a n c e s

c a n b e u s e d a s monomers f o r d e v e l o p i n g humus m o l e c u l e s whose q u a l i t y determines s o i l f e r t i l i t y . 4.

The p h e n o l s u b s t a n c e s a r e o f t e n u s e d a s g r o w t h s t i m u l a t o r s

f o r many p l a n t s .

P-hydroxybenzoic a c i d i n the f i r s t stage o f oat.

g e r m i n a t i o n a n d g r o w t h seems t o f u l f i l l t h i s r o l e .

However,

t h e ex-

c e s s o f t h i s s u b s t a n c e i n t h e medium i s n o t p r o f i t a b l e f o r f u r t h e r p l a n t growth.

Therefore,

i n t h e main sample where p - h y d r o x y b e n z o i c

a c i d e x c e s s was r e d u c e d b y c o m p l e x f o r m a t i o n , t i o n c o n d i t i o n s were c r e a t e d ,

5.

more n a t u r a l v e g e t a -

and t h e p l a n t s grew b e t t e r .

The o b t a i n e d r e s u l t s show t h a t a r e a l p o s s i b i l i t y e x i s t s

o f a t l e a s t p a r t i a l r e c u l t i v a t i o n o f t h e s o i l d e s t r o y e d by t h e i n d u s t r i a l e m i s s i o n o f p h e n o l s u b s t a n c e s and excesses o f i n s o l u b l e iron.

Also,

t h e s o i l s b u r n t by a h i g h t e m p e r a t u r e s e t t i n g o f s u i t a b -

l e s t r a i n s o f Actinomycetes,

w i t h t h e i n t e n t i o n a l a d d i t i o n o f phe-

n o l s u b s t a n c e s and m e t a l l i c i o n s ,

can a c c e l e r a t e t h e p e r i o d o f t h e

soils natural reconstruction function significantly.

REFERENCES

1

A. Leonowicz and J.M. E o l l a g , Laccases i n s o i l and t h e f e a s i b i l i t y o f t h e i r e x t r a c t i o n , S o i l E i o l . E i o c h e m . , 1 9 ( 1 9 8 7 ) 237-

2

E . M a l a r c z y k , I. K o r s z e n - P i l e c k a a n d A . L e o n o w i c z . P r o d u c t i o n o f a f e r r o - p h e n o l i c complex by N o c a r d i a opaca and m e t a b o l i s m o f p h e n o l i c a c i d s i n b a c t e r i a l c u l t u r e s , P h y t o c h e m i s t r y , 28 (1989) 4 1 5 - 4 1 8.

242.

This Page Intentionally Left TBlank

MICROBIAL TREATMENT OF INDUSTRIAL WASTES L.I.

Vorobjevs,

and E. V.

L.V.

Modyanova,

P.B.

Terentijev,

F.M.

Chasaeva

Dovgilevich Moscow, USSR

Moscow S t a t e U n i v e r s i t y ,

B i o c h e m i c a l wastes t r e a t m e n t w i t h a c t i v a t e d s l u d g e as s m a j o r a g e n t i s e f f e c t i v e f o r t h e d e t o x i f i c a t i o n o f d e t o x i f i c a t i o n o f dom e s t i c wastes.

I n d u s t r i a l wastes d i f f e r from d o m e s t i c ones because

t h e y h a v e a n u n s t a b l e c o m p o s i t i o n e n d c o n t a i n many compounds i n c l u d i n g those t h a t are o x i d i z e d with d i f f i c u l t y .

I n those conditions

the o x i d i z i n g a c t i v i t y o f t h e microorganisms o f sludge i s repressed and t h e s c r e e n i n g o f a c t i v e d e s t r u c t o r 6 i s n e c e s s a r y . I n t h e S o v i e t Union, t h e m i c r o b i a l d e s t r u c t i o n o f hexamethyle-

nedismine, dioxine,

several surfsctanta,

aromatic dicarbonic acids,

n i t r o z o b e n z o l e have a t e c h n i c e l s o l u t i o n ( 1 - 5 ) .

dimethyl-

Recently,

s method o f wastes t r e a t m e n t o f a c e t a l d e h y d e and r u b l e r i n d u s t r i e s

was recommended. More t h a n 50 a c t i v e s t r a i n s o f m i c r o o r g a n i s m s w e r e i s o l a t e d from a c t i v a t e d s l u d g e and waste w a t e r s t h a t were c a p a b l e o f d e s t r o y i n g t h e t o x i c compounds o f t h e a e i n d u s t r i e s : n i c a l d e h y d e and t o l u o l ( 5 ) . mones e n d B a c i l l u s . 2-3

I n 5-6

croto-

The s t r a i n s b e l o n g t o t h e g e n u s Pseudo-

days t h e b a c t e r i a Bac.

pumillus u t i l i z e

After the hydratetion o f crotonic sl-

g/1 o f c r o t o n i c aldehyde.

dehyde,two

methylstyrola,

m o l e c u l e s o f a c e t a l d e h y d e a r e formed,

which are then oxi-

d i s e d t o a c e t i c a c i d and i n v o l v e d i n anabolism: CH3

-

CH

=

CH

-

-

CHO

CH3CH0

-

CH3COOH

A l m o s t a l l i s o l a t e d s t r a i n s o f pseudomonada u t i l i z e s u c h s u b s t a n c e s as t o l u o l , b i p h e n y l ,

n s p h t a l i n and a r e r e a i s t s n t t o h i g h

c o n c e n t r e t i o n o f Hg ( u p t o 200 m g / l ) .

Many o f t h e a e a t r a i n s c o n t a i n

p l s s m i d s o f b i o d e g r a d a t i o n t h a t d e t e r m i n e t h e i r g r o w t h on t h e aromst i c compounds.

Pseudomones s t r a i n s were c a p a b l e o f g r o w i n g a n d u t i -

l i z i n g or-methylstyrol quickly.

A f t e r 48 h r s .

u

-

methylatyrole i n

the i n i t i a l concentration 4 g / l

222 -

d i s s i p a t e d f r o m t h e g r o w t h medium.

The d e g r a d a t i o n p r o c e s s c a n l e a d t o t h e m i n e r a l i z a t i o n o f s u b s t r a t e s or t o t h e a c c u m u l a t i o h o f i n t e r m e d i a t e s w h i c h a r e m o r e t o x i c than,

t h e i n i t i a l compounds.

So t h e i n v e s t i g a t i o n o f c a t a b o l i c

p r o c e s s e s i s i m p o r t a n t i n t h e o r e t i c a l and p r a c t i c a l a s p e c t s . Eight products o f d - m e t h y l s t y r o l

d e g r a d a t i o n have been i d e n t i -

f i e d and t w o d i f f e r e n t ways o f d e g r a d a t i o n were p r o p o s e d ( F i g .

1).

I SH2

C- COOH

0' J

a!; CH\3,CH2

&TA

- CLEAVAGE

T

F i g . 1. P a t h w a y s f o r t h e d e g r a d a t i o n o f a - m e t h y l a t y r o l monas a e r u q i n o s a s t r a i n s .

b y Pseudo-

-

223

-

The m a i n way t h a t o x i d a t i o n b e g i n s i s f r o m t h e h y d r o x y l a t i o n of

t h e a r o m a t i c r i n g a n d t h e f o r m a t i o n o f compound I a n d 1 1 , f o l l o w -

i n g t h e m e t a s p l i t and f o r m a t i o n o f t h r e e a l i f a t i c a c i d s w i t h c a r b o n c h a i n s from C7

ato Cg

( t h e acids q u i c k l y involved i n the metabolism).

The m i n o r p a r h w a y l a y s t h r o u g h a - p h e n o l a c r y l i c phenylmethylglycol.

a c i d and

a,a -

A c e t o p h e n o n e was a l s o f o r m e d a n d t h e f o r m a t i o n

o f a w h o l e s p e c t r u m o f compounds i s t y p i c a l f o r m i c r o b i a l o x i d a t i o n o f xenobiotics.

B y u s i n g b a c t e r i a l s t r a i n s c a p a b l e o f d e s t r o y i n g a r o m a t i c com-

p o u n d s and c r o t o n i c a l d e h y d e and a c c u m u l a t e Hg,

a new t e c h n o l o g y

was d e v e l o p e d t h a t p r o v i d e s a p r e l i m i n a r y t r e a t m e n t o f w a s t e s ,

con-

t a i n i n g h i g h c o n c e n t r a t i o n s o f t o x i c compounds b y i m m o b i l i z e d m i c r o o r g a n i s m s b e f o r e t h r o w i n g t h e m i n t o t h e a e r o t a n k where s p o n t a n e o us m i c r o f l o r a m i n e r a l i z e less t o x i c

and more e a s i l y m e t a b o l i z e d com-

pounds. A t t h e U n i v e r s i t y o f K a s a n s t r a i n s were i s o l a t e d t h a t a r e a c -

t i v e d e s t r u c t o r s o f i s o m e r s o f p h t h a l i c a c i d u s e d as monomers i n the production o f polymeric materials.

Among o t h e r s ,

a flocculant

s t r a i n o f P h o d o c o c c u s was i s o l a t e d t h a t was c a p a b l e o f u t i l i z i n g a l l three isomers o f arylcarboxylates. t e s treatment o f terephtalic

T h i s t r a i n i s u s e d i n t h e was-

acid industry

2).

(Fig.

I n t h e U S S R a b i o t e c h n o l o g y o f w a s t e w a t e r t r e a t m e n t was d e v e l o p e d which c o n t a i n e d an a r o m a t i c x e n o b i o t i c 2 , 4 , 6 - t r i n i t r o t o l u o l ( u p t o 200 m g / l ) s u p p l i e s 94-100

(4).

and n i t r a t e s ( u p t o 650 m g / l ) .

This biotechnology

% water cleaning from t r i n i t r o t o l u o l s

and n i t r a t e s

The t r e a t m e n t i s r e a l i z e d b y u s i n g t w o i m m o b i l i z e d a s s o c i a t i o n s

o f bacteria.

One a s s o c i a t i o n f u n c t i o n s i n a c o n d i t i o n o f n i t r a t e and.

t h e o t h e r i n t h e c o n d i t i o n o f oxygen r e s p i r a t i o n .

By c h a n g i n g t h e

a n a e r o b i c a n d a e r o b i c c o n d i t i o n s o f b a c t e r i a c u l t i v a t i o n i t i s pos s i b l e t o transform t h e mutagenic xenobiotic no-4,6-dinitrotoluol

( t r i n i t r o t o l u o l , 2-ami-

and 4 - a m i n o - 2 , 6 - d i n i t r o t o l u o l )

i n t o nonmutage-

n i c 2 , 4 - d i a m i n o - 7 - n i t r o t o l u o l t h a t i s u s e d by b a c t e r i a a s a s o l e source o f nitrogen.

A t t h e Moscow U n i v e r s i t y some s t r a i n s o f m i c r o o r g a n i s m s were i s o l a t e d t h a t a r e capable o f d e g r a d i n g p y r i d i n e bases.

P y r i d i n e and

i t s d e r i v a t i v e s belong t o t h e t o x i c substances which a r e produced during fuel processing.

P y r i d i n e bases,

l i k e d e r i v a t i v e s o f benzol,

a r e examples o f h i g h c h e m i c a l i n e r t n e s s and t h e i r a c c u m u l a t i o n i n w a s t e s and s o i l s i n s i g n i f i c a n t q u a n t i t i e s as i n d u s t r i a l w a s t e s , p e s t i c i d e s and f e r t i l i z e r s r e p r e s e n t a s e r i o u s t h r e a t

f o r many l i v -

i n g things.

Thus,

224

-

the i n v e s t i g a t i o n o f microbial degradation o f the-

se x e n o b i o t i c s i s v e r y a c t u a l .

U n t i l recently only oxygen-containing ke n i c o t i n i c a c i d ,

p y r i d i n e d e r i v a t i v e s li-

p y r i d o x i n a n d o t h e r s were s t u d i e d ,

while the mic-

r o b i a l m e t a b o l i s m o f p y r i d i n e and i t s a l k y l - d e r i v a t i v e s well-known

was n o t

a n d c o r r e s p o n d i n g b i o t e c h n o l o g y was n o t d e v e l o p e d .

We h a v e shown t h a t t h e b a c t e r i a l s t r a i n s N o c a r d i a sp., monas s p . ,

Arthrobacter

Pseudo-

g l o b i f o r m i s and A r t h r o b a c t e r c r y s t a l o p o i e t e s

a r e c a p a b l e o f u t i l i z i n g p y r i d i n e a n d i t s mono a n d d i m e t h y l d e r i v a tives.

COOCH,

coo-

COOCH,

COOCH3

coo-

coo-

coo-

0- 0-0Y . r Gem- 0""OH

coo-

OH

coo-

a

@OH OH

coo-

coo.- p C m -

OH

0

coo-

6oH

-0OC - C H Z - C O - C H ~ - C H ~ -COO-

F i g . 2. Pathways f o r t h e d e g r a d a t i o n o f t e r e p h t a l a t e , i s o p h t a l a t e a n d d i m e t h y l p h t a l a t e b y P h o d o c o c c u s r u b r o p e r t i n c t u s M3.

-

-

225

I t i s curious t o n o t i c e t h a t Arthrobacter g l o b i f o r m i s growing i n a medium w i t h

a - p i c o l i n e p r o d u c e d much more r i b o f l a v i n e t h a n

when g r o w i n g i n a medium w i t h g l u c o e s e a s a s o l e s o u r c e o f c a r b o n . So i t was recommended t o p r o d u c e b i o m a s s c o n t a i n i n g l o t s o f v i t a m i n

B2

a-picoline.

by u s i n g t o x i c

T h i s w o r k was t h e b e g i n n i n g o f o u r

i n v e s t i g a t i o n s o f m i c r o b i a l d e g r a d a t i o n o f p y r i d i n e bases.

2- a n d 3 - m e t h y l p y r i d i n e s was d i s -

The m e t a b o l i s m o f p y r i d i n e , cussed e a r l y

(6,7). The i s o l a t i o n o f o x y g e n - c o n t a i n i n g s u b s t a n c e s

and t h e s i g n i f i c a n t u t i l i z a t i o n o f oxygen b y b a c t e r i a l s u s p e n s i o n s u s i n g p y r i d i n e s i n d i c a t e t h e p a r t i c i p a t i o n o f oxygen a t t h e f i r s t stages of p y r i d i n e s u t i l i z a t i o n , of

a l t h o u g h e a r l i e r a reduced-pathway

p y r i d i n e m e t a b o l i s m was s u g g e s t e d (7). We a l s o s t u d i e d t h e d e g r a d a t i o n o f 2 - m e t h y l - 5 - e t h y l - p y r i d i n e .

The c o n v e r s i o n o f 2 - m e t h y l - 5 - e t h y l p y r i d e has been demonstrated, the aromatic ring.

and f u r t h e r

t o h y d r o x y a l k y l compounds

oxidation l e a d t o cleavage o f

Thus t h e m e t a b o l i s m o f t h i s compound was a l s o

c o n n e c t e d w i t h o x i d a t i o n (8).

I t was d e m o n s t r a t e d t h a t s u b s t r a t e s p e c i f i c i t y o f s t r a i n s - d e s t r u c t o r s i s d e p e n d e n t on t h e i r p r e l i m i n a r y c o n t a c t w i t h c o r r e s ponding xenobiotics,

b u t we h a v e i s o l a t e d a s t r a i n A r t h r o b a c t e r

c r y s t a l l o p o i e t e s w i t h a wide s u b s t r a t e s p e c i f i t y . pable of

T h i s s t r a i n i s ca-

2- a n d 4 m e t h y l p y r i d i n e

u t i l i z i n g 2,6-dimethylpyridine,

t o g e t h e r as w e l l a s s e p a r a t e l y ,

and o f u s i n g them as a s o l e s o u r c e

of

(9,101.

carbon,

n i t r o g e n and energy

F u l l u t i l i z a t i o n o f t h e mix-

t u r e o f t h e above m e n t i o n e d s u b s t a n c e s i n c o n c e n t r a t i o n s o f 0,25 0,lO

% and 0,04

%,

?6 r e s p e c t i v e l y i s c o m p l e t e d i n 9 0 - 9 5 h o u r s i n t h e

w i d e t e m p e r a t u r e r a n g e t i n g f r o m 22-40

OC.

I t was e s t a b l i s h e d t h a t t h e f i r s t s t e p o f m e t a b o l i s m o f a l l s t u d i e d compounds i s a n o x i d a t i o n o f h e t e r o c y c l i c r i n g w i t h t h e f o r mation of-2-

and 3 - h y d r o x y

and t r i h y d r o x y p y r i d i n e s .

derivatives,

which f u r t h e r y i e l d s d i -

The c l e a r a g e o f t h e p y r i d i n e r i n g o f d i -

a n d t r i h y d r o x y p y r i d i n e s l e a d s t o a f o r m a t i o n o f l o w m o l e c u l a r compounds:

ammonia,

ketoacids,

mono-

and d i c a r b o n i c a c i d .

Thus,

we h a -

ve t h e n e x t p r o p o s e d p a t h w a y s o f 2 , 6 - d i m e t h y l p y r i d i n e

and 4-methyl-

p y r i d i n e degradation by Arthrobacter c r y s t s l l o p o i e t e s

(Fig.

Strains-destructor

A.

t e r s t h a t c o n t a i n e d benzopyrene (20-200 (40-90 m g / l ) ,

methanol (0,2-5,0

c y a n i d e (0,5-5,0 tions,

A.

3,4).

c r y s t a l l o p o i e t e s w a s t e s t e d i n w a s t e wa-

9/11,

% ) , pH 6,O-8,O; T o

-

mkg/l),

p y r i d i n e bases

ammonium s a l t 25-28

OC.

(10-100 m g / l ) ,

Under t h e s e c o n d i -

c r y s t a l l o p o i e t e s d e s t r u c t s p y r i d i n e bases a t s h i g h r a t e :

-

226

$COOH

-

+

NHZ-C, // 0 CH3

CH3 0 F i g . 3 . Pathway f o r t h e d e g r a d a t i o n o f 2 , 6 - d i m e t h y l p y r i d i n e Arthrobacter crystallopoietes.

6-

by

112 02

H

H

F C O O H COOH

F i g . 4 . Pathway for t h e d e g r a d a t i o n o f 4 - m e t h y l p y r i d i n e bacter crystallopoietes.

by A r t h r o -

- 227

-

i n 6 h o u r t h e i r c o n t e n t d e c r e a s e s f r o m 90 m g / l

t o 0,9 mg/l

and a f -

t e r 24 h o u r s a f u l l s u b s t r a t e m i n e r a l i s a t i o n i s a c h i e v e d . A consortium o f microorganisms capable o f e f f e c t i v e l y destroy-

i n g 2,4-dimethylpyridine 4 bacterial strains:

nes and

P.

putida.

a substrate.

was a l s o i s o l a t e d .

Pseudomonas d i m i n u t a ,

A consortium consists o f

P.

P.

syringa,

alcalige-

Each i n d i v i d u a l s t r a i n c o u l d n o t grow and u t i l i z e

The g r o w t h waa a l s o a b s e n t i n t h e a s s o c i a t i o n o f t w o

or t h r e e s t r a i n s .

Degradation o f 2,4-dimethylpyridine

only i n spontaneously i s o l a t e d strains-association. hed t h a t t h e P.

was r e a l i z e d

I t was e s t a b l i s -

d i m i n u t a was a d o m i n a n t s t r a i n o f t h e a s s o c i a t i o n .

F u l l u t i l i z a t i o n o f s u b s t r a t e ( 2 9/11

i s c o m p l e t e d i n 36 h o u r s .

The i n v e s t i g a t i o n o f t h e m e t a b o l i c p a t h w a y o f 2 , 4 - d i m e t h y l p y r i d i n e d e g r a d a t i o n showed t h a t t h e h y d r o x y l a t i o n o f t h e p y r i d i n e r i n g as w e l l as t h e o x i d a t i o n o f m e t h y l groups t a k e s p l a c e ,

so t h e

m e t a b o l i c pathway o f d i m e t h y l p y r i d i n e d e g r a d a t i o n by mixed c u l t u r e d i f f e r s f r o m a pathway c a r r i e d o u t by m o n o c u l t u r e o f A. poietes (Fig.

crystallo-

5).

:~cooul-

COOH

COOH H O O C A

COOH HOOC&OH

F i g . 5 . Pathway f o r t h e d e g r a d a t i o n o f 2 , 4 - d i m e t h y l p y r i d i n e xed c u l t u r e .

by m i -

-

228

-

The r e s u l t s o f o u r i n v e s t i g a t i o n s g i v e s b a s i s f o r t h e c o n s t r u c t i o n o f b i o t e c h n o l o g i c a l d e g r a d a t i o n o f p y r i d i n e bases i n i n d u s t r i a l wastes. REFERENCES

1 M. 2

3 4

5 6

7

8

9

10

R o t m i s t r o v , P. G v o s d j a k a n d 5. S t a v s k a j , M i c r o b i a l w a t e r c l e a i n i n g . Russ. K i e v : Naukova Dumka, 1 9 7 8 . 5. S t a v s k a j , B i o l o g i c a l d e s t r u c t i o n o f a n i o n i c s u r f a c t a n t s . Russ. K i e v : N a u k o v a Dumka, 1 9 8 3 . R. Naumova, M i c r o b i a l m e t a b o l i s m o f x e n o b i o t i c s . Russ. K a s a n j , 1986. R . Naumova e t e l . B i o o x i d a t i o n o f o z o p h t h a l a t e i n c o n n e c t i o n w i t h t h e t e c h n o l o g y o f sewage d i s p o s a l i n m i c r o b i a l m e t h o d s o f e n v i r o n m e n t a l c o n t r o l . Russ. P u s h i n o , 1 9 6 8 . R . A l i e v a , M i c r o b i a l a s p e c t s o f i n d u s t r i a l sewsge d i s p o s e 1 f r o m t o x i c compounds. Russ. / / 4 t h Symp. o f S o c i a l i s t c c o u n t r i e s o n b i o t e c h n o l o g y , 1986. P. 86. K o s t , P.B. T e r e n t i j e v , L.A. K o r o s t e l e v a , L . I . V o r o b j e v a , A.N. S h i b i l k i n a , The r o l e o f m i c r o o r g a n i s m s L.V. Modyanova a n d O.K. i n p r e s e r v a t i o n o f t h e e n v i r o n m e n t / / P r o c . INCHEBA, 1978. B r a t i s l a v a . P. 81. K o s t , L . I . V o r o b j e v s , P.8. T e r e n t i j e v , L.A. K o r o s t e l e v a , A.N. Kulikov, Microbiological degradation L.V. Modyanova a n d N.S. o f p y r i d i n e and 3 - m e t h y l p y r i d i n e // P r i k l . biochem., m i c r o b i o l . 1981. V . 1 7 , N 3 . P. 380. A.H. K o s t , P.B. T e r e n t i j e v , M . B . K u p l e t s k a y a , L.V. Modyanova, A.S. Oemina, M.P. C h o v r i t c h e v and E.V. Shushenschevs, M i c r o b i o l o g i c a l t r a n s f o r m a t i o n o f 2 - m e t h y l - 5 - e t h y l p y r i d i n e / / DAN S S S R . 1 9 7 4 . V . 214, N 4. P. 947-950. F.M. K h s s a e v a , L.A. K o r o s t e l e v a , L . I . V o r o b j e v s , L.V. Modyenova a n d P.B. T e r e n t i j e v , D e g r a d a t i o n o f p y r i d i n e , mono- a n d d i m e t h y l p y r i d i n e s by m i c r o o r g a n i s m s / / P r o c . 4 t h E u r o p . C o n g r e s s on B i o technol., 14-19 j u n e . V. 2 , P. 286. Amsterdam. L . I . V o r o b j e v a , F.M. K h a s a e v a , S.D. T a p t y k o v a a n d L.V. Modyanova, U n i q u e p r o p e r t i e s o f new i s o l a t e s o f a r t h r o b a c t e r / / P r o c . 3d Symponium o f E u r o p . Group. o f A c t i n o m i c e t o l o g i s t s , 2-4 s e p t e m b e r , 1 9 8 8 , B u d a p e s t . P. 6.

H I G H E R FUNGI A S A POTENTIAL FEED AND FOOD SOURCE FROM LIGNOCELLULOSIC WASTES

A.

LEONOWICZ, M.

.I.ROGALSKI a n d J.

WOJTAS-WASILEWSKA,

LUTEREK

B i o c h e m i s t r y Department, M a r i a Curie-Sklodowska U n i v e r s i t y , M. C u r i e - S k l o d o w a k a S q u a r e 3 , 2 0 - 0 3 1 L u b l i n ( P o l a n d )

INTRODUCTION I n t h e age o f

demand f o r

food,

fast

p o p u l a t i o n g r o w t h and c o n s e q u e n t l y a g r e a t e r

especially p r o t e i n containing products,

man a t t e m p t s

t o s o l v e t h e p r o b l e m o f f o o d s h o r t a g e s i n a number o f ways. f r o m t h e c o n v e n t i o n a l manners o f p r o t e i n p r o d u c t i o n , a r e b e i n g examined.

Among o t h e r s ,

t h e m i c r o b i o l o g i c a l m e t h o d s seem

t o be h o p e f u l f o r two b a s i c reasons:

f a s t biomass g r o w t h and t h e po-

s s i b i l i t y o f i n d u s t r i a l waste u t i l i z a t i o n (e.g. paper i n d u s t r i e s ) .

Apart

new a p p r o a c h e s

f r o m f o o d o r p u l p and

The i d e a o f f e e d o r f o o d b i o m a s s p r o d u c t i o n b y t h e

submerged c u l t u r e i n t h e a g i t a t e d a n d a e r a t e d b a f f l e t t a n k s r e s u l t s f r o m t h e e x p e r i m e n t s c a r r i e d o u t on p e n i c i l l i n a n d o t h e r a n t i b i o t i c fermentation processes.

I n t h i s csse,

l o w - c o s t m a t e r i a l s m i g h t b e u-

s e d as s u b s t r a t e s f o r f u n g a l m y c e l i u m p r o d u c t i o n , me,

a n d a t t h e same t i -

t h e r e d u c t i o n o f t h e b i o l o g i c a l oxygen r e q u i r e m e n t by waste by

p r o d u c t s t o an a c c e p t a b l e

l e v e l c o u l d be a c h i e v e d .

I t r e s u l t s from

t h e f a c t t h a t h i g h e r f u n g i a r e e q u i p p e d w i t h e f f i c i e n t e n z y m a t i c appar a t u s w h i c h c a n a t t a c k t h e s u b s t r a t e s n o t a c c e p t a b l e f o r y e a a t and bacteria.

Solid-stationary

o r submerged and a g i t a t e d c u l t u r e s have

b e e n c a r r i e d i n o r d e r t o o b t a i n e i t h e r b i o m a s s o r some n u t r i t i v e s u b stances such as v i t a m i n s ,

aminoacids or monosaccharides.

The i n t e r e s t i n h i g h e r

f u n g i a s a s o u r c e o f f o o d p r o t e i n comes

f r o m t h e f a c t t h a t t h e y h a v e b e e n u s e d f o r many t h o u s a n d s o f y e a r s . T h e i r t . a s t e a n d aroma a r e p l e a s a n t f o r man,

and u n l i k e y e a s t and bac-

t e r i a t h e y c o n t a i n f e w e r n u c l e i c a c i d s w h i c h s l l o w them t o b e c o n sumed w i t h o u t s i d e e f f e c t s .

A c c o r d i n g t o Torew,

(cited i n ref.

1)

t h e o v e r a l l c o n t e n t s o f n u c l e i c a c i d s i n b a c t e r i a amount t o 1 7 . 0 0 0

m g % a s c a l c u l a t e d i n i t s d r y maas; f u n g i o n l y 500 mg% ( e . g .

i n y e a s t o v e r 5.000

Aqaricua campestris).

However,

mgX a n d i n even t h i s

- 230 -

c o n s i d e r a b l y low amount o f n u c l e i c a c i d s s u b s t a n t i a l l y r e d u c e s t h e n u t i r t i o n value of fungal biomass.

T h e r e f o r e , modern b i o t e c h n o l o g y

aims a t t h e i r c o m p l e t e e l i m i n a t i o n ( r e f .

O u t o f a b o u t 2.000

2).

species of edible fungi, only three a r e cul-

t i v a t e d on a l a r g e , c o m m e r c i a l s c a l e u n d e r a r t i f i c i a l c o n d i t i o n s e.g.

Aqaricus campestris (Europe and North America), C o r t i n e l l u s

shii-take

(Japan) and Volvaria volvacea (China and South E a s t Asia)

( r e f . 3 ) . I n Poland and a l s o i n o t h e r c o u n t r i e s P l e u r o t u s o s t r e a t u s i s s u c c e s s f u l l y c u l t i v a t e d , however,

on a s m a l l e r scale compared t o

A q a r i c u s c a m p e s t r i s ( r e f . 4 ) . A v a s t m a j o r i t y of o t h e r e d i b l e f u n g i i a t i o n f o r t h e f r u c t i f i c a t i o n mechanism still

defies classical cul'

r e m a i n s l a r g e l y unknown.

I t is b e l i e v e d t h a t c e r t a i n endogenic sub-

s t a n c e s o f a hormone c h a r a c t e r p l a y a c r u c i a l r o l e i n t h e p r o c e s s . Their n a t u r e , however,

h a s n o t b e e n f u l l y d e t e r m i n e d so f a r .

An a l t e r n a t i v e seems t o b e o f f e r e d n o t b y t h e c u l t u r e o f f r u i t i n g b o d i e s b u t r a t h e r by c u l t i v a t i o n of v e g e t a t i v e m y c e l i u m .

I t exhi-

b i t s i d e n t i c a l biochemical p r o p e r t i e s and compostition t o f r u i t i n g The v e g e t a t i v e m y c e l i u m c o n t a i n s a f a i r l y g o o d a m o u n t o f p r o -

bodies.

t e i n (up t o 50%) p a r t i c u l a r l y r i c h i n l i s i n e and d i c a r b o x y l i c aminoacids.

The q u a l i t y a n d amount o f p r o t e i n a n d o t h e r i m p o r t a n t compo-

n e n t s c a n b e r e g u l a t e d by m e a n s o f v a r i o u s c u l t u r e c o n d i t i o n s .

The a -

mount o f v i t a m i n s ( e s p e c i a l l y o f g r o u p B) e q u a l s t h a t o f y e a s t .

The

mycelium i s a l w a y s m u l f i c e l l u l a r a n d l a r g e l y r a m i f i e d which a l l o w s for the aggregation.

T h i s form u s u a l l y f i l l s t h e whole volume of t h e

culture. K I N D S OF T H E F U N G A L C U L T U R E The e f f i c i e n c y a n d q u a l i t y o f b i o m a s s d e p e n d s o n t h e f u n g a l T h e r e a r e two known k i n d s o f s h a l l o w s t a t i o n a r y and submerged, mixed and a e r a t e d i n t h e l i q u i d p h a s e . T h e l a t t e r i s m o r e a d v a n t a g e o u s b e c a u s e o f t h e f o l 0-

s p e c i e s and methods o f c u l t u r e growth. cultures:

wing f a c t o r s :

-

f a s t v e g e t a t i o n ( a b o u t 3 times as f a s t a s i n t h e c a s e o f t h e shallow stationary),

- r e l a t i v e l y e a s y s e p a r a t i o n o f m y c e l i u m from t h e c u l t u r e m e d i u m

-

p o s s i b i l i t y t o grow a s a c o n t i n u o u s c u l t u r e o c c u p y i n g a r e l a t i v e l y small area,

- p o s s i b i l i t y o f t h e medium s t e r i l i z a t i o n i n t h e g r o w i n g f e r m e n t o r and keeping t h e c u l t u r e a s e p t i c ,

- p o s s i b i l i t y t o c o n t r o l c u l t i v a t i o n conditions during t h e fermentation process.

- 231

However,

-

t h i s k i n d o f c u l t u r e demands more w o r k a n d e n e r g y t h a n

the shallow stationary culture. P a t e n t l i t e r a t u r e p r o v i d e s numerous e x a m p l e s o f b i o m a s s p r o d u c t i o n using the vegetative mycelium c u l t u r e . s u b s t a n t i a l research i n t h i s aspect.

Poland a l s o c a r r i e s out

I t r e s u l t s i n many P o l i s h p a -

t e n t s d e s c r i b i n g t h e way o f m y c e l i u m c u l t i v a t i o n f o r b i o m a s s p r o d u c t i o n u s i n g v a r i o u s b y p r o d u c t s e.g. d i s t i l l e r y brew ( r e f s . sawdust ( r e f .

s u l f i t e waste l i q u o r s ( r e f .

9 1 , whey ( r e f s .

6 - 8 ) , molasses ( r e f .

13) and s t r a w ( r e f s .

14,15).

Also,

t h e a u t h o r s from

o t h e r c o u n t r i e s r e s t r i c t e d t h e i r own i n v e s t i o n s i n P o l a n d . p l e s may b e t h e p a t e n t o f

51,

10-121, The exam-

the English authors concerning the process

o f m y c o p r o t e i n p r o d u c t i o n from t h e F u s a r i u m qraminosum c u l t u r e ( r e f . 1 6 ) a n d t w o J a p a n e s e p a t e n t s d e s c r i b i n g c o n d i t i o n s o f t h e submerged cul.ture o f Basidiomycete fungi

(refs.

17,181.

SOME H I S T O R I C A L D A T A

R e s e a r c h on t h e p o s s i b i l i t i e s o f v e g e t a t i v e m y c e l i u m c u l t u r e was i n i t i a t e d on a w o r l d w i d e s c a l e more t h a n 4 0 y e a r s a g o ( r e f . The f i r s t campestris

i n c u b a t e d u n d e r t h e submerged,

agitated conditions i n a

s y n t h e t i c n u t r i e n t medium c o n t a i n i n g g l u c o s e , (ref.

19).

successful attempt pertained t o the c u l t u r e o f Aqaricus

u r e a , and m i n e r a l s a l t s

20). U n t i l 1959 t h e r e a p p e a r e d s e v e r a l d o z e n s i m i l a r p i l o t w o r k s ,

a l l d i s c u s s e d a t l e n g t h i n a m o n o g r a p h by R o b i n s o n a n d D a v i d s o n ( r e f . 21). far

The a u t h o r s e s t i m a t e d t h e v a l u e o f t h e r e s e a r c h c a r r i e d o u t a 0 rnd f o u n d i t e n c o u r a g i n g f o r t h e i n i t i a l c o m m e r c i a l p r o d u c t i o n

o f mvcelium as food.

( r e f s . 1,3,22) t e d so f a r

A n o t h e r more c o m p r e h e n s i v e m o n o g r a p h i c a n a l y s i s

i s a compendium o f m u l t i d i r e c t i o n a l r e s e a r c h c o m p l e -

and a s u r v e y o f t h e used f u n g a l s t r a i n s and t h e k i n d s o f

n u t r i e n t media ( s y n t h e t i c and waste)

as w e l l as t h e p h y s i c a l and

chew c a l p a r a m e t e r s o f t h e c u l t u r e s .

The f u n g a l s p e c i e s ,

referred t o

i n t h e monographs as o p t i m a l p r o d u c e r s o f biomass f o r n u t r i t i o n a r e the following:

*,

Aqaricus blazei,

Collybia velutipes,

Morchella rimosipes,

Aqaricus campestris,

Cantharellus cibarius,

Boletus inde-

Morchella hybrida,

T r i c h o l o m a nudum a n d X y l a r i s p o l y m o r p h a .

They

may grow o n t h e m e d i s c o n t a i n i n g s u c h c s r b o h y d r a t e s 8 s g l u c o s e , tose,

lactose,

spent molasses,

mal.

a n d t h e w a s t e m a t e r i a l s o r b y p r o d u c t s s u c h a s whey, s u l f i t e waste l i q u o r ,

wastes from t h e p r o d u c t i o n o f

soybean o i l and f r o m c o r n and pumpkin p r e s e r v a t i o n .

8

- 232

-

COMMERCIAL P R O D U C T I O N The c u l t u r e s on a m i c r o - ,

semi-

and f u l l t e c h n o l o g i c a l s c a l e s

a r e c a r r i e d i n t h e f e r m e n t o r s o f some t o s e v e r a l h u n d r e d a n d e v e n t h o u s a n d l i t r e s i n volume. fermentor o f 400 revs/min

Humfeld e t a l .

20) u s e d a 2 0 l i t r e

(ref.

a t the aeration r a t e o f 1-3 l i t r e s o f a i r

p e r l i t r e o f medium p e r m i n u t e .

A f t e r reaching the optimal y i e l d ,

t h e A q a r i c u s c a m p e s t r i s c u l t u r e was i n c u b a t e d 1 - 3 order t o obtain strong,

p l e a s a n t aroma.

Finally,

15 k g o f m y c e l i u m ( 1 3 . 6 % o f d r y mass)

incubation,

1 5 0 l i t r e s o f medium,

days l o n g e r i n a f t e r 93 hours o f were o b t a i n e d f r o m

I n o t h e r mic-

a n d t h e aroma was s a t i s f a c t o r y .

r o - a n d s e m i - t e c h n o l o g i c a l c a s e s T r i c h o l o m a nudum s t i r r e d a n d t h e a e r a t e d c u l t u r e c a r r i e d o u t by R e u s s e r e t a l . optimum a f t e r 9 5 h o u r s .

(ref.

231, reached i t s

The a e r a t i o n r a t e was o f 0 . 3 a i r p e r l i t r e

o f t h e c u l t u r e p e r m i n u t e a t a s t i r r i n g r a t e o f 400 r e v s / m i n . t h i s case, (ref.

p o r k l a r d was u s e d a s an a n t i f o a m a g e n t .

I n

RehaEek e t a l .

24) incubated Boletus e d u l i s i n a 10 l i t r e fermentor.

The me-

d i u m was i n o c u l a t e d w i t h 5 0 0 m l o f 7 day o l d h o m o g e n i z e d m y c l l i u m a n d a e r a t e d w i t h 0.7

l i t r e o f a i r p e r l i t r e o f t h e c u l t u r e per minu-

t e a t t h e a g i t a t i o n speed o f 430 r e v s / m i n .

Maximum y i e l d o f m y c e l i u m

was r e a c h e d a f t e r 1 2 0 h o u r s o f i n c u b a t i o n .

However,

a f t e r 72 hrs,

t h e c u l t u r e r e a c h e d t h e maximum y i e l d o f p r o t e i n ( 5 7 . 5 % ) .

An A m e r i -

2 5 ) was t h e b a s i s f o r t h e c o m m e r c i a l c u l t i v a t i o n o f

can p a t e n t ( r e f .

M o r c h e l l a e s c u l e n t a by S p e c i a l P r o d u c t s ,

Inc.,

C r e a m e r y Company ( S p r i n g f i e l d ,

USA)

c o n t a i n i n g glucose,

Missouri,

ammonium p h o s p h a t e ,

n a t e a n d w i t h s i l i c o n a s an a n t i - f o a m .

D i v i s i o n o f Producers

i n t h e l i q u i d medium

corn extract,

calcium carbo-

The m y c e l i u m f r o m t h e a g a r

s l a n t s was t r a n s f e r r e d s e q u e n t i a l y o r i n s e q u e n c e t o 500 m l c o n i c a l flasks,

10 l i t r e glass vessels,

l i t r e tanks.

500 l i t r e t a n k s and f i n a l l y t o 10.000

A f t e r 3-4 days o f c u l t i v a t i o n a t 21-24

r e a c h e d t h e f o r m o f b e a d s w i t h 2 cms i n d i a m e t e r . translocation, y i e l d of of

yield,

The

l i t r e t a n k r a n g e d on t h e a v e r a g e f r o m 1 . 2 t o 2 t o n s

which a f t e r c e n t r i f u g a t i o n (1.000

r e v s p e r m i n ) was

d r i e d i n a way t o i m p r o v e i t s f r a g r a n c e a n d f l a v o u r

4 3 . 5 OC).

t h e mycelium

t h e m a t e r i a l undergoes s t e r i l e homogenization.

a 10.000

wet biomass,

OC

I n the process o f

(optimal at

I t has been assessed t h a t c u l t u r e a e r a t i o n i n t e s i f i e s t h e

e s p e c i a l l y when t h e a i r i s a d m i t t e d i n t o t h e f e r m e n t a t i o n t a n k

while the culture i s being stirred.

The e f f i c i e n t g r o w t h o f M o r c h e l h

m y c e l i u m was a c h i e v e d b y t h e a e r a t i o n r a t e o f 0 . 1 mM 0 / l i t r e / m i n u t e .

2 I t i s worth n o t i n g t h a t t h e n u t r i t i o u s value o f t h e product surpasses t h a t o f t h e M o r c h e l l a f r u i t i n g b o d i e s ,

found i n t h e i r n a t u r a l

22).

environment ( r e f .

233

21)

A c c o r d i n g t o Robinson and Davidson ( r e f .

t h e M o r c h e l l a c u l t u r e may b e d e v e l o p e d f r o m t h e c o l o n y on a s l a n t . The medium i n a 2 5 0 m l f l a s k was i n o c u l a t e d w i t h t h e s t e r i l e homog e n a t e and i n c u b a t e d a t s h a k i n g ( 1 0 0 r e v s p e r m i n u t e ) .

Lower speeds

c a u s e d l a r g e a g g l o m e r a t i o n s and t h e i r c o l l e c t i n g i n t h e f l a s k n e c k o f t h e mycelium;

b u t h i g h e r speeds d i d n o t y i e l d g r a n u l e s .

3 days o f i n c u b a t i o n ,

t e t h e medium o f a 7 l i t r e b o t t l e .

Then t h e m a t e r i a l was f u r t h e r

u s e d t o i n o c u l a t e t h e medium o f a 20 l i t r e f e r m e n t o r .

400 and 75.000

l i t r e f e r m e n t o r s were i n o c u l a t e d .

b i g f e r m e n t o r s l a s t e d 3 d a y s a t 25 b e s t r e s u l t s and s u b s t a n t i a l l y r i n g incubation, consumption. se,

After

t h e c o n t e n t o f t h e f l a s k was u s e d t o i n o c u l a -

OV.

Consequently,

The i n c u b a t i o n i n

T h i s t e m p e r a t u r e gave t h e

reduced t h e danger o f i n f e c t i o n .

Du-

t h e pH o f t h e c u l t u r e l o w r e d t o g e t h e r w i t h s u g a r t h e pH b e g a n t o r i -

When t h e r e was n o more s u g a r l e f t ,

and t h a t p r o v e d t o b e t h e b e s t moment t o c o l l e c t t h e m y c e l i u m .

G l o b u l e s o f 0.5-2.5

cm i n d i a m e t e r w e r e r e c e i v e d .

l i t r e f e r m e n t o r c o n t a i n i n g 5.700 o f wet m y c e l i u m were o b t a i n e d .

From t h e 7 . 5 0 0

l i t r e s o f t h e medium,

1.5-2.0

o r d e r t o remove t h e s m e l l c o m i n g f r o m t h e f e r m e n t e d s u p p o r t . t a i n e d m y c e l i u m was d r i e d ,

tons

The p r o d u c t was r i n s e d w i t h w a t e r i n

powdered o r p r e s s e d ,

and f i n a l l y

The obsold.

A n o t h e r example o f c o m m e r c i a l p r o d u c t i o n o f m y c e l i u m i s t h e F u s a r i u m graminearum c u l t u r e c a r r i e d o u t i n England ( r e f s .

2,161.

The b i o m a s s

p r o d u c e d i n t h e c o n t i n u o u s c u l t u r e c o n t a i n e d 45% p r o t e i n o f a v e r y a d v i a b l e a m i n o a c i d c o m p o s i t i o n and a v e r y l o w l e v e l o f f a t a n d c h o lesterol.

An a p p r o p r i a t e m e t h o d ,

t e l y h e a t e d up t o 64' limination (refs. are inactivated

for

2,16).

i n w h i c h t h e m y c e l i a a r e immedia-

20 m i n . ,

a l l o w s f o r t h e n u c l e i c a c i d s e-

U n d e r t h e s e c o n d i t i o n s p r o t e o l y t i c enzymes

s o t h a t t h e r e i s n o b r e a k i n g down o f p r o t e i n w h i l e

t h e r m o s t a b l e n u c l e u s e s a r e s t i m u l a t e d t o be a c t i v e .

The d e g r a d a t i o n

p r o d u c t s o f n u c l e i c a c i d s c a n b e washed o f f t h e c e l l s .

i n t h e c o n t i n u o u s c u l t u r e a t 30

OC

The y e a r l y

The f u n g u s was g r o w n

p r o d u c t , i o n o f b i o m a s s a m o u n t e d up t o 1 0 0 t o n s .

i n t h e 1.300

l i t r e fermentation

t a n k s i n t h e medium c o n t a i n i n g g l u c o s e s y r u p a n d ammonia a s t h e s o u r c e o f c a r b o n and n i t r o g e n r e s p e c t i v e l y .

The i n c o m i n g m y c o p r o -

t e i n was t h e f i r s t p r o d u c t w h i c h c o u l d b e u s e d f o r human c o n s u m p t i o n .

POLISH CONTRIBUTION P i l o t a t t e m p t s t o c u l t i v a t e f u n g i on a s e m i t e c h n i c a l s c a l e have a l s o been s u c c e s s f u l i n o u r l a b o r a t o r y ( r e f s . t i o n t a n k s o f 700 and 1.250 l i t r e s were used.

26,27).

Fermenta-

Innonotus obliquous

-

234

-

m y c e l i u m was c u l t i v a t e d f o r 48 h o u r s i n t h e medium c o n t a i n i n g d i s t i l l e r y brew (3.71% d r y mass),

0.2% b e e t m o l a s s e s a n d 0.2% c a l c i u m

n i t r a t e a t t h e a e r a t i o n r a t e o f 10 l i t r e s p e r h o u r f o r one l i t r e o f the culture.

The s t i r r i n g r a t e was 1 0 0 r e v s p e r m i n u t e .

The y i e l d

was 250 k g o f w e t m y c e l i u m p e r 1 . 0 0 0 l i t r e o f t h e medium i . e . k g o f d r y mass i n c l u d i n g 6 . 2

kg o f pure protein.

t h e m y c e l i u m grew i n t h e f o r m o f b e a d s ,

13.4

During c u l t i v a t i o n ,

which i n t h e process o f

growth underwent spontaneous comminution,

so t h e r e was n o n e e d f o r

h o m o g e n i z a t i o n when t r a n s f e r r i n g t h e i n o c u l u m .

After

d r y i n g a t 65 O C ,

t h e c o m p o s i t i o n o f t h e a m i n o a c i d s was d e t e r m i n e d a n d n u t r i t i o n t e s t s on a n i m a l s were made.

The h y d r o l i z a t e i n c l u d e d a m i n o a c i d s i m p o r t a n t

f o r n u t r i t i o n such as m e t h i o n i n e ,

t i o n t e s t s made on m i c e ,

rats,

l y s i n e and p h e n y l a l a n i n e .

hamsters,

Nutri-

and c h i c k e n s i n our l a b o r a -

t o r y h a v e shown f u l l a p p l i c a b i l i t y o f b i o m a s s a s p r o t e i n s u p p l e m e n t i n g animal feed.

No s i d e e f f e c t s o n h e a l t h o r g r o w t h r a t e w e r e ob-

s e r v e d i n p i g s w h i c h r e a d i l y consumed t h e p r o d u c t ( r e f s .

26,271.

The r e s u l t s o f l a b o r a t o r y r e s e a r c h c a r r i e d o u t b y some o t h e r authors i n Poland are a l s o very encouraging.

I t was r e p o r t e d t h a t

t h e submerged c u l t u r e o f P l e u r o t u s o s t r e a t u s i n t h e l i q u i d m i n e r a l s a c c h a r o s e medium,

e n r i c h e d w i t h whey a n d s u l f i t e w a s t e l i q u o r ,

pro

d u c e d 1 4 g o f d r y mass o f m y c e l i u m p e r l i t r e o f t h e medium d u r i n g 3 days.

( r e f . 28).

I t was a l s o r e p o r t e d t h a t T r a m e t e s v e r s i c o l o r

T y r o m y c e s a l b e l l u s w e r e submerged

f o r 120 h o u r s ,

r a t e d c u l t u r e s on whey a m o u n t e d t o 21.7 p e r l i t r e o f t h e medium r e s p e c t i v e l y .

and

f r o m w h i c h t h e ae-

a n d 1 6 . 2 grams o f d r y mass

I t i s important t o note t h a t

an a p p r o p r i a t e d i l u t i o n o f whey p l a y e d a c r u c i a l r o l e f o r t h e mycelium y i e l d ( r e f .

29).

F A C T O R S INFLUENCING FUNGAL G R O W T H AND BIOMASS QUALITY Carbon s o u r c e The c a r b o n u t i l i z a t i o n b y h i g h e r f u n g i d e p e n d s n o t o n l y on i t s s o u r c e b u t on t h e o t h e r c o m p o n e n t s o f t h e medium a s w e l l .

The p r e -

s e n c e ~~fv i t a m i n s a n d t h e r e l a t i o n o f c a r b o n t o n i t r o g e n i n t h e med i u m seems t o b e o f s p e c i a l i m p o r t a n c e . A c c o r d i n g t o P e r l m a n ,

(ref.

3 0 ) g l u c o s e u t i l i z a t i o n b y P o l y p o r u s a n c e p s a n d c o n s e q u e n t l y mycel i u m g r o w t h i s much g r e a t e r ( a b o u t f o u r t i m e s ) thiamine.

i n the presence o f

The P e r l m a n ' s e x p e r i m e n t a l s o p r o v e d t h a t t h i s f u n g u s u t i -

l i z e s s t a r c h t w i c e a s much a s f r u c t o s e .

I t can be seen from Table 1 t h a t m a n n i t o l i s t h e b e s t c a r b o n s o u r c e f o r b o t h biomass and p r o t e i n p r o d u c t i o n b y A q a r i c u s c a m p e s t r i s .

-. 2 3 5 -

I t i s a l s o i n t e r e s t i n g t o n o t e t h a t l a c t o s e and saccharose p r o d u c e d

worse r e s u l t s . TABLE 1 The e f f e c t o f v a r i o u s c a r b o n s o u r c e s o n b i o m a s s arid p r o t e i n y i e l d i n t h e submerged c u l t u r e o f A g a r i r u s c a m p e s t r i s ( r e f . 31) C a r b o n source

P r o t e i n content

M y c e l i u m dry mass

c g’ll

M a n n i to1 Glucose

[%I 31 . O 28.2 27.1 28.0

3.8

4.4 3.2 2.6 2.4 1.9 1.4

D(+)-Xylose D(+)-Fructose Maltose Lactose Saccharone

27.5 27.0 24.0

r

T h o u g h t h e l a b o r a t o r y e x p e r i m e n t s p r o v i d e d some i n f o r m a t i o n o f how t o f i t t h e b e s t c o n d i t i o n s f o r

the cultivation of funqi,

can n o t be used on a wider s c a l e because o f t h e h i g h p r i c e .

they To a v o i d

the high cost o f low molecular carbon sources the conceptions are evaluated t o u t i l i z e natural i n d u s t r i a l byproducts l i k e l i g n o c e l l u -

l o s e s and w a s t e s o f o r g a n i c o r i g i n .

These e x p e r i m e n t s w i l l be des-

c r i b e d i n d e t a i l i n t h e r e m a i n i n g part, o f t h e paper. Nitroqen source U t i l i z a t i o n o f v a r i o u s s o u r c e s o f n i t r o g e n depends o n i n d i v i d u a l f e a t u r e s o f t h e fungus.

I t was e s t a b l i s h e d t h a t o n l y a f e w

s t r a i n s o f h i g h e r f u n g i can u t i l i z e i n o r g a n i c n i t r o g e n s o u r c e s (e.9. Lentinus lepidus,

Ptychoqaster r u b e s c e s ) .

n i c n i t r o g e n sources,

grow b e t t e r on aminoacids, (e.9.

Other f u n g i p r e f e r orga-

t h o u g h w i t l i some d i f f e r e n c e s : m o r e o f t h e m b u t some p r e f e r o r g a n i c ammonium s a l t s

Fomes a n n o s u s w h i c h g r o w s t h e b e s t i n t h e p r e s e n c e o f ammonium

tartrate).

Among i n o r g a n i c n i t r o g e n s o u r c e s ,

t o be t h e b e s t t o u t i l i z e , n i c one ( r e f .

ammonium p h o s p h a t e s e e m s

b u t t o a much l o w e r e x t e n t t h a n t h e o r g a -

31).

Some n a t u r a l p r o d u c t s ,

l i k e yeast extract,

mand f o r c a r b o n a n d n i t r o g e n s o u r c e s .

i n f l u e n c e f u n g a l de-

I t depends on t h e carbon and

n i t r o g e n r a t i o w h i c h p r o v e s t o b e t h e m o s t f a v o r a b l e a t 20-3O:l (ref.

32).

The y e a s t e x t r a c t p r o b a b l y s e r v e s a s a v i t a m i n s o u r c e .

The y i e l d o f A q a r i c u s c a m p e s t r i s b i o m a s s ,

grown on asparagine,

n y l a l a n i n e o r p r o l i n e as a source o f n i t r o g e n , t h i a m i n e o r y e a s t e x t r a c t was a b o u t 4 - 1 0

phe-

i n the presence o f

times greater

than t h a t

-

236

devoided o f v i t a m i n s a t a l l ( r e f .

-

33).

The m i x t u r e s o f v a r i o u s n i -

t r o g e n sources a r e sometimes e f f i c i e n t e . g . w e l l on ammonium s u l p h a t e ,

T r i c h o l o m a nudum g r o w s

ammonium c h l o r i d e ,

urea,

ammonium t a r -

The b e s t r e s u l t s f o r p r o t e i n p r o d u c t i o n a r e ob-

t r a t e and n i t r a t e s .

t a i n e d w i t h t h e m i x t u r e o f ammonium s u l p h a t e ,

t a r t r a t e and c h l o r i d e

( r e f . 2 3 ) . However, t h e b e s t s o u r c e o f n i t r o g e n a r e a m i n o a c i d s , e s pecially valine,

glutamine,

a c i d or arginine. sources ( r e f .

asparagine,

L i s i n e and

31).

-alanine

glutamic

acid,

aspartic

p r o v e t o b e weaker n i t r o g e n

As i n t h e c a s e o f c a r b o n ,

a t t e m p t s a r e made t o

use cheaper b u t e q u a l l y e f f e c t i v e n i t r o g e n s o u r c e s .

The whey p r o t e -

i n s a n d d i s t i l l e r y b r e w may s e r v e a s e x a m p l e s ( r e f s .

6-8,

10-12).

Some o t h e r b y p r o d u c t s o f t h e f o o d i n d u s t r y w e r e a l s o e f f e c t i v e l y used i n our l a b o r a t o r y

(refs.

5,13,15).

M i n e r a l components The m i n e r a l c o m p o n e n t s o f t h e medium l i k e p h o s p h o r u s ,

sulphur,

magnesium a n d p o t a s s i u m i n f l u e n c e n o t o n l y t h e y i e l d o f t h e f u n g a l biomass b u t a l s o i t s q u a l i t y e . g .

fragrance.

I t s h o u l d be n o t e d

t h a t optimum f o r biomass p r o d u c t i o n u s u a l l y does n o t c o i n c i d e w i t h t h a t f o r aroma.

F o r example A q a r i c u s c a m p e s t r i s c u l t u r e grows b e s t

i n t h e medium w i t h 50 m g / l ma,

however,

400 m g / l ) .

o f phosphorus

I n t h e case o f potassium,

3).

The o p t i m u m a r o (300-

t h e optimum c o n c e n t r a t i o n i n

t h e medium f o r b i o m a s s y i e l d was 50 m g / l . component,

(ref.

r e q u i r e s a much h i g h e r p h o s p h o r u s c o n c e n t r a t i o n

I n t h e absence o f t h i s

t h e m y c e l i u m y i e l d d e c r e a s e s by 1 0 % . The b e s t f r a g r a n c e

was o b t a i n e d a t t h e p o t a s s i u m c o n c e n t r a t i o n b e t w e e n 1 0 0 a n d 3 0 0 m g / l d e p e n d i n g on t h e c u l t i v a t i o n t i m e . t h e c o n c e n t r a t i o n o f 200 m g / l tion.

S u l p h u r g a v e t h e b e s t aroma a t

o f medium a f t e r e i g h t d a y s o f c u l t i v a -

Magnesium a t t h e c o n c e n t r a t i o n o f 2 0 m g / l

d e d t h e h i g h e s t amount o f m y c e l i u m ( r e f .

i n t h e medium y i e l -

3).

P l a n t o i l s and t h e i r c o n s t i t u e n t s The r e s p e c t i v e r e s e a r c h shows t h a t some n a t u r a l o i l s , flower,

cottonseed,

t h e c o n c e n t r a t i o n o f a b o u t 0.21 s u b s t a n t i a l l y nary c u l t u r e growth. f a t t y acids.

sun-

3).

improved the s t a t i o -

The s i m i l a r e f f e c t was o b t a i n e d w i t h e s t e r s o f

Free acids,

ged c u l t u r e ( r e f .

e.g.

s o y b e a n o r o l i v e a d d e d t o t h e f u n g a l medium a t

however,

i n h i b i t e d t h e g r o w t h o f t h e submer-

I t i a interesting that the positive effect o f

p l a n t o i l s waa o b s e r v e d a l s o d u r i n g l i g n i n a s e p r o d u c t i o n .

This en-

zyme was h i g h l y s t i m u l a t e d b y s u n f l o w e r a n d o l i v e o i l s ( r e f . 3 4 ) .

-

237

-

Temperature The h i g h e r f u n g i u s u a l l y r e q u i r e s a l o w e r t e m p e r a t u r e f o r v e g e t a t i o n t h a n t h e y e a s t and m o u l d ( r a n g i n g 25-28

9).I t

was e s t a b -

l i s h e d t h a t even s l i g h t t e m p e r a t u r e changes s u b s t a n t i a l l y i n f l u e n ced t h e mycelium g r o w t h ( r e f .

32).

A t e m p e r a t u r e h i g h e r t h a n 30 OC

u s u a l l y i n h i b i t s the fungal growth, 1 5 O C s l o w s i t down. of 0

OC

Below 1 5

OC

while a temperature lower than A temperature

f u n g i s t i l l grow.

u s u a l l y does n o t k i l l h i g h e r f u n g i ,

b u t t h e i r growth i n

t h i s case i s extremely poor. The o p t i m u m t e m p e r a t u r e f o r A q a r i c u s c a m p e s t r i s r a n g e s f r o m 25-30 OC ( r e f s .

32,331,

M o r c h e l l a h y b r i d a 20-25 (ref.

361,

f o r B o l e t u s e d u l i s 25 'C OC

(ref.

f o r Trametes v e r s i c o l o r ,

t u s o s t r e a t u s 27

OC

(ref.

31, for

3 5 ) f o r P h l e b i a r a d i a t a 28 O C P h o l i o t a m u t a b i l i s and P l e u r o -

( r e f . 37).

M The h i g h e r f u n g i u s u a l l y r e q u i r e l o w e r pH o f t h e c u l t u r e t h a n bacteria, o f 4.0

but n o t as l o w as moulds.

and 7.5.

For example,

I t u s u a l l y r a n g e s b e t w e e n a pH

pH o p t i m u m f o r A q a r i c u s c a m p e s t r i s

submerged c u l t u r e i s b e t w e e n pH 5 . 1 a n d 7 . 5

Morchella 4.5

-

5.5

h y b r i d a pH 4.0 (ref.

-

6.2

(ref.

351,

(refs.

31,32,38),

-

3 ) a n d f o r P o l y p o r u s a n c e p s pH 4.0

Wood r o t t i n g f u n g i l i k e T r a m e t e s v e r s i c o l o r , Pleurotus ostreatus,

for

f o r B o l e t u s e d u l i s pH 7.0

(ref.

30).

Pholiota mutabilis,

P h l e b i a r a d i a t a o f Phanerochaete chrysosporium

p r e f e r a s l i g h t l y l o w e r pH r a n g i n g f r o m 3.5

-

5.5

( r e f s . 34,36,39).

A e r a t i o n and s t i r r i n g H i g h e r f u n g i u s u a l l y r e q u i r e a l o w e r i n t e n s i t y o f a e r a t i o n and s t i r r i n g t h a n y e a s t and moulds. Sometimes t h e y even p r e f e r t h e s t a t i o n a r y s h a l l o w c u l t u r e t o t h e submerged and a g i t a t e d one g i v i n g , i n t h i s case,

b e t t e r aroma a n d p r o d u c i n g more enzymes ( r e f

40).

The r e s u l t s o b t a i n e d d u r i n g t h e f e r m e n t o r c u l t i v a t i o n o f

Mar-

h e l l a h y b r i d a show t h a t i t r e q u i r e s a r a t h e r l o w s p e e d o f s t i r r i n g (below 100 revs/min) O2

a n d v e r y weak a e r a t i o n ( 0 . 0 8

-

0.15

micromole

p e r l i t r e o f t h e medium p e r m i n u t e ) i n c o m p a r i s o n w i t h t h e o t h e r

investigated fungi.

F o r example, AQaricus c a m p e s t r i s grows t h e b e s t

i n a f e r m e n t o r when t h e s p e e d o f t h e c u l t u r e s t i r r i n g is 1 8 0 - 3 5 0

r e v s / m i n and a e r a t i o n a b o u t h u n d r e d p e r c e n t more i n t e n s i v e t h a n i n t h e case o f M o r c h e l l a ( r e f .

41).

I n some e x p e r i m e n t s ,

o f t h e fermentor c u l t u r e loweLed t h e mycelium y i e l d , t i o n was o n l y a p p l i e d ( r e f .

41).

the s t i r r i n g t h e r e f o r e aera-

- z3rj

-

MYCELIAL BIOMASS COMPOSIIION P r o t e i n and aminoacids The p r o t e i n c o n t e n t v a r i e s w i t h i n e x t r e m e l y w i d e l i m i t s u n d e r d i f f e r e n t c u l t u r a l c o n d i t i o n s f o r any organism.

I t r a n g e s from seve-

r a l t o s e v e r a l s c o r e s p e r c e n t o f m y c e l i u m d r y mas. No p r e c i s e d a t a on t h e p r o t e i n c o n t e n t a r e a v a i l a b l e d u e t o d i f f i c u l t i e s i n complet i n g t h e e x t r a c t i o n o f t h e p r o t e i n from t h e mycelium and t h e c o n t e n t o f chitin.

Thus a n a r b i t r a r y f i g u r e o f N x 6.25

is f a l l a c i o u s l y em-

ployed t o determine t h e p r o t e i n codntent i n mycelium.

T h e r e f o r e some

works s u g e s t t h a t t h e n i t r o g e n c y c o e f f i c i e n t s h o u l d range from 4.05 t o 4.45

or even lower.

For example, i n t h e case o f Aqaricus bisporus,

t h e s u g g e s t e d v a l u e is 3.0; choloma e q u e s t r a 2.84

a n d f o r A r m i l l a r A J l a m e l l e a 3.44

However, F i z p a t r i c e t a l . tein calculation, 8.48.

f o r C a n t h a r e l l u s u b e r i u s 2.8;

Tri-

for

(ref.

1).

( r e f . 4 2 ) p r o p o s e d i n some e x a m p l e s o f p r o -

t o r e p l a c e t h e commonly u s e d c o e f f i c i e n t 6 . 2 5 by

i.e.

I n s u c h c a l c u l a t i o n s t h e l e v e l o f d i g e s t i v e f a c t o r (NPU,

n i t r o g e n p r o t e i n u t i l i z a t i o n c e o f f i c i e n t f o r fungal mycelium) f o r p a r t i c u l a r f u n g a l p r o t e i n s s h o u l d be t a k e n i n t o c o n s i d e r a t i o n . T h i s v a l u e f o r B o l e t u s e d u l i s e q u a l s 6 4 . 9 2 and i s 100 p e r c e n t a s h i g h a s t h a t of Aqaricus campestris. Despite t h e f a c t t h a t the protein of B o l e t u s e d u l i s and Aqaricus c a m p e s t r i c a r e l e s s v a l u a b l e t h a n t h e other vegetable proteins, products, (ref.

i t was e s t a b l i s h e d t h a t when a d d e d t o c o r n

they s u b s t a n t i a l l y improve t h e b i o l o g i c a l value o f g l u t e n

4 3 ) . A c c o r d i n g t o o u r e x p e r i m e n t s i t was p o s s i b l e t o r e p l a c e

t h e c o n v e n t i o n a l p r o t e i n s o u r c e o f a n i m a l f e e d , by t h e f u n g a l mycelium (ref. 4 4 ) . The c h e m i c a l c o m p o s i t i o n o f t h e c u l t u r e medium h a s n o b e a r i n g on t h e p r o t e i n q u a l i t y , t h o u g h i t d o e s have a n e f f e c t on its cont e n t s i n t h e mycelium. 50.4% o f p r o t e i n ,

For example Aqaricus c a m p e s t r i s accumulates

when n i t r o g e n c o m e s f r o m c a s e i n h y d r o l i s a t e ,

and

o n l y 4 5 . 0 % w h e n ammonium n i t r a t e i s t h e s o u r c e o f n i t r o g e n ( r e f .

38).

Trametes v e r s i c o l o r a c c u m u l a t e s 2 9 - 3 6 . 9 % p r o t e i n i n t h e m y c e l i u m w i t h t h e same amino a c i d c o m p o s i t i o n r e g a r d l e s s o f t h e k i n d of medium. The p r o t e i n c o n t e n t i n t h e c u l t u r e s g r o w n o n m o l a s s e s wastes r a n g e d f r o m 30-58% ( r e f . contains

2 4 ) . The m y c e l i u m o f t h e

submerged c u l t u r e u s u a l l y

more p r o t e i n t h a n c a n b e f o u n d i n f r u i t i n g b o d i e s .

g e s t i b i l i t y i s a l s o much h i g h e r

(ref.

Its di-

1 ) . For example t h e f r u i t i n g

b o d i e s o f _ P l e u r o t u s o s t r e a t u s c o n t a i n 25% p r o t e i n , a n d i t s d i g e a t i b i l i t y i s 54X, w h e r e a s f o r mycelium t h e s e v a l u e s are 76.2% a n d 84.7% r e s p e c t i v e l y ( r e f . 1 ) . The p r o t e i n h y d r o l i z a t e s o f v e g e t a t i v e myce-

-

239

-

l i u m c o n t a i n a l m o s t a l l a m i n o a c i d s w h i c h can be found i n o t h e r p l a n t materials (refs.

45-48).

Indispensable aminoacids u s u a l l y occur i n

a n a c c e p t a b l e amount ( r e f .

49).

The a m i n o a c i d c o m p o s i t i o n i s compa-

r a b l e t o t h a t o f e g g s o r y e a s t and s o m e t i m e s ,

particularly in rela

t i o n t o l y s i n e and t r y p t o p h a n e i t i s e v e n b e t t e r ( T r i c h o l o m a nudum and Jrametes

versicolor)

(refs.

40,46,50).

The c o n t e n t s o f a m i n o a -

c i d s undergo changes d u r i n g f u n g a l c u l t u r e g r o w t h .

F o r example t h e

m e t h i o n i n e was n o t d i s c o v e r e d a t a n e a r l y s t a g e o f B o l e t u s e d u l u s c u l t i v a t i o n t h o u g h i t a p p e a r e d l a t e r on ( r e f .

24). A f t e r 24 h r s o f

i n c u b a t i o n a s u b s t a n t i a l i n c r e a s e o f v a l i n e c o n c e n t r a t i o n was n o t i ced,

though i t d i d n o t appear a t t h e b e g i n n i n g ( r e f .

24).

Some e x p e -

r i m e n t a l data f o r m y c e l i a l aminoacid composition i n comparison with t h a t f o u n d i n e g g s o r y e a s t a n d t h o s e recommended by FA0 a r e d i s played i n Table 2 . TABLE 2 The a m i n o a c i d c o m p o s i t i o n o f some f u n g a l m y c e l i a i n c o m p a r i s o n w i t h o t h e r p r o t e i n s o u r c e s a n d FA0 r e c o m m e n d a t i o n ( r e f s . 2 4 , 4 0 , 4 5 - 4 9 , 5 1 1 .

Amino

Trick

Inon:

oloma

otus

nudm

obliqw

acid

Trum etrr

Cha, t .

omium

uerst- c e l u l o color lyticwn

Trich; odermn

FA0

Egg

2.1

3.2

0.3

2.6

1.5

2.2

-

-

2.0

3.4

5.2

4.7

3.5

6.0

6.3

9.3

7.5

6.0

2.9

5.3

6.8

1.6

1.7

1.7

3.4

3.2

3.4

3.6

3.2

-

-

-

4.2

6.1

-

3.0

4.9

-

1.5

recon aenda tion

viride

-

4.2

Yeant

0.6

-

2.0

2.8

-

6.0

5.5

4.2

5.8

9.0

8.3

4.8

4.4

6.3

6.8

4.2

2.3

1.4

3.1

2.6

2.2

4.6

3.0

3.7

6.0

4.5

2.0

5.6

6.1

4.9

-

5.0

5.0

2.8

5.8

4.4

1.7 7.4

0.8 5.9

1.4 4.2

-

Nucleic acid However. t h e i m p o r t a n t ,

negative f a c t o r which l i m i t s t h e a p p l i -

c a t i o n o f m i c r o o r g a n i s m p r o t e i n s as a source o f f e e d o r f o o d i s t h e content o f nucleic acids. a f f e c t s kidney diseases,

T h e i r consumption i n l a r g e r q u a n t i t i e s e.g.

u r i n a r y gout.

As m e n t i o n e d e a r l i e r ,

-

240

-

t h e amount o f n u c l e i c a c i d s i n t h e c e l l s o f h i g h e r f u n g i i s s u b s t a n t i n a l l y s m a l l e r t h a n i n b a c t e r i a and y e a s t . shown i n T a b l e 3 ,

According t o t h e data

i t i s a b o u t 1 0 a n d 50 t i m e s s m a l l e r i n y e a s t a n d

bacteria respectively. TABLE 3 C o m p a r i s o n o f n u c l e i c a c i d c o n t e n t s i n d r y mass o f some f u n g a l m y c e l i a , y e s s t a n d b a c t e r i a c e l l s ( r e f . 1).

A#aricur birporw l w cibariw

Ccmthar.1

Ncluel la c r i s p Ycamt T o r u l a E s c h o r i c h i a col i

On t h e o t h e r h a n d ,

i c acid metabolites. "meat"

rig%!

Nuclcic acid content

Organin.

RNA

DNA

604.5

116.1

616.6

123.7

395.8

119.6

3.950.0

310.0

135.000.0

3.500.0

a p o s i t i v e e f f e c t i s o b s e r v e d i n some n u c l e -

V a r i o u s p u r i n e s and n u c l e o t i d e s cause s p e c i f i c

aroma o f m y c e l i u m ,

w h i c h makes f u n g i m o r e a t t r a c t i v e a s f o o d

compared w i t h o t h e r m i c r o o r g a n i s m s ( r e f .

23).

Carbohydrates Carbohydrates appear i n f u n g i as aminopolysaccharide c e l l w a l l constituents, rides, well.

i n the form o f m a t r i x glycoproteins,

some o l i g o s a c c h a r i d e s ,

monosaccharides and sugar a l c o h o l s as

The m a i n c e l l w a l l c o m p o n e n t ,

constitued entirely o f

f r e e polysaccha-

A-1.4

chitin,

i s a l i n e a r molecule

l i n k e d N-acetylglucosamine residues.

The l o n g c h a i n s o f s u c h u n i t s may a c h i e v e a m o l e c u l a r w e i g h t o f cellulose

(ref.

52).

des a l s o appear, lactosamine enzymes)

(ref.

I n the fungal c e l l w a l l other aminopolysacchari-

which are d i f f e r e n t 53).

f r o m c h i t i n e.g.

appear i n t h e form o f g l y c o p r o t e i n .

o f aminosugar,

mannan,

polvsaccharide moiety,

p o l y m e r o f ga-

Almost a l l f r e e fungal p r o t e i n s ( i n c l u d i n g g l u c a n and p r o t e i n .

They o c c u r a s a c o m p l e x

I n the hydrolizate o f

o f N e u r o s p o r a c r a s s a l a c c a s e p r o t e i n mannose

a n d g l u c o s a m i n e were f o u n d i n l a r g e a n d s m a l l s m o u n t s r e s p e c t i v e l y (ref.

54).

The s t r u c t u r e o f g l y c o p r o t e i n c o m p l e x e s i s unknown,

but i

i t h a s b e e n s u g g e s t e d t h a t g l u c o s a m i n e s e r v e s as a l i n k b e t w e e n p r o t e i n and p o l y s a c c h a r i d e s ( r e f . llusose (refs. (ref.

56,571,

55).

g l u c a n s (e.9.

58) and l e n t i n o n ( r e f .

Among f r e e p o l y s a c c h a r i d e s , glicogen (ref.

5 9 1 1 , mannans ( r e f .

ce-

5 6 1 , pachyman

60) and p o l y u r o n i d s

( r e f . 6 1 ) a r e t h e main.

-

241

Among o l i g o s a c c h a r i d e s t h e most p o p u l a r i s

t r e h a l o a e ( r e f . 621, which a p p e a r s i n f r u i t t i n g b o d i e s and mycelium a s well i n 90% o f h i g h e r f u n g i ( r e f . 6 3 ) . Monosaccharides l i k e g a l a c tose (ref.

561, glucose

( r e f . 5 6 ) , mannose ( r e f .

refs.

56,64),

fructose (ref.

5 6 1 , mannose

5 6 ) a n d s e d o h e p t u l o a e ( r e f . 6 5 ) were i d e n -

t i f i e d i n various fungal mycelia.

Polyhydroxylalcohols (sugar alco-

h o l s ) a r e r e p r e s e n t e d by m a n n i t o l ( d i s c o v e r e d f o r t h e f i r s t time i n Aqaricus i n t e q e r ( r e f

56), very popular i n higher fungi ( r e f .

6 2 ) ) , v o l e m i t o l ( i s o l a t e d from L a c t a r i u a volemus ( r e f . 5611, s o r b i t o 1 (found i n B o l e t u s bovinus ( r e f . 6 2 ) ) , e r y t h r i o l ( A l m i l s r i e l l a

mellea ( r e f . 6611, a r a b i t o l ( F r u s t u l i n a h e p a t i c a and B o l e t u s b o v i n u s (refs.

67,68)), i n o a i t o l (Romariopsis croccea ( r e f .

l i t o l (Agaricus campestris (ref.

5 6 ) ) and ksy-

69)).

The t o t a l a m o u n t o f c a r b o h y d r a t e s i n h i g h e r f u n g i r a n g e s f r o m 28% d r y mass o f L a c t a r i u a d e l i c i o s u s t o 7 6% i n A r m i l l a r i e l l a m e l l e a . The d r y mass o f A q a r i c u s c a m p e s t r i s c o n t a i n e d 2 4 . 9 % c a r b o h y d r a t e s , w h e r e g l y c o q e n , c e l l u l o s e , aome r e d u c i n g s u g a r a n d m a n n i t o l were dominant ( r e f . 70). Alcohols w i t h t h e i r r e s p e c t i v e k e t o n e s , espec i a l l y octene-1-on-3 f u n g a l aroma.

are p a r t l y r e s p o n s i b l e f o r

and octene-2-on-3,

These s u b s t a n c e s are s t i l l p e r c e p t i b l e a t v e r y low

concentrations reaching

and

lo-’

mg% r e s p e c t i v e l y ( r e f .

71).

Fats The m y c e l i u m o f e d i b l e f u n g i c o n t a i n s v a r i o u s f a t c o m p o u n d s o l e i c and l i n o l e i c a c i d s are t h e main c o n s t i t u e n t s I t i s s u g g e s t e d t h a t t h e f u n g a l aroma d e p e n d s p a r t l y o n

where p a l m i t i c , (ref. 72).

the autooxidation of these unsaturated fatty acids (ref.

71).

The

c o m p o s i t i o n o f mycelium l i p i d e s c h a n g e s d u r l n g t h e c u l t u r e growth. The a m o u n t o f t r i g l y c e r i d e a i n y o u n g c u l t u r e s i s 2 0 % , w h e r e a s i n t h e o l d e r ones decreaaea t o 1.7% ( r e f .

71).

V i t amina The h i g h e r f u n g i a r e a r e l a t i v e l y g o o d s o u r c e o f so m e v i t a m i n s . I t h a s b e e n e s t a b l i s h e d f o r e x a m p l e t h a t 1 0 0 g o f f r e s h f u n g i may p r o v i d e 20% o f t h e d a i l y human d e m a n d o f r i b o v l a v i n e a n d 2 5 % o f n i a c i n ( r e f . 7 0 ) . The a m o u n t o f r i b o f l a v i n e a n d n i a c i n i n f u n g i p l a c e s t h e m b e t w e e n h i g h e r p l a n t a n d y e a s t a n d i n some c a s e s ( e . 9 . e d u l i s ) even higher ( r e f .

Boletus

1 ) . A vegetative mycelium is e s p e c i a l l y

r i c h i n vitamins o f group 8. According t o Bell

,

(ref.

73) 5% o f

Morchella esculenta mycelium i n a s y n t h e t i c d i e t f u l l y s a t i s f i e s t h e

-

242

Nine vitamins are contained i n

demand f o r t h e g r o u p B v i t a m i n s . t h i s group, tol.

as w e l l as n i a c i n ,

-

panthotenic acid,

c h o l i n e and i n o a i -

T a b l e 4 shows t h e v i t a m i n c o n t e n t i n t h e m y c e l i a o f some f u n g i

in comparison w i t h t h a t o f yeast. TABLE 4 The v i t a m i n c o n t e n t i n some f u n g a l m y c e l i a a n d y e a s t ( r e f .

A#UriCW

Vitamins

campstris

P b r c h l La osculonta

T r i cho L oma nudrar

74)

Yeaat CToruld

MS/g Of d r y ma88

Biotin Polic acid Niacin Pantothenic acid Pyridoxin Riboflavin Thiamine

-

I

-

0.8

3.5

146.0

690.

-

1.8 2.8

82.0

150.0

500.0

8.7

145.0

130.0

-

-

5.8

34.0

24.6

53.0

49.0

2.0

3.9

11.0

6.2

F r a q r ance Aroma i s one o f t h e moat c h a r a c t e r i s t i c f e a t u r e s o f e d i b l e fungi,

b u t due t o t h e l a c k o f a p r e c i a e m e t h o d o f i t s d e t e r m i n a t i o n ,

many c o n f u s i o n s r e s u l t . "not plessant".

Usually fragrance i s c a l l e d "pleasant"

or

I n o r d e r t o a c h i e v e t h e s t r o n g e r aroma i t was p r o -

v e d t h a t t h e c u l t u r e m u s t b e g r o w n one or t w o d a y a l o n g e r t h a n need e d t o r e a c h t h e maximum f u n g a l g r o w t h ( r e f .

70).

I t was a l s o i n t e n -

s i f i e d by m i x i n g m y c e l i u m w i t h NaCl and i n c u b a t i n g i t a t 4 t e r one d a y o f s u c h t r e a t m e n t , ved,

OC.

Af-

aome i m p r o v e m e n t o f aroma was o b a e r -

b u t t h e b e s t r e s u l t s were a c h i e v e d a f t e r aeven days ( r e f .

75).

P o s i t i v e e f f e c t a w e r e a l s o o b s e r v e d when l e c i t i n o r v e g e t a b l e o i l

or m i l k was a d d e d t h e g r o w t h c u l t u r e ( r e f . g o o d aroma,

t h e c o n c e n t r a t i o n o f N,

P,

K,

70). S,

I n order t o achieve

Fe a n d Zn a h o u l d b e

f a r g r e a t e r t h a n t h a t n e e d e d t o o b t a i n t h e maximum y i e l d o f t h e mycelium (ref.

20).

I n concluaion,

i t can be s a i d t h a t a more concen-

t r a t e d medium a n d s l o w e r g r o w t h o f t h e c u l t u r e e n s u r e a s t r o n g e r a n d more p l e a s a n t aroms.

Research s h o u l d aim a t f i n d i n g a mutant

which could, w h i l e growing feet,

p r o d u c e a d e s i r e d aroma.

-

243

-

Toxicity A l t h o u g h numerous f u n g a l s p e c i e s have been u s e d as f o o d f o r t h o u s a n d s o f y e a r s w i t h o u t any s i d e e f f e c t s ,

i t i s h a r d t o assume

t h a t t h e mycelium o f e d i b l e f u n g i grown i n t h e submerged c u l t u r e i s equally safe.

(ref.

So f a r ,

i t s t o x i c i t y has n o t been observed.

73,761.

LIGNOCELLULOSIC BYPRODUCTS A S A SUPPORT FOR FUNGAL BIOMASS PRODUCTION As m e n t i o n e d ,

i t i s possible t o c u l t i v a t e higher fungi not

o n l y f r o m e x p e n s i v e s y n t e h t i c m e d i a b u t a l s o f r o m b y p r o d u c t s or even waste m a t e r i e l s . s e a r c h f o r cheep,

I t i s important,

therefore,

t o proceed i n the

abundant and e a s i l y a c c e s s i b l e s u p p o r t s w h i c h can

s i m u l t a n e o u s l y m e e t t h e p h y s i o l o g i c a l demands o f f u n g i .

One s h o u l d

mention t h e cheapest o r g a n i c substance which c o u l d be t r a n s f o r m e d i n t o p r o t e i n by f u n g i ,

that i s lignocellulose

g r e a t number o f i n d u s t r i a l w a s t e s , wood i n d u s t r i e s .

-

a component o f a

e s p e c i a l l y t h o s e o f paper and

According t o Bellamy ( r e f .

77),

t h e world produc-

t i o n o f c e l l u l o s e b y e l l p l a n t s on t h e e a r t h a m o u n t s t o 2 4 t o n s p e r capita

8

year.

A m e a n i n g p a r t o f t h a t mass u s e d i n t h e p a p e r i n -

d u s t r y i s r e s p o n s i b l e f o r waste ( m o a t l y l i g n i n weate) a b o u t 500 b i l l i o n t o n s a y e a r on a w o r l d w i d e s c a l e .

r a n g i n g up t o Therefore,

i t

w o u l d be d e s i r a b l e t o f i n d o r g a n i s m s w h i c h c o u l d u t i l i z e t h e l i g n o cellulose,

e s p e c i a l l y t h e i r l i g n i n component a n d a t t h e same t i m e

e l i m i n a t e any c o n s t r a i n t s i n t r e a t i n g t h e m a t e r i e l a s e s o u r c e o f f e e d biomas.

In fact,

these

r e q u i r e m e n t s a r e f u l f i l l e d by a r b o r e s l

f u n g i f o r which l i g n o c e l l u l o s e i s t h e n a t u r a l growth environment. A l r e a d y i n t h e 308,

t h e f i r s t a t t e m p t s w e r e made t o d e t e r m i n e

t h e p r o c e s s o f wood d e l i g n i f i c a t i o n c a u a e d b y t h e a c t i v i t y o f v a r i o u s white rot Baaidiomycetes. sicolor

(ref.

wood d e l i g n i f i e r s . (ref. it

WBS

I t h s s b e e n ahown t h a t T r e m e t e s v e r -

78) end Trsmetes p i n i ( r e f .

79) a r e t h e most a c t i v e

The s e c o n d o f t h e above m e n t i o n e d p i o n e e r w o r k a

79) i n t h i s f i e l d i s o f p a r t i c u l a r i m p o r t a n c e f o r ua because prepared by a Pole.

Several years l a t e r ,

n a l y s i s was o f f e r e d on b e e c h - t r e e species 8s Polyporus e b i e t i n u s , donius (refs.

80,Bl).

a m o r e d e t a i l e d a-

sawdust d e l i g n i f i c s t i o n by such

S t e r e u m ruqosum a n d M a r s s i m i u s s c o r o -

These w o r k a f o r t h e f i r s t t i m e s t r e s s e d t h e

f a c t t h a t d e l i g n i f i c a t i o n end c e l l u l o a e d e c o m p o s i t i o n were p a r a l l e l processes.

I t t o o k 9 months end t h e consumption o f b o t h components

r a n g e d f r o m 4 5 t o 65% d e p e n d i n g o n t h e s p e c i e s .

This r e l a t i v e l y low

-

244

-

l i g n o c e l l u l o s e complex d e g r a d a t i o n r e s u l t s from l i g n i n r e s i s t a n c e t o

I t h a s b e e n s t a t e d t h a t i n t h e wood, c e l l u l o forming a three-dimensional n e t w o r k w h i c h p r o t e c t s c e l l u l o s e f r o m c e l l u l o l y t i c e n z y m e s A s a res u l t o f s u c h " l i g n i n b a r r i e r ' ' a p p e a r a n c e , wood c a r b o h y d r a t e s a r e n o t d e c o m p o s e d by t h e m a j o r i t y o f c a r b o h y d r a t e d e g r a d i n g m i c r o b e s the biological attack. se

f i b e r s are surrounded with l i g n i n ,

(ref. 82). The p r o b l e m o f " l i g n i n b a r r i e r " h a s b e e n t h e f o c a l p o i n t o f a numbe r o f b i o t e c h n o l o g i c a l s t u d i e s .

T h e r e are two p o s s i b l e ways o f

s o l v i n g i t . The f i r s t c o n s i s t s i n u s i n g a s a s u p p o r t , k f o r t h e f u n g a l c u l t u r e , c e l l u l o s e waste or i t a h y d r o l i s a t e s which can be che-

m i c a l l y o b t a i n e d o r d e r i v e d from t h e c e l l u l o s e complex a c t v i t y i s o l a t e d from c e l l u l o s e - d e g r a d i n g

f u n g i . The s e c o n d c o n s i s t s

l i g n o c e l l u l o s e waste as a suport f o r fungi possesing both

n using igno-

and c e l l u l o l y t i c a c t i v i t y ( i n t h e case o f o n l y c e l l u l o l y t i c f u n g i , t h e e x t e n t o f d e g r a d a t i o n was l i m i t e d a n d t h e p r o d u c t w i l l a l w a y s contain indigested lignin). More i n f o r m a t i o n a b o u t t h e f i r s t way c a n b e f o u n d i n many r e views and p a p e r s ( r e f s . 77,83-100).

T h e r e f o r e we s h a l l l i m i t our

p r e s e n t a t i o n t o t h e most popular and t y p i c a l examplex o f biotechnol o g i c a l methods a l r e a d y a p p l i e d or i n t e n d e d t o be a p p l i e d soon. M u r r a y Moo-Young

of t h e Canadian Waterloo University, develo-

p e d a m e t h o d by w h i c h p a p e r i n d u s t r y waste i s c o n v e r t e d i n t o g l u c o -

ae ( r e f . 101). Thia c o p y r i g h t p r o c e s s i s c a r r i e d o u t i n s p e c i a l f e r mentation tanks f o r s e l e c t e d lower fungus species.

The p r o d u c t i s

f u r t h e r u s e d as a s u p p o r t f o r y e a s t c u l t i v a t i o n and f i n a l l y t h e val u a b l e feed biomass is o b t a i n e d . ( r e f .

102).

S w e d i s h r e s e a r c h e r s h e a d e d by P r o f . E r i k s s o n o f t h e F o r e s t P r o d u c t s L a b o r a t o r y i n S t o c k h o l m d e s c r i b e d a m e t h o d o f p a p a r m i l l waste u t i l i z a t i o n by m e a n s o f S p o r o t r i c h u m p u l v e r u l e n t u m . produced biomass c o n t a i n i n g 14% p r o t e i n .

This culture

To i m p r o v e t h e y i e l d a n d

a synergic c u l t u r e of Sporotrichum pulvelurentum a n d C a n d i d a u t i l i s was s u g g e s t e d ( r e f . 1 0 3 ) . A n o t h e r s p e c i e s , Trichoderma v i r i d e c o u l d v e g e t a t e e f f i c i e n t l y on straw a u t o c l a v e d p r e q u a l i t y of biomass,

v i o u a l y with 5% natrium lhydroxide.

A f t e r t h e 5 day growth, t h e pro-

t e i n c o n t e n t i n t h e p r o d u c t was 2 5 % a n d t h e d e g r a d a t i o n o f s t r a w c e l l u l o s e r e a c h e d 75% ( r e f .

104).

A m e t h o d o f P a e c i l o m y c e s v a r i o t t i c u l t u r e o n s u l f i t e w a s t e liq u o r s f r o m t h e c e l l u l o s e p r o d u c t i o n waa p a t e n t e d i n F i n l a n d a n d i n t r o d u c e d o n a c o m m e r c i a l s c a l e ( t h e p r o c e s s "PEKILO").

I n t h i s method,

-

245

-

c a r b o h y d r a t e f r a c t i o n s o f t h e s u l f i t e w a s t e s a r e m o s t l y consumed by t h e mycelium.

The y e a r l y y i e l d o f b i o m a s s ( c o n t a i n i n g 55% p r o -

t e i n ) amounts t o 1 0 . 0 0 0 (refs.

t o n s and i s a p p r o p r i a t e d f o r a n i m a l f e e d

105,106).

A n American p a t e n t w h i c h p o i n t s t o t h e p o s s i b i l i t y o f t h e sub-

merged c u l t u r e on s u l f i t e wastes l i q u o r o f s u c h h i g h e r f u n g i a s T r i c h o l o m a nudum,

Callybia velutipes,

-b l a z e i i s o f a p i o n e e r i n g c h a r a c t e r .

L e p i o t a neuryna and A q a r i c u s

An a g a r m y c e l i u m i s t r a n s f e r r e d

i n t o t h e medium c o n t a i n i n g s u l f i t e w a s t e l i q u o r e n r i c h e d w i t h 0.1% ammonium p h o s p h a t e .

Then i t i s moved a g n i n i n t o t h e s u l f i t e w a s t e

o f a concentrstion i n the c i n g !3ugsrs,

medium c o r r e s p o n d i n g t o 15 g / l

o f redu-

n e t u r s l i z e d b y c a l c i u m c a r b o n a t e t o pH 5 e n d e n r i c h e d

w i t h 0.1% ammonium p h o s p h a t e .

as i n t h e p r e v i o u s case, o f the aerated culture,

A f t e r fl h o u r s

t h e y i e l d was f a i r l y s m a l l a n d r a n g e d u p t o

10 g o f dYy mass p e r 1 l i t e r o f t h e c u l t u r e ( r e f .

107).

P r e t r e a t m e n t o f c e l l u l o s e wastes w i t h c e l l u l s s e complex prepar a t i a n s i s a very useful starting-paint specialized fungi. strains)

for further cultivation of

T r i c h o d e r m a v i r i d e ( e s p e c i a l l y Q M 9123 a n d OM 9 4 1 4

i s known a s a n e f f i c i e n t c e l l u l a s e p r o d u c e r ( r e f .

108),

so

i t s e n z y m a t i c s y s t e m waa u s e d t o o b t a i n t h e g l u c o s e s y r u p f r o m p a p e r m i l l waste.

This syrup ( f r e e o f fungal metabolites)

s e r v e d a s a sup-

p o r t i n biomass p r o d u c t i o n f o r a p p r o p r i a t e s p e c i e s o f f u n g i ,

o r was

used d i r e c t l y as a s u b s t r a t e i n r e f i n e r i e s o r o t h e r i n d u s t r i e s ( r e f s .

loe-llo). ning,

B e c a u s e p a p e r m i l l w a s t e c o n t a i n s a c e r t a i n amount o f l i g -

which renders

c e l l u l o l y t i c enzyme a c c e a a d i f f i c u l t ,

a prelimi-

n a r y c u l t i v a t i o n was s u g g e s t e d o n t h o s e w a s t e m a t e r i a l s o f T r s m e t e s versicolor,

w i d e l y known a s a l i g n o l y t i c s p e c i e s .

The p a r t l y d i g e s -

t e d waste s u b s t r a t e by Trametea v e r a i c o l o r no l o n g e r c o n t a i n s l i g n i n a n d t h u s becomes m o r e a c c e s s i b l e f o r c e l l u l o l y t i c enzymes o f T r i c h o derma v i r i d e ( r e f .

111).

Some a u t h o r s s u g g e s t t h e a p p l i c a t i o n o f c s l l u l o l y t i c f u n g u s a s a d i r e c t p r o t e i n biomass p r o d u c e r ( r e f .

101)

The t h e r m o r e s i s t a n t

Ghaetomium c e l u l o l y t i c u m s t r a i n p r o v e s t o b e s u c h a s p e c i e s ( r e f . 112).

I t surpasses Trichoderma v i r i d e by 100 % a s f a r as t h e vegeta-

t i o n r a t e i s concerned,

and has t h e p o s s i b i l t y t o grow a t a r e l a -

t i v e l y h i g h t e m p e r a t u r e (37

OC).

This temperature serves aa a b a r r i e r

a g a i n s t t h e i n v a s i o n o f many f u n g a l s p e c i e s w h i c h w o u l d i n f e c t t h e culture. The a t t e m p t s d e s c r i b e d so f a r d o n o t a u t h o r i t a t i v e l y s o l v e t h e p r o b l e m o f t h e l i g n i n component o f l i g n o c e l l u l o s i c s u p p o r t a .

The

-

-

246

r e m a i n i n g un d i g e s t e d l i g n i n s u b s t a n t i a l l y t r i e n t value.

r e d u c e s t h e b i o m a s s nu-

I t i s necessary t o look f o r appropriate organisms or

enzymatic systems d i r e c t l y degrading l i g n i n .

Another problem i s t o

f i n d an a d e q u a t e m o d e l o f t h e l i g n i n p o l y m e r .

Such a m o d e l w o u l d

s e r v e e i t h e r as a s o u r c e o f carbon for f u n g i or as a s u b s t r a t e f o r t h e l i g n o l y t i c enzmye s y s t e m .

I n t h e f i r s t case,

t o a c h i e v e a more a c c e p t a b l e o r b e t t e r s u p p o r t

Moreover,

t h e f u n g a l biomass

b u t i n t h e s e c o n d i t w o u l d be p o s s i b l e

would be d i r e c t l y o b t a i n e d ,

for fungal growth.

t h e f u n g i g r o w i n g d i r e c t l y on a l i g n i n model would produce

t h e i n d u c i b l e enzymatic system u s e f u l

i n t h e second case.

g r a d a t i o n p r o d u c t s o b t a i n e d i n t h e e n z y m a t i c way,

L i g n i n de-

would s e r v e as a

The s o c a l l e d l i g n o s u l p h o n i c

valuable substrate f o r biotechnology.

a c i d s can s e r v e as t h e adequate l i g n i n model.

They a p p e a r a s t h e

b y p r o d u c t when c e l l u l o s e i s o b t a i n e d b y t h e s u l f i t e m e t h o d .

I n this

c a s e t h e w h o l e amount o f l i g n i n i n t h e f o r m o f c a l c i u m l i g n o s u l f o n a t e s s o l u b l e i n water is t r a s f e r r e d .

S u l f i t e waste l i q u o r s a r e

u s u a l l y a c c o m p a n i e d by s i m p l e s a c c h a r i d e s i n t h e f o r m o f f r e e h e x o s e s a n d h i g h m o l e c u l a r p e n t o s a n s w h i c h c a n be e l i m i n a t e d b y means o f yeasting.

The c u l t u r e o f B s s i d i o m y c e t e s ,

metes p i n i ,

and P l e u r o t u s o s t r e a t u s )

i n i n g medium was a s u b j e c t

(Trametes v e r s i c o l o r ,

Tra-

i n the lignosulphonates conta-

o f the Polish patent ( r e f

5).

The r e s e -

a r c h e r s i n C z e c h o s l o v a k i a showed t h a t t h e m e l i u m s t e r i l i u m s t r a i n , which they i s o l a t e d ,

wss a b l e t o d e g r a d e l i g n o a u l f o n i c a c i d e f f e c -

t i v e l y a n d y i e l d 32-35:;

p r o t e i n c o n t a i n i n g biomass ( r e f .

113).

The

f u n g a l u t i l i z a t i o n o f s u l f i t e waste l i q u o r p r e v i o u s l y d e p r i v e d o f c a r b o h y d r a t e s b y y e a s t i n g was s u c c e s s f u l l y a c h i e v e d i n N i e d o m i c e , Poland,

on a s e m i - t e c h n o l o g i c a l

scale.

n i n g medium was e n r i c h e d w i t h whey, superphosphate,

The l i g n o s u l p h o n a t e c o n t a i -

yeast e x t r a c t ,

some m i c r o e l e m e n t s a n d a n e u r i n e .

c u l t u r e was g r o w n i n a 4 . 0 0 0

1 f e r m e n t a t i o n t a n k and t h e s u p p o r t

was s t e r i l i z e d b y o v e r h e a t e d w a t e r steam. of

the culture,

1 l i t r e (ref.

natrium n i t r a t e ,

Trametes v e r s i c o l o r

A f t e r a 4 day i n c u b a t i o n

t h e y i e l d o f m y c e l i u m r e a c h e d 4 0 g o f d r y mass p e r

114).

The l i g n o c e l l u l o s e c o m p l e x ( v a r i o u s k i n d s o f s a w d u s t a n d s t r a w ) was d i r e c t l y u t i l i z e d u s i n g t h e f u n g a l c u l t u r e s i n o u r l a b o r a t o r y . P r e l i m i n a r y s c r e e n i n g t e s t s p r o v e d Trametes v e r s i c o l o r ,

Pleurotus

o s t r e a t u s a n d Chaetomium p i l u l i f e r u m g r e w t h e b e s t on l i g n o c e l l u l o s e supports. optimized.

The c o m p o s i t i o n o f t h e s u p p o r t s f o r t h e s e f u n g i was f u r t h e r Under t h e o p t i m a l c o n d i t i o n s ,

3 0 a n d 40% o f l i g n i n a n d

c e l l u l o s e d e g r a d a t i o n were a c h i e v e d r e s p e c t i v e l y

( r e f . 1 1 4 ) . The p o s s i

-

-

247

b i l i t y o f b a r k u t i l i z a t i o n f o r f u n g a l biomass p r o d u c t i o n s h o u l d a l so be mentioned.

A c i d i c p i n e b a r k e x t r a c t s were used t o grow about

200 p u r e f u n g a l c u l t u r e s .

Out o f a l l t e s t e d s p e c i e s ,

a f a i r l y r a p i d vegetation process,

Trametes v e r s i c o l o r and P o l y p o r u s b r e n n i s , ratory,

38 e x h i b i t e d

smong them t h e l i g n i n d e g r a d e r s (ref.

115).

I n our labo-

d e l i g n i f i c a t i o n o f t h e p i n e b a r k was a c h i e v e d i n t h e a t a -

t i o n a r y c u l t u r e o f aome B a s i d i o m y c e t e s . Inonotus obliquus,

The m o s t a c t i v e d e g r a d e r was

w h i c h r e m o v e d f r o m t h e s u p p o r t 23.3% o f l i g n i n

and 29.51 o f c e l l u l o s e .

Some i n d u s t r i a l w a s t e s w h i c h s e r v e d aa a

n i t r o g e n source s t i m u l a t e d t h e g r o w t h o f t h e fungus and d e g r a d a t i o n o f support s i g n i f i c a n t l y ( r e f . Lignin,

as p o i n t e d o u t ,

116).

i s extremely r e s i s t a n t t o the attack o f

microorganisms. I n t h e l a s t two decades a s u b s t a n t i a l p r o g r e s s has b e e n made t o w a r d s t h e f u l l e x a m i n a t i o n o f i t s b i o l o g i c a l d e g r a d a t i o n

( r e f s . 117-120).

First of all,

f o r t h a t has s u b s t a n t i a l l y

a number o f o r g a n i s m s r e s p o n a i b l e

i n c r e a s e d and i t h a s been s t a t e d t h a t n o t

only white r o t fungi are responaible f o r l i g n i n degradation, soft r o t fungi (ref. as w e l l ( r e f s .

121),

123,124).

brown r o t f u n g i ( r e f .

Recently,

a number o f enzymes a c t i v e i n l i g -

n i n d e g r a d a t i o n were m a r k e d l y e n l a r g e d . r e d a s a m a i n l i g n i n d e g r a d i n g enzyme) biose:

quinone oxidoreductase

1 3 2 1 , manganese - d e p e n d e n t

(ref.

Despite ligninaae (conside (refs.

35,

125-1301,

cello-

131), c e l l o b i o a e oxidase ( r e f .

peroxidaae (refs.

133,

oxidase ( r e f .

135),

formaldehyde diamutase ( r e f .

oxidase ( r e f .

137),

g l i o x a l oxidase ( r e f .

139-141) and methylase ( r e f .

but

122) and b a c t e r i a

1341, m e t h a n o l 136),

glucose-2-

1381, demethylaee ( r e f s .

1 4 2 ) were d i s c o v e r e d .

Knowledge a b o u t

t h e r o l e o f t h e o t h e r enzymes s u c h a s l a c c a s e ( r e f s .

143,1441,

pe-

roxidase (refs.

118,145,146),

144,1471,

dio-

xygenaae ( r e f s .

148,149)

markedly increased.

glucose oxidase (refs.

p a r t i c i p a t i n g i n l i g n i n d e g r a d a t i o n haa

The c o o p e r a t i o n among t h e s e enzymes i s t a k e n i n -

t o consideration (refs.

118,131,144,147,150).

I t t h i a case,

aimul-

t a n e a u a t r a n s f o r m a t i o n o f c e l l u l o s e a n d l i g n i n b y means o f c e l l u l a ae a n d o x y g e n a a e c o m p l e x e s may o c c u r ( r e f s .

120,131,147).

The e x i a -

t e n c e o f t h e enzyme c o n n e c t i n g t h e t w o m e t a b o l i c a l s e q u e n c e i . e . cel1obiose:quinone (ref.

oxidoreductaae

1 4 7 ) was shown.

(ref.

1 3 1 ) or g l u c o a e o x i d a s e

A l l these achievements g i v e a h o p e f u l o u t l o o k

f o r a s o l u t i o n t o the " l i g n i n b a r r i e r " problem i n the near future. REFERENCES

1

R . C r z y b o w s k i , The f r u i t i n g b o d i e s a n d m y c e l i a o f h i g h e r f u n g i s s a f o o d s o u r c e , Przem. Spoz, 32 ( 1 9 7 8 ) 1 3 - 1 6 .

2 3 4 5

6

7

8 9 10 11

12 13 14 15 16 17 18 19 20 21 22 23 24

248

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v e r s i c o l o r , A c t a Chem. Scand., B 4 1 ( 1 9 8 7 ) 766-769. A . K a n t e l i n e n , R. W a l d n e r , M.-L. N i k u - P a a v o l a and M.S.A. Leis o l a , Comparison o f two l i g n i n - d e g r a d i n g f u n g i : P h l e b i a r a d i a t a and Phanerochaete c h r y s o s p o r i u m , Appl. M i c r o b i o l . B i o t e c h n o l . , 28 ( 1 9 8 8 ) 1 9 3 - 1 9 8 . T.K. K i r k , M. T i e n , P . J . K e r s t e n , M.D. Mozuch a n d B . K a l y a n a r a man, L i g n i n a s e o f P h a n e r o c h a e t e c h r y s o s p o r i u m , M e c h a n i s m o f i t s degradation o f the non-phenolic a r y l g l y c e r o l - h - a r y l ether s u b s t r u c t u r e o f l i g n i n , B i o c h e m . J., 236 ( 1 9 8 6 ) 279-287. K . M i k i , V . R e n g a n a t h a n , M.B. M a y v i e l d a n d M.H. G o l d , A r o m a t i c r i n g cleavage o f a &biphenyl e t h e r dimer catalysed by l i g n i n p e r o x i d a s e o f P h a n e r o c h a e t e c h r y s o s p o r i u m , FEBS L e t t . , 210 (1987) 199-203. M. Shimada, 1. H a t t o r i , T . Umezawa, T . H i g u c h i a n d K . U z u r a , R e g i o s p e c i f i c oxygenations d u r i n g r i n g cleavage o f a secondary m e t a b o l i t e 3,4-dimethoxybenzyl a l c o h o l c a t a l y z e d by l i g n i n per o x i d a s e , FEES L e t t . , 2 2 1 ( 1 9 8 7 ) 3 2 7 - 3 3 1 U . W e s t e r m a r k a n d K.-E. Eriksson, Cellobiose: quinone oxidored u c t a s e , a new w o o d - d e g r a d i n g enzyme f r o m w h i t e - r o t f u n g i , A c t a Chem. Scand., B 28 ( 1 9 7 4 ) 209-214. A.R. Ayers, S.B. A y e r s and K.-E. Eriksson, C e l l l o b i o s e oxidase, p u r i f i c a t i o n l a n d p a r t i a l c h a r a c t e r i z a t i o n o f a hemoprotein E u r . J. B i o c h e m . , 9 0 ( 1 9 7 8 ) f r o m S p o r o t r i c h u m pulverulen-. 171-181. J . K . G l e n n a n d M.H. Gold, P u r i f i c a t i o n and c h a r a c t e r i z a t i o n o f an e x t r a c e l l u l a r Mn(I1)-dependent p e r o x i d a a e from t h e l i g n i n degrading Basidiomycete, Phanerochaete chrysosporium, Arch. B i o chem. B i o p h y s . , 242 (1985) 329-341. T . J o h a n s s o n a n d P . O . Nyman, A manganese ( 1 1 ) - d e p e n d e n t e x t r a c e l l u l a r p e r o x i d a s e from t h e w h i t e - r o t fungus Trametes v e r s i c o lor, A c t a Chem. Scand., B 4 1 ( 1 9 8 7 ) 7 6 2 - 7 6 5 . A. N i s h i d a and K.-E. E r i k s s s o n , Formation, p u r i f i c a t i o n and p a r t i a l c h a r a c t e r i z a t i o n o f methanol oxidase, a H202-producing Appl. B i o enzyme i n P h a n e r o c h a e t e c h r y s o s p o r i u m , B i o t e c h n o l chem., 9 ( 1 9 8 7 ) 3 2 5 - 3 3 8 . N. K a t o , S . M i z u n o , Y . Imada, M. Shimao a n d C. Sakazawa, F o r mate p r o d u c t i o n f r o m m e t h a n o l b y f o r m a l d e h y d e d i s m u t a s e coup l e d w i t h a methanol o x i d a t i o n system, Appl. M i c r o b i o l . B i o technol., 27 ( 1 9 8 8 ) 5 6 7 - 5 7 1 . K.-E. E r i k s s o n , B. P e t t e r s o n , J. V o l c a n d V . M u s i l e k , Format i o n and p a r t i a l c h a r a c t e r i z a t i o n o f g l u c o s e - 2 - o x i d a s e , a H202 p r o d u c i n g enzyme i n P h a n e r o c h a e t e c h r y a o s p o r i u m , A p p l . M i c r o b i o l . B i o t e c h n o l . , 23 ( 1 9 8 6 ) 257-262. P . J . K e r s t e n a n d T . K . K i r k , I n v o l v e m e n t o f a new enzyme, glyoxal oxidase, i n e x t r a c e l l u l a r H 0 p r o d u c t i o n by Phaneroc h a e t e c h r y s o s p o r i u m , J. B s c t e r i o 1 . f ? 6 9 ( 1 9 8 7 ) 2195 2201. T.D. F r i c k a n d R.L. C r a w f o r d , M e c h a n i s m s o f m i c r o b i a l demeH i g u c h i , H.-m. Chang t h y l a t i o n o f l i g n i n model polymers, i n : T K i r k (Eds.), Recent Advances i n L i g n i n B i o d e g r a d a t i o n and T.K. R e s e a r c h , U n i . P u b l i s h e r s Co., L t d . Chuo-ku, T o k y o 1 0 3 Japan, 1983, pp. 1 4 3 - 1 5 2 . A.I. Hatakka, D e g r a d a t i o n o f v e r a t r i c a c i d and o t h e r l i g n i n - r e l a t e d a r o m a t i c sompounds b y t h e w h i t e - r o t f u n g u s P y c n o p o r u s c i n n a b a r i n u s , Arch. M i c r o b i o l . , 1 4 1 (1985) 22-28. M.H. G o l d , M.B. M a y f i e l d , T . M . Cheng, K K r i a n a n g k u r a , M. S h i made, A. E n o k i a n d J . K . Glenn, A Phanerochaete c h r y s o s p o r i u m mutant d e f e c t i v e i n l i g n i n d e g r a d a t i o n as w e l l as s e v e r a l o t h e r secondary m e t a b o l i c f u n c t i o n s , Arch. M i c r o b i o l . , 132 (1982) 1 1 5 - 1 22. A.C. F r a z e r , I. B o s s e r t a n d L . Y . Young, E n z y m a t i c a r y l - 0 - m e t h y l -

-

143 144 145 146

147

148 149

150

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14C l a b e l i n g o f m o d e l l i g n i n monomers, A p p l . E n v i r o n . M i c r o b i o l . , 5 1 ( 1 9 8 6 ) 80-83. A . L e o n o w i c z , G. S z k l a r z a n d M. W o j t a a - W s s i l e w s k a , The e f f e c t o f f u n g a l l a c c s s e on f r a c t i o n a t e d l i g n o s u l p h o n a t e s ( P e r i t a n - N a ) , P h y t o c h e m . , 24 ( 1 9 8 5 ) 393-396. G. S z k l a r z a n d A . L e o n o w i c z , C o o p e r a t i o n b e t w e e n f u n g a l l a c c a a e and g l u c o s e o x i d a s e i n t h e d e g r a d a t i o n o f l i g n i n d e r i v a t i v e s , Phytochem., 25 ( 1 9 8 6 ) 2537-2539. J. L o b a r z e w s k i , J. T r o j a n o w s k i a n d M. W o j t a s - W a s i l e w s k a , The e f f e c t o f f u n g a l p e r o x i d a s e on N a - l i g n o s u l p h o n a t e s , H o l z f o r s c h . , 36 ( 1 9 8 2 ) 173-176. P . J . K e r s t e n , 6 . K a l y a n a r a m s n , K . E . Hammel a n d T . K . Kirk, H o r s e r a d i s h p e r o x i d s s e o x i d i z e s , 1,2,4,5-tetramethoxybenzene b y a c a t i o n r a d i c a l mechanism, i n : E . O d i e r ( E d . ) , P r o c . I n t . Sem i n a r on L i g n i n E n z y m i c a n d M i c r o b i a l D e g r a d a t i o n , P a r i s , A p r i l 23-24, 1987, I N R A P u b l i c a t i o n s , P a r i s , 1 9 8 7 , p p 7 5 - 7 9 . A. L e o n o w i c z , J . R o g a l s k i , J . S z c z o d r s k and J . F i e d u r e k , The p o s s i b l e k e y r o l e o f g l u c o s e o x i d s s e i n t r a n s f o r m a t i o n o f l i g n o c e l l u l o s e , i n : Swedish F o r e s t P r o d u c t s Research Labor a t o r y a n d The S w e d i s h A s s o c i a t i o n o f P u l p a n d P a p e r E n g i n e e r s ( E d s . ) , P r o c . 3 r d I n t e r n . C o n f . E i o t e c h n o l . F u l p P a p e r Ind., S t o c k h o l m , Sweden, J u n e 1 6 - 1 9 , 1 9 8 6 , p p . 1 6 0 - 1 6 2 . M. W o j t a s - W a s i l e w s k a a n d J . L u t e r e k , The e f f e c t o f f u n g a l p r o t o c a t e c h u a t e 3,4-dioxygenase on sodium l i g n o s u l p h o n a t e f r a c t i o n s , P h y t o c h e m . , 26 ( 1 9 8 7 ) 2 6 7 1 - 2 6 7 4 . M . W o j t a s - W a s i l e w a k a , J. L u t e r e k , A . D a w i d o w i c z a n d A . L e o n o wicz, D e a r o m a t i z a t i o n o f l i g n i n d e r i v a t i v e s by f u n g a l p r o t o c a t e c h u a t e 3 , 4 - d i o x y g e n a s e i m m o b i l i z e d on p o r o s i t y g l a s s , B i o t e c h n o l . B i o e n g . , 29 ( 1 9 8 7 ) i n p r e s s . 3. L o b s r z e w s k i e n d P a s z c z y n s k i , L i g n o c e l l u l o s e b i o t r a n s f o r m a t i o n with i m m o b i l i z e d c e l l u l a s e , D-glucose o x i d a s e and f u n g a l p e r o x i d a s e a , Enzyme M i c r o b . T e c h n o l . , 7 ( 1 9 8 5 ) 5 6 4 - 5 6 6 .

ACKNOWLEDGEMENT T h i s w o r k was s u p p o r t e d b y t h e P o l i s h S c i e n t i f i c P r o j e c t s Nos.

C P E P 04.11.2.33,

CPEP 0 4 . 0 2 / 2 . 2

a n d CPER 3 . 1 3 . 2 . 1 . 1 8 .

This Page Intentionally Left TBlank

II. Bioenergy and Environment

This Page Intentionally Left TBlank

IMMOBILIZED P H O T O S Y N T H E T I C S Y S T E M S F O R THE P R O D U C T I O N

OF F U E L S AND CHEMICALS D.O.

HALL,

K.K.

R A O and I . H .

PARK

D i v i s i o n o f Biosphere Sciences, King’s L o n d o n WE 7AG, U . K .

C o l l e g e London

ABSTRACT The e f f i c i e n c y o f s o l a r e n e r g y c o n v e r s i o n b y p h o t o s y n t h e s i s i n t h e n a t u r a l ecosystem seldom exceeds 1 % on an annual b a s i s , r e p r e s e n t s s t o r e d energy.

but t h i s

B i o m i m e t i c systems w i t h i s o l a t e d components

o f t h e p h o t o s y n t e h t i c l i g h t e n e r g y h a r v e s t i n g a n d c o n v e r s i o n appar a t u s have been d e s i g n e d w i t h t h e a i m o f a c h i e v i n g h i g h e r s o l a r e n e r gy c o n v e r s i o n e f f i c i e n c e s .

Chloroplasts,

t h y l a k o i d membranes a n d P S I

p a r t i c l e s f r o m p l a n t s and c y a n o b a c t e r i a have been used t o g e t h e r w i t h r e d o x c a t a l y s t s a n d enzymes f o r t h e p h o t o p r o d u c t i o n o f H 2 , NADPH2.

H202 and

T h y l a k o i d s and P S I 1 membranes h a v e a l s o b e e n u s e d f o r t h e

g e n e r a t i o n o f H2 and p h o t o c u r r e n t s r e s p e c t i v e l y w i t h w a t e r a s t h e e l e c t r o n source.

C y a n o b a c t e r i a a r e good “ b i o c a t a l y s t s ”

NH3,

d u c t i o n o f H2,

H202,

organic acids etc.

p o t e n t i a l sources f o r g l y c e r o l ,

h c a r o t e n e and o t h e r p l a n t p i g m e n t s .

Microalgae a r e a l s o prime p o t e n t i a l organisms

CO

f o r photopro-

Other microalgae are

f o r the absorption o f

from t h e environment since they r e q u i r e only i n o r g a n i c n u t r i e n t s

2

and water

for

growth.

Immobilization o f the microalgae i n gels,

v i n y l a n d p o l y u r e t h a n e foams,

h o l l o w f i b r e membranes e t c .

poly-

stabilises

t h e i r p h o t o s y n t h e t i c a c t i v i t y and f a c i l i t a t e s t h e i r m a n i p u l a t i o n i n photobioreactora.

1

INTRODUCTION

1.1

Importance o f photosynthesis

A l l our f o s s i l ( o i l ,

coal,

gas)

and b i o l o g i c a l

are derived v i a the process o f photosynthesis.

(biomass)

The amount o f c a r b o n

f i x e d a n n u a l l y by p h o t o s y n t h e t i c p r o c e s s e s i s a p p r o x i m a t e l y 10 tonnes. num,

fuels

11

The e n e r g y c o n t e n t o f t h i s f i x e d c a r b o n i s 3 ~ 1 0 ’ J~ p e r an-

which i s about 10 times the world’s

present annual consumption

o f energy.

260

-

Photosynthetic carbon f i x a t i o n u s i n g s o l a r r a d i a t i o n i s

a s i g n i f i c a n t c o n t r i b u t o r t o our energy resource:

i t has been and

w i l l c o n t i n u e t o be one o f t h e m a j o r s o u r c e s o f e n e r g y on E a r t h .

A t p r e s e n t p h o t o s y n t h e s i s i s t h e o n l y p r o c e s s w h i c h removes v a s t

C O P from t h e atmosphere; t h i s u n i q u e r o l e p l a y e d by pho-

amounts o f

t o s y n t h e s i s i s v i t a l i n r e d u c i n g t h e a t m o s p h e r i c C02 c o n t e n t and t h u s p r e v e n t i n g e x c e s s i v e g l o b a l warming.

P h o t o s y n t h e s i s and C O P

r e c y c l i n g has r e c e n t l y a t t r a c t e d t h e a t t e n t i o n o f p o l i c y makers concerned w i t h energy p l a n n i n g , depollution

g l o b a l w a r n i n g and e n v i r o n m e n t a l

[ll.

I n a d d i t i o n t o C02 f i x a t i o n t o carbohydrates,

photosynthetic

p r o c e s s e s can a l s o be used t o p r o d u c e l i p i d s ( f a t s and o i l s ) , nia,

amino a c i d s ,

proteins,

hydrogen,

hydrogen peroxide,

The t h e o r e t i c a l maximum e f f i c i e n c y energy conversion,

etc.

for photosynthetic

solar

d e f i n e d as t h e r a t i o o f s t o r e d c h e m i c a l e n e r g y

t o t h e i n c i d e n t s o l a r r a d i a t i o n on t h e p h o t o s y n t h e s i z e r , l a t e d t o b e a b o u t 1 3 %; However,

X (J.R.

B o l t o n and D.0

t h i s maximum c o n v e r s i o n e f f i c i e n c y

chieved i n the f i e l d ; o n an a n n u a l b a s i s .

i s calcu-

t h e p r a c t i c a l maximum c o n v e r s i o n e f f i c i e n c y

under optimal c o n d i t i o n s i s 9 blished).

ammo-

Hall,

unpu-

i s n e v e r a-

f o r most p l a n t s p e c i e s i t seldom exceeds 1

X

There i s t h e r e f o r e a g r e a t i n t e r e s t i n d e v e l o -

ping photobiological/photochemical

systems w h i c h would mimic t h e

energy conversion process o f n a t u r a l photosynthesis,

hopefully with

better efficiency.

1.2 P h o t o m i m e t i c s y s t e m s There a r e two t y p e s o f n a t u r a l p h o t o s y n t h e s i s v i z o x y g e n i c (water-splitting) and anoxygenic

c a r r i e d o u t by p l a n t s ,

a l g a e and c y a n o b a c t e r i a

( w h e r e w a t e r i s n o t u s e d aa e l e c t r o n s o u r c e )

t i n g i n photosynthetic bacteria.

opera-

I n both types the essential featu-

r e s are: l i g h t energy a b s o r p t i o n b y antenna pigments, t r a n s f e r o f absorbed energy t o

a reaction centre with special chlorophylls,

charge s e p a r a t i o n and t r a n s f e r t o quinones v i a p h e o p h y t i n s and f i n a l l y e n e r g y c o n v e r s i o n t o ATP a n d NAD(P)H t h r o u g h r e d o x p r o t e i n s .

A l l t h e s e p r o c e s s e s o c c u r a t a f a s t r a t e t h r o u g h components l o c a t e d i n membrsnes.

P h o t o b i o l o g i s t s and p h o t o c h e m i a t s a r e i n v o l v e d i n un-

d e r s t a n d i n g and m i m i c k i n g v a r i o u s s e g m e n t s o f t h i s p h o t o s y n t h e t i c energy c o n v e r s i o n process. (a)

These i n c l u d e :

s y n t h e s i s and s t r u c t u r e a n a l y s i s o f Mn complexes m i m i c k i n g

t h e P S I 1 o x y g e n e v o l u t i o n c o m p l e x 121,

(b)

261

-

s y n t h e s i s and s t u d y o f r e a c t i o n r a t e s and k i n e t i c s o f mul-

ticomponent carotenoid-porphyrin-quinone l i g h t a b s o r p t i o n and energy t r a n s f e r ria.

Molecular t r i s d s ,

tetrads,

complexes m i m i c k i n g

i n photosynthetic bacte-

and r e c e n t l y a p e n t a d have been

assembled [ 3 1 , ( c ) synthesis o f e l e c t r o n donor-acceptor t h e mechanism o f e l e c t r o n t r a n s f e r

compounds t o u n d e r s t a n d

141,

w i t h pho-

( d ) d e s i g n o f p r o t e o - l i p i d m o l e c u l a r a s s e m b l i e s (e.9. tosensitizer

dyes e n t r a p p e d i n l i p i d v e s i c l e s )

t o study elec-

t.ron m i g r a t i o n and c h a r g e a e p s r s t i o n i n v e s i c l e s [ 5 1 , ( e ) b u i l d i n g p h o t o c h e m i c s l e n d p h o t o e l e c t r o c h e m i c a l a r r a y s comprising photosensitizer, electron relay,

and r e d o x c a t a l y s t s

f o r w a t e r s p l i t t i n g o r f o r C02 r e d u c t i o n 161, ( f ) i s o l a t i o n o f t h y l a k o i d membranes, particles

i n d i v i d u a l photosystem

( P S I a n d PS 1 1 ) o r b a c t e r i a l c h r o m s t o p h o r e a a n d deaystems f o r t h e p h o t o p r o d u c t i o n o f energy

signing " i n vitro" r i c h chemicals,

and

( g ) c u l t u r i n g c y a n o b a c t e r i a o r algae under " s t r e s s "

f o r the

e x c r e t i o n o f s p e c i f i c m e t a b o l i t e s 1 7 1. Our l a b o r a t o r y h a s m a i n l y b e e n i n t e r e s t e d i n t h e l a s t t w o ( f and 9 )

2

t o p i c s a n d e m p h a a i s w i l l b e p l a c e d on t h e s e p r o c e s s e s .

IMMOBILIZATION I m m o b i l i z a t i o n i s t h e p r o c e s s o f a t t a c h i n g c e l l s o r t h e i r con-

s t i t u e n t b i o c a t a l y a t s t o a s o l i d m a t r i x s o t h a t t h e y do n o t move i n d e p e n d e n t l y when p l a c e d i n a f l u i d e n v i r o n m e n t . b e c a r r i e d o u t b y p h y s i c a l means, i n a g e l o r foam m a t r i x , binding.

o f t e n e n h a n c e d r e d u c t i o n r a t e s due t o c o n c e n t r a -

t i o n o f c a t a l y s t surface, permeability,

are

1OSS

such 5s a d s o r p t i o n o r entrapment

o r by c h e m i c a l methods such a s c o v a l e n t

The a d v a n t a g e s o f i m m o b i l i z a t i o n a r e s t a b i l i z a t i o n o f c a -

talytic activity,

products,

I m m o b i l i z a t i o n can

i n c r e a s e d c e l l metabolism and c e l l w a l l

ease o f s e p a r a t i o n and r e u s e o f c a t a l y s t s f r o m f l u i d

and p r o c e s s c o n t r o l i n a b i o r e a c t o r . o f a c t i v i t y during immobilization,

Main disadvantagea

t h e enzyme a c t i v i t i e s

a r e e x t r s c e l l u l a r a n d l i m i t a t i o n o f r e a c t i o n r a t e s due t o d i f f u s i o n resistance.

Gels (acrylamide,

glutaraldehyde-albumin

agar,

sponges,

elginate,

carrageenan,

r e t i c u l a t e foams ( p o l y v i n y l

chitosen), end po-

l y u r e t h a n e ) p o l y p r o p y l e n e and p o l y a u l f o n e h o l l o w f i b r e s and c e r a m i c s (silica)

a r e t h e u s u a l m a t e r i e l s u s e d aa t r a n s p a r e n t o r r e f l e c t i n g

supports f o r immobilization [8,91.

-

262

Entrapment i n porous g e l s o r

-

foam m a t r i x i s t h e f a v o u r e d t e c h -

nique f o r the immobilization o f chloroplasts, and a l g a l c e l l s ,

chromatophores,

plant

e t c a s i t d o e s n o t m o d i f y o r c a u s e any damage t o

t h e o r g a n e l l e or c e l l .

Since l i g h t i s a d i r e c t substrate f o r photo-

s y n t h e s i z e r s t h e i m m o b i l i z a t i o n m a t r i x must be reasonably transpar e n t as w e l l as b e i n g p o r o u s f o r p e n e t r a t i o n o f t h e c e l l s .

I n our

i m m o b i l i z a t i o n w o r k w i t h c h l o r o p l a s t s a n d c y a n o b a c t e r i a we h a v e used agar and a l g i n a t e t e l s ,

p o l y u r e t h a n e a n d p o l y v i n y l foams,

and

membrane h o l l o w f i b r e s . A l q i n a t e i s a p o l y m e r o f mannuronic a c i d and g u l u r o n i c a c i d and i s i s o l a t e d from m a r i n e a l g a e .

A l g i n a t e bead i m m o b i l i z a t i o n i s

performed by mixing a suspension o f c h l o r o p l a s t s or a l g a l c e l l s w i t h an e q u a l v o l u m e o f 2 % s o d i u m a l g i n a t e i n b u f f e r a n d d i s p e n s i n g t h e m i x t u r e i n d r o p s t o a 0 . 1 M CaC12 s o l u t i o n k e p t s t i r r e d i n an i c e bath.

Calcium a l g i n a t e g e l beads w i t h e n t r a p p e d p h o t o s y n t h e t i c

m a t e r i a l a r e o b t a i n e d [ 10

I.

-

U r e t h a n e i s f o r m e d b y t h e r e a c t i o n b e t w e e n an i s o c y a n a t e and a h y d r o x y l group: a r y l group.

RNCO

+ R’-OH

urethane polymer.

are toluene 2 , 4 diisocyanate ( T D I )

(MDI).

where R i s an a l k y l or

Condensation with o t h e r isocyanates,

produces a c r o s s - l i n k e d

t e

RNHCOOR’

a l c o h o l s o r amines

P r i n c i p a l i s o c y a n a t e s used

and d i p h e n y l methane d i i s o c y a n a -

The h y d r o x y l compounds g e n e r a l l y u s e d a r e p o l y e t h e r o r

p o l y e s t e r based. i s evolved.

D u r i n g h y d r o l y t i c c o n d e n s a t i o n s o f u r e t h a n e s C02

The o v e r a l l t e x t u r e o f p o l y u r e t h a n e m a t r i x i s d e p e n d e n t

on t h e c h e m i c a l n a t u r e o f t h e p o l y o l ,

t h e t e m p e r a t u r e o f condensa-

t i o n and t h e presence o f added c a t a l y s t . C e l l i m m o b i l i z a t i o n can be p e r f o r m e d e i t h e r by entrapment,

i.e.

by s t i r r i n g an a q u e o u s s u s p e n s i o n o f c y a n o b a c t e r i a l c e l l s i n t o a ur e t h a n e p r e p o l y m e r ( e g H y p o 1 2002,

W.R.

Grace L i m i t e d ,

London) and

g e n e r a t i n g t h e p o l y m e r by c o n d e n s a t i o n or by a d s o r p t i o n o f t h e c e l l s on t o s m a l l p i e c e s o f p o l y m e r

foams b y c u l t u r i n g .

Entrapment r e s u l t s

i n a homogeneous d i s t r i b u t i o n o f t h e c e l l s w i t h i n t h e m a t r i x ;

howe-

v e r loss o f c e l l v i a b i l i t y h a s b e e n r e p o r t e d a s a r e s u l t o f e n t r a p ment. P o l y v i n y l foams l y v i n y l alcohol.

a r e u s u a l l y p r e p a r e d by t h e c o n d e n s a t i o n o f po-

These foams w h i c h r e t a i n r e a c t i v e OH g r o u p s ,

t r a n s p a r e n t and q u i t e s t a b l e .

are

Immobilization o f c e l l s i s carried out

m a i n l y by a d s o r p t i o n . Membrane h o l l o w f i b r e r e a c t o r s h a v e b e e n u s e d f o r a l o n g t i m e for

t h e i m m o b i l i z a t i o n and p r o d u c t i o n o f a n i m a l c e l l s ;

t h e y a r e now

-

263

-

finding application i n the immobilization o f plant, terial cells. fone,

polypropylene, etc.)

polymers.

a l g a l and bac-

These membranes a r e made f r o m h y d r o p h o b i c

They p r o v i d e a h i g h s u r f a c e a r e a a n d c a n a c t a s a m o l e c u -

l a r sieve.

P o t e n t i a l advantages o f h o l l o w - f i b r e b i o r e a c t o r s a r e h i g h

c e l l r e t e n t i o n , more e f f i c i e n t c o n t i n u o u s p r o c e s s i n g , paration o f products, b i l i t y t o s c a l e up. ticulate-free ons,

a n d amena-

Major l i m i t a t i o n s are the requirement o f a par-

media,

f i b r e r u p t u r e , maintenance o f s t e r i l e c o n d i t i e s p e c i a l l y gases

A p o l y s u l f o n e h o l l o w f i b r e membrane b i o r e s c t o r u s e d f o r

t h e p r o d u c t i o n o f NH;, i n Fig.

a u t o m a t i c se-

r e c o v e r y and reuse o f b i o c a t a l y s t s ,

and d i f f i c u l t y i n t h e s u p p l y o f n u t r i e n t s ,

111,121.

(polyaul-

or h y d r o p h i l l i c (rayon, c e l l u l o s e acetate

w i t h i m m o b i l i z e d Anebaena a z o l l a e ,

1 (Wang and H a l l , B i o m a s s ,

i a shown

i n press).

Serum a l b u m i n p o l y m e r s c r o s s l i n k e d w i t h q l u t a r a l d e h y d e i s a favoured technique f o r chemical i m m o b i l i z a t i o n o f c h l o r o p l a s t s , c h r o m a t o p h o r e s a d a p t e d by Thomas e t a 1

[el.

C r i t e r i a f o r successful

i m m o b i l i z a t i o n a r e maintenance o f p h o t o s y n t h e t i c a c t i v i t y measured as o x y g e n e x c h a n g e o f cells,

fluorescence,

and i n t h e case o f i m m o b i l i z e d

t h e maintenance o f v i a b i l i t y and g r o w t h a s d e t e r m i n e d by

c h l o r o p h y l l c o n c e n t r a t i o n o r enzyme a c t i v i t y

(eg n i t r o g e n a a e

or hy-

droyenase).

F i g . 1. S c h e m a t i c i l l u s t r a t i o n o f a membrane h o l l o w f i b r e r e a c t o r f o r t h e c o n t i n u o u s p r o d u c t i o n a n d m o n i t o r i n g o f NHJ f r o m i m m o b i l i zed A . a z o l l a e .

- 264

3

-

APPLICATIONS T y p i c a l e x a m p l e s o f t h e u s e o f i m m o b i l i z e d s y s t e m s f o r t h e pho-

t o p r o d u c t i o n o f f u e l s and c h e m i c a l s a r e shown i n T a b l e 1. D e t a i l s c a n b e f o u n d i n r e c e n t r e v i e w s and monographs. I l 3 - 1 6 1 TABLE 1 P r o d u c t s f r o m i m m o b i l i z e d P h o t o s y n t h e t i c Systems

Matrix used and Material Immobilized

Biotechnological Application

AGAR

Mastigocladus, Phormidium

HZ production

A n a b a e ~7363

H2, fuel cells

Chromatiumand Rhohspirillum rubrwn chloroplasts+ catalysts Photosystem I +catalysts thylakoid membranes

H2production

N2 + NADPH2 H2 HzO2

ALBUMIN-GLUTARALDEHYDE

thylakoid membranes Scenedesmur chromatophores PSI1 membranes

H2, A" biochemical studies ATP

ATP

ALGINATE GEL

Botryococcus Chlorella emersoni Dunaliella sp Anatmena 27893 Mastigocladus laminosus synechococcus 40607 Rhodobacter capsdata @a) thylakoid membranes

hydrocarbons (oils) glycollate glYCW! arnrmn? ammDllla

glutamate HZ ?PZ

PoLYVINYLANDmLYmEm4NEmAMs Porphyridiwn and Chlamydomnas sp Anabaenaawllae

Phonnidium Variouscyanobacteria Capsicumfrutescens

exocellularpolysaccharides ammonia and HZ H202 H2 and NADPHZ Capsaicin (alkaloid)

MEMBRANEHOLLOWWRES A. azollae

$andammonia

-

265

-

3 . 1 Hydrogen Hydrogen i s t h e i d e s 1 llgreen"

fuel,

i t s combustion product

( w a t e r ) i s n o n - p o l l u t i n g end i s renewable. ( w a t e r ) i s cheap and abundant.

Besides t h e feed stock

There i s p r e s e n t l y renewed i n t e r e s t

i n t h e development o f s o l a r hydrogen p r o d u c t i o n t e c h n o l o g i e s .

3.la

Photosynthetic Bscteria

H2

Hydrogen i s a m e t a b o l i c p r o d u c t o f p h o t o s y n t h e t i c b a c t e r i a , e v o l u t i o n b e i n g c a t s l y s e d b y t h e n i t r o g e n a s e enzyme.

Feasibility

o f H 2 p r o d u c t i o n has been demonstrated w i t h i m m o b i l i z e d c e l l s o f R h o d o s p i r i l l u m rubrum ( a g a r ) , capsulsta (8s alginate).

Chromatium sp ( a g a r ) and Rhodobacter

1171 L a b o r a t o r y - s c a l e b i o r e a c t o r s packed

w i t h i m m o b i l i z e d c e l l s produced H2 a s e f f i c i e n t l y as f r e e - l i v i n g cells. etc.)

S u b s t r a t e s f o r c a r b o n and e l e c t r o n s o u r c e ( m a l a t e , a r e a v a i l a b l e i n o r g a n i c wastes from a g r o i n d u s t r y ;

lactate, simulta-

neous d e p o l l u t i o n o f i n d u s t r i a l wastes can be a c h i e v e d t o g e t h e r w i t h

H p production.

I f e n e r g y i n p u t f r o m s u b s t r a t e s is d i s r e g a r d e d t h e

e f f i c i e n c y o f H2 p r o d u c t i o n c o u l d be n e a r u n i t y .

The d i s a d v a n t a g e

o f t h e process i s t h a t t h e p h o t o s y n t h e t i c b a c t e r i a cannot use water as s u b s t r a t e a n d t h a t t h e p h o t o b i o r e a c t o r h a s t o b e m a i n t a i n e d anaerobic t o prevent oxygen-inhibition o f nitrogenase.

3.lb

Cyanobacteris Nitrogen f i x i n g cysnobacteria (blue-green

successfully

a l g a e ) have been

i m m o b i l i z e d and used f o r t h e p h o t o p r o d u c t i o n o f energy,

H 2 e v o l u t i o n is c a t e l y s e d m a i n l y by n i t r o g e n a s e and t o a l e s s e r ext e n t by hydrogenaae.

C y a n o b a c t e r i a a r e good h a r v e s t e r s o f s u n l i g h t

and a r e easy and e c o n o m i c a l t o grow. b e e n p e r f o r m e d w i t h A n a b s e n s spp;

A.

Most H2 e v o l u t i o n s t u d i e s have c y l i n d r i c a and A.

azollse.

Se-

v e r a l marine c y a n o b a c t e r i a have a l s o been screened f o r t h e i r pot e n t i a l as s o l a r H 2 p r o d u c e r s [181. l e t o immobilization.

C y s n o b a c t e r i s a r e q u i t e arneneb-

An o u t d o o r b i o p h o t o l y t i c s y s t e m u s i n g A .

cy-

l i n d r i c a 8 6 2 9 s u s p e n d e d i n g l a s s b e a d s was shown t o p r o d u c e H 2 c o n t i n u o u s l y f o r o v e r t h r e e weeks.

Continuous H2 e v o l u t i o n l a s t i n g

f o r o v e r 5 m o n t h s was o b s e r v e d f r o m A . lyvinyl

szollee immobilized i n p o -

o r p o l y u r e t h a n e foams [ 1 9 1 . The v i a b i l i t y o f t h e enzymes

end p h o t o c a t a l y s t s i s p r e s e r v e d f o r m o n t h e . a f t e r

A t the present time,

immobilized cyanobacterisl

t h e most p r o m i s i n g source f o r

immobilizetion. c e l l s appear t o be

H2 generation i n photobioresctors.

3.lc

-

266

Green A l g a e Hydrogenase a c t i v i t y can be i n d u c e d i n g r e e n a l g a e by g r o w i n g

them a n a e r o b i c a l l y ;

t h e a l g a e w i l l t h e n e v o l v e H 2 and O 2 s t o i c h i o -

m e t r i c a l l y by water p h o t o l y s i s . efficiencies

(3 t o 12

X)

Maximum l i g h t e n e r g y c o n v e r s i o n

were r e p o r t e d w i t h t h i n f i l m s o f a l g a e en-

t r a p p e d on f i l t e r p a p e r s 1 2 0 1 .

However t h e e x t r e m e o x y g e n s e n s i t i v i -

t y o f hydrogenase l i m i t s t h e use o f t h e s e organisms f o r b i o p h o t o l y t i c H2 p r o d u c t i o n .

3.1d

C h l o r o p l a s t membranes ( t h y l a k o i d s ) E l e c t r o n s and p r o t o n s g e n e r a t e d b y l i g h t - i n d u c e d

water s p l i t t i n g

a t Photosystem I 1 can be t r a n s p o r t e d t o P S I where t h e y can reduce an e x o g e n o u s e l e c t r o n m e d i a t o r .

The r e d u c e d m e d i a t o r c a n b e c o u p l e d

t o hydrogenase o r P t t o produce H2.

S e v e r a l l a b o r a t o r i e s have s t u -

d i e d H 2 p r o d u c t i o n b y t h i s r o u t e w i t h t h y l a k o i d s a n d c a t a l y s t s immob i l i z e d i n various types o f matrices.

Although immobilization resul-

t e d i n some s t a b i l i z a t i o n o f t h e s y s t e m ,

problems o f p h o t o i n h i b i t i o n

o f t h y l a k o i d components and oxygen i n h i b i t i o n o f c a t a l y s t s c o u l d n o t be s o l v e d c o m p l e t e l y

[ l o ] . The p h o t o b i o r e a c t o r c o u l d n o t f u n c -

t i o n f o r m o r e t h a n a few h o u r s

-

we h a v e t o l e a r n m o r e a b o u t p h o t o -

i n h i b i t i o n b e f o r e s v i a b l e system can be developed.

An alternative

would be t o bypass Photosystem I 1 and use o n l y Photosystem I a c t i vity

f o r H2 production.

This,

o f course,

w o u l d be more e x p e n s i v e s i n -

ce i t would n e c e s s i t a t e t h e c o n s u m p t i o n o f P S I e l e c t r o n d o n o r s ,

as

i n photosynthetic bacteria. 3.2

HYDROGEN P E R O X I D E H y d r o g e n p e r o x i d e s i s an e n e r g y - r i c h

compound,

i t l i b e r a t e s mo-

r e t h a n 1 0 0 k J p e r m o l when i t decomposes t o w a t e r a n d 0 2 . I s o l a t e d c h l o r o p l a s t s a r e a b l e t o p h o t o r e d u c e O 2 t o H 2 0 2 by t h e s o c a l l e d "Mehler"

r e a c t i o n "211. The r a t e o f H 2 0 2 f o r m a t i o n c a n b e e n h a n c e d

by t h e a d d i t i o n o f P S I e l e c t r o n a c c e p t o r s s u c h a s m e t h y l v i o l o g e n , f l a v i n nucleotides, reduction o f H 0

2 2

quinones,

etc.,

a l l o f which can c a t a l y z e t h e

O 2 t o H202 v i a s u p e r o x i d e . L i g h t - i n d u c e d f o r m a t i o n o f

m e d i a t e d by r i b o f l a v i n ,

h a s b e e n shown w i t h s p i n a c h t h y l a k o i d

i m m o b i l i z e d i n d i f f e r e n t g e l s a n d foams entrapped c h l o r o p l a s t s [221.

-

b e s t y i e l d was f r o m a g a r -

The r e a c t i o n was

'short

lived'

w i t h wa-

t e r as e l e c t r o n s o u r c e ( p o s s i b l y d u e t o l i g h t a n d O 2 i n h i b i t i o n o f PSII);

however,

w i t h P S I d o n o r s H202 p r o d u c t i o n c o n t i n u e d f o r h o u r s .

P r o d u c t i o n o f H 2 0 2 w i t h i n i t i a l r a t e s o f 5 0 0 N m o l H202 p e r mg c h l o -

rophyll.h,

267

-

from m i c r o a l g a e i l l u m i n a t e d i n t h e presence o f methyl v i o -

l o g e n has been r e p o r t e d 1 2 3 1 . We h a v e r e c e n t l y s u c c e e d e d i n d e s i g n i n g a p h o t o b i o r e a c t o r

for

c o n t i n u o u s H 2 0 2 p r o d u c t i o n w i t h p o l y v i n y l foam i m m o b i l i z e d P h o r m i d i u m laminosum c e l l s as p h o t o s y n t h e s i z e r and m e t h y l v i o l o g e n as P S I e l e c t r o n acceptor. (Park,

The a r r a n g e m e n t o f t h e ' r e a c t o r '

Rao a n d H a l l ,

i s shown i n F i g .

2

unpublished).

jq

c )

PI .... ................... ... ............... . ........... . ..... ........ . .. . . .. ................... . ............

PHOTOPRODUCTION

A

IMMOB. CELL

WATER

LIGHT SOURCE

4

t

.

ORANGE COLOUR FILTER F i g . 2 . Column r e a c t o r f o r t h e p h o t o p r o d u c t i o n o f H202 u s i n g P V i m m o b i l i z e d Phormidium laminosum c e l l s .

3 . 3 A T P a n d NAD(P)H2 These m o l e c u l e s a r e f o r m e d d u r i n g p h o t o s y n t h e t i c e l e c t r o n t r a n s p o r t and p h o s p h o r y l a t i o n end s r e h i g h v a l u e , micals.

energy-rich,

bioche-

I n v i t r o systems c o n s i s t i n g o f c h l o r o p l a s t s or b a c t e r i a l c h r o -

matophores i m m o b i l i s e d i n m a t r i c e s such as agar, rum a l b u m i n foam,

polyscrylamide,

se-

b a r i u m a l g i n e t e o r p o l y u r e t h a n e have a l l been used

f o r photophosphorylation y i e l d i n g ATP.

I n a l l o f the reported studies

immobilization resulted i n s t a b i l i z a t i o n o f photosynthetic a c t i v i t i e s o f the i s o l a t e d organelle

1241.

C y a n o b a c t e r i a l c e l l s i m m o b i l i z e d on p o l y u r e t h a n e foams p h o t o r e d u c e d NADP t o N A D P H 2 i n t h e p r e s e n c e o f c a t a l y t i c a m o u n t s o f t h e e n z y me f e r r e d o x i n - N A D P

dehydrogenase

1251.

P h o t o c h e m i c a l r e d u c t i o n o f NAD

t o NADH c a n b e a c c o m p l i s h e d b y b a n d gap i l l u m i n a t i o n o f T i 0 2 c o l l o i d a

i n s o l u t i o n s o f NAD i n t h e p r e s e n c e o f r h o d i u m t r i s b i p y r i d y l c o m p l e x , n o n e n z y m a t i c r e d u c t i o n o f NAD 1261.

4

-

268

AMMONIA Nitrogen f e r t i l i s e r s

up t o 50

(nitrate,

ammonium,

urea,

etc.)

contribute

X o f t h e energy i n p u t i n modern a g r i c u l t u r e . Development

o f photobiological or photochemical N2 f i x a t i o n processes could sign i f i c a n t l y r e d u c e t h e b u r n i n g o f f o s s i l f u e l s o r wood ( t h u s i n c i d e n t a l l y r e d u c i n g C02 e m i s s i o n ) cing nitrogen fertilisers.

i n t h e chemical p l a n t s p r e s e n t l y produ-

Ammonia i n s d d i t i o n i s a v a l u a b l e c h e m i -

cal fuel.

I t i s e s t i m a t e d t h a t a s much s s 10'

t o n n e s o f N 2 p e r annum i s

c o n v e r t e d t o ammonia b y b i o l o g i c a l p r o c e s s e s ( c y a n o b a c t e r i s a n d r h i zobia-legume

symbiosis).

The e n e r g y demand f o r t h i s N 2 f i x a t i o n i s

u l t i m a t e l y met b y t h e s u n v i e p h o t o s y n t h e s i s .

A l t h o u g h l a r g e amounts

o f N 2 a r e f i x e d by c y a n o b a c t e r i a l n i t r o g e n a s e o n l y i n a few cases can t h i s f i x e d N 2 b e d i r e c t l y u t i l i s a b l e ss s c r o p f e r t i l i s e r . s p e c i a l case i s t h e Anabaena-Azolla

One s u c h

symbiotic a s s o c i a t i o n which i s

e x t e n s i v e l y used as a n a t u r a l f e r t i l i s e r

i n r i c e f i e l d s and w h i c h i s

c a p a b l e o f h i g h r a t e s o f N2 f i x a t i o n ( u p t o 3 k g o f N f i x e d p e r h e c t s r e per day).

I n t h i s a s s o c i a t i o n t h e N2 f i x e d by t h e cyanobscteria

i s e x c h a n g e d f o r p h o t o s y n t h a t e s p r o d u c e d by t h e A z o l l a f e r n

-

any

u n u s e d ammonia i s e x c r e t e d i n t o t h e medium C 2 7 1 . We h a v e c h o s e n Anabaena a z o l l a e t o d e v e l o p an i m m o b i l i z e d p h o -

t o s y n t h e t i c r e a c t o r f o r t h e c o n t i n u o u s p r o d u c t i o n o f smmonia.

A.

azo-

c e l l s were i m m o b i l i z e d i n h y d r o p h i l i c p o l y u r e t h a n e o r p o l y v i n y l foam p i e c e s o r i n p o l y s u l p h o n e h o l l o w f i b r e s . constructed with t h e foam-immobilized

A p h o t o b i o r e a c t o r was

c y a n o b a c t e r i a packed i n t o a

f l u i d i s e d b e d c o l u m n w i t h c o n s t s n t c i r c u l a t i o n o f g r o w t h medium.

The

medium was o c c a s i o n a l l y g i v e n p u l s e s o f L-methionine-D-L-sulfoximine, an i n h i b i t o r o f g l u t a m i n e s y n t h e t a s e ,

which minimised the conversion

o f ammonia t o g l u t a m i n e a n d o t h e r m e t a b o l i t e s . e f f l u e n t was d e t e r m , i n e d ,

The ammonia i n t h e

either colorimetrically or

A p h o t o b i o r e a c t o r w i t h A.

polarographically.

a z o l l a e i m m o b i l i z e d i n p o l y v i n y l foam gene-

r a t e d one mmol NH3 p e r l i t r e e f f l u e n t p e r h o u r w h i c h i s e q u i v a l e n t o n a n a r e a b a s i s t o t h e f i x a t i o n o f 900 k g o f N p e r h e c t a r e p e r annum.

This r a t e s h o u l d be compared t o t h e t y p i c a l N f i x a t i o n r a t e s o f 400 k g p e r h a p e r annum i n l e g u m e s a n d 1 1 0 k g p e r h a p e r annum i n t h e n a t u r a l Anabaena-Azolla

symbiosis.

The o n l y l i m i t a t i o n i n t h i s i m m o b i l i z e d

s y s t e m i s t h a t t h e ammonia i n t h e e f f l u e n t i s r a t h e r d i l u t e ( 0 . 1 however,

mM);

t h i s w i l l n o t b e a drawback i f t h e a f f l u e n t i s c h a n n e l l e d

t o i r r i g a t i o n water f o r t h e crop.

5

269

-

OTHER FUELS a n d CHEMICALS Hydrocarbons ( o i l s and l i p i d s ) a r e produced as m a j o r m e t a b o l i c

products o f several green algae.

F o r example,

c a n s y n t h e s i z e h y d r o c a r b o n s up t o 8 0

Botryococcus brauni

YO o f i t s d r y mass. The h y d r o -

c a r b o n s can b e e x t r a c t e d by t r e a t m e n t o f t h e c e l l s w i t h hexane w i t hout a f f e c t i n g t h e i r photosynthetic a c t i v i t y . and y i e l d s were s u b s t a n t i a l l y

increased,

Hydrocarbon recovery

relative t o free cells,

b y i m m o b i l i z a t i o n o f t h e a l g a i n p o l y u r e t h a n e foams 1 2 8 1 . Polyurethane-immobilized

forphyridium

cruentum (a r e d alga)

have produced s u l p h a t e d p o l y s a c c h a r i d e s f o r l o n g p e r i o d s i n a tubul a r p h o t o b i o r e a c t o r s t t h e r a t e o f 20 g p e r sq m e t r e p e r d a y . b i l i z e d g r e e n a l g a e ( C h l o r e l l a a n d Chlamydomonas)

Immo-

secretes extra-

c e l l u l a r p o l y s a c c h a r i d e s u s e d i n f o o d a n d a g r i c u l t u r e , The g r e e n algs Dunaliella,

i m m o b i l i z e d i n Cs a l g i n a t e ,

p r o d u c t i o n o f g l y c e r o l and

R carotene.

can be used f o r t h e

Immobilized m i c r o a l g a l sys-

tems i s a r a p i d l y e x p a n d i n g f i e l d o f b i o t e c h n o l o g y

6

"291.

ENVIRONMENTAL DEPOLLUTION Two o f t h e c h a l l e n g i n g p r o b l e m s f a c i n g s c i e n t i s t s c o n c e r n e d

w i t h t h e environment a r e t h e accumulation o f "greenhouse"

gases i n

t h e atmosphere and t h e s c c u m u l a t i o n o f i n o r g a n i c f e r t i l i s e r s

(nitra-

t e s a n d p h o s p h a t e s ) i n t h e b i o s p h e r e c a u s i n g e u t r o p h i c a t i o n i n wat e r reservoirs,

l a k e s and r i v e r s .

What c o n t r i b u t i o n c a n p h o t o b i o l o -

g i s t s make t o w a r d s t h e a l l e v i a t i o n ,

i f n o t complete a b o l i t i o n ,

of

t h e s e p o l l u t i o n problems? 6 . 1 l e r t i s r y Treatment o f Water The e f f l u e n t s f r o m p r i m a r y a n d s e c o n d a r y u r b a n w a s t e w a t e r t r e a t m e n t s may s t i l l c o n t a i n u n a c c e p t a b l e and P(P03-)

4

l e v e l s o f N(NO;,

compounds f o r u a e i n d o m e s t i c w a t e r s u p p l y .

NO;,

NHd)

These i n o r -

ganics are responsible f o r t h e e u r o t r o p h i c a t i o n o f water bodies prod u c i n g m i c r o a l g a l blooms w h i c h a r e v e r y d i f f i c u l t t o remove from t h e water.

T e r t i a r y t r e a t m e n t o f w a t e r w i t h i m m o b i l i z e d m i c r o a l g a e may

p r o v i d e a a o l u t i o n t o t h i s p o l l u t i o n problem.

Preliminary studies

h a v e shown t h a t p a s s a g e o f s e c o n d a r y t r e a t e d w a t e r t h r o u g h c h i t o s a n i m m o b i l i z e d Phormidium laminosum ( a cyanobacterium) a n d 60

r e m o v e d 98

X N

X P compounds i n 24 h. The m i c r o a l g a e consumes t h e s e n u t r i e n t s

d u r i n g t h e i r growth i n t h e c h i t o s a n m a t r i x [ 301.

Periodically the

a l g a e can be s t r i p p e d o u t o f t h e m a t r i x and s t o r e d o r used as b i o mass.

We h a v e now s u c c e a s f u l l y

270

-

i m m o b i l i z e d Phormidium laminosum i n

p o l y v i n y l foams a n d u t i l i s e d t h e i m m o b i l i z e d c e l l s f o r p h o t o s y n t h e t i c n i t r a t e uptake ( G i l , hed).

Garbisu,

Hall,

S e r r a and B a z i n ,

unpublis-

N i t r a t e u p t a k e was m a i n t a i n e d f s r 3 m o n t h s w i t h v e r y l i t t l e

e v i d e n c e o f c e l l l e a k a g e f r o m t h e foams. 6.2

Abatement o f

t h e C02-induced

tl

greenhouse e f f e c t " .

The b u i l d up o f C02 i n t h e a t m o s p h e r e ( 2 7

X increase t o date,

w i t h h a l f t h e r i s e o c c u r r i n g o v e r t h e l a s t 30 y e a r s )

l e c o n t r i b u t i o n o f t h e o t h e r so c a l l e d

'qreenhouse gases'

v e r a l l w a r m i n g o f t h e e a r t h ( a b o u t a 0.5' century)

i s now w i d e l y r e c o g n i s e d .

accompanying s e a l e v e l r i s e s ,

The

and t h e p o s s i b t o t h e o-

increase over the l a s t

'greenhouse e f f e c t '

with i t s

c h a n g i n g r a i n f a l l p a t t e r n s and p o s s s i b -

l e i n c r e a s i n g p l a n t p r o d u c t i v i t y a r e s u b j e c t s o f much s t u d y i n t r y i n g t o u n d e r s t a n d t h e p r e s e n t s i t u a t i o n and where t h e i n c r e a s i n g a t m o s p h e r i c e m i s s i o n s may l e a d us i n t h e f u t u r e . ture

( n e x t 100 years)

The s h o r t - t e r m

fu-

a p p e a r s t o d e p e n d on how we u s e f o s s i l f u e l s

a n d how t h e c h a n g i n g a t m o s p h e r e a f f e c t s c l i m a t e a n d t h e p h o t o s y n t h e t i c p r o d u c t i v i t y o f p l a n t s and microorganisms,

e s p e c i a l l y on s r e g i o -

n a l basis. Unfortunately,

i t i s t h e r a t e o f change w h i c h w i l l d e t e r m i n e

w h e t h e r we c a n a d a p t t o c h a n g i n g c i r c u m s t a n c e s .

Humans p r e s e n t l y

d e t e r m i n e t h e r a t e o f b u i l d up o f g r e e n h o u s e g a s e s i n t h e a t m o s p h e -

re because t h e y c o n t r o l f o s s i l f u e l use and most d i s t u r b a n c e s t o the biota. Over t h e p a s t t w o c e n t u r i e s up u n t i l t h e 1 9 2 0 s - 1 9 4 0 ~ ,b i o s p h e r i c e m i s s i o n s and ocean b u f f e r i n g have been t h e m a j o r c o n t r o l s o f a t m o s p h e r i c C02 l e v e l s . century

A b o u t a t h i r d t o h a l f way t h r o u g h t h i s

( t h e t i m i n g i s s t i l l debated)

f o s s i l f u e l C02 e m i s s i o n s b e -

csme t h e p r e d o m i n a n t s o u r c e o f a t m o s p h e r i c relative contribution so c o n t e n t i o u s .

COP; the extent o f the

from f o s s i l f u e l and b i o s p h e r e s o u r c e s i s a l -

N o t w i t h s t a n d i n g t h i s debate,

i t must be a p p r e c i a t e d

t h a t t h e r a t e o f i n c r e a s e i n t h e use o f f o s s i l f u e l s o v e r t h e l a s t 15 y e a r s has f l u c t u a t e d g r e a t l y , t o forecast trations.

t h u s making i t e s p e c i s l l y d i f f i c u l t

f u t u r e e n e r g y t r e n d s and hence s t m o s p h e r i c

C O P concen-

Thus i n t h e p e r i o d 1 9 6 7 - 7 3 c o m m e r c i a l f u e l use i n c r e a -

s e d b y a b o u t 6 . 4 % p e r annum, o n l y 0 . 1 % p e r annum,

f r o m 1973-83

t h e r a t e o f i n c r e a s e waa

due t o t h e t w o " e n e r g y c r i s e s " ,

t h e i n c r e a s e h a s b e e n 2.9

b u t s i n c e 1983

% p e r annum a s t h e w o r l d a d j u s t s t o h i g h e r

e n e r g y p r i c e s and l e a r n s t o use e n e r g y more e f f i c i e n t l y .

A brief

-

-

271

summation o f c a r b o n f l u x e s and s i n k s i s c o m p i l e d i n T a b l e 2

[311.

I f t h e C02 c o n t e n t o f t h e a t m o s p h e r e were t o d o u b l e o v e r t h e n e x t c e n t u r y t o r e a c h a b o u t 5 4 0 ppm ( p r e - i n d u s t r i a l

l e v e l about

270

ppm) i t i s p r e d i c t e d t h a t t h e E a r t h w i l l e x p e r i e n c e an a v e r a g e w a r m i n g up o f a b o u t 3' house e f f e c t "

over p r e i n d u s t r i a l temperatures. This "green-

c o u l d be p r o d u c e d s o l e l y as a r e s u l t o f COP e m i s s i o n s

o r a c o m b i n a t i o n o f C02 a n d o t h e r " g r e e n h o u s e g a s e s " s u c h a s C H 4 , CFCs ( c h l o r o f l u o r o c a r b o n s ) ,

N 2 0 a n d 03.

6 . 3 A l g a e a n d C y a n o b a c t e r i a i n C02 a b a t e m e n t One p o s s i b l e s t r a t e g y f o r l a r g e - s c a l e

C02 f i x a t i o n w o u l d i n -

v o l v e t h e a p p l i c a t i o n o f p r e s e n t p h o t o b i o l o g i c a l knowledge i n a l g a l and c y a n o b a c t e r i a l systems.

P h o t o b i o r e a c t o r s c o u l d be a t t a c h e d t o

C O - e m i t t i n g s o u r c e s , f o r d i r e c t c o n v e r s i o n o f C02 t o f u e l s o r s t o 2 r a g e compounds. F o r e x a m p l e , p r o d u c t i o n o f h y d r o c a r b o n f u e l s a n d o t h e r o i l s by B o t r y o c o c c u s , N a n n o c h l o r o p s i s ,

etc.

c a n be e n v i s a g e d

i n l a r g e open p o n d s i n t o w h i c h t h e C02 o u t p u t s t r e a m i s d i r e c t l y injected.

Alternatively,

closed photobioreactors which exclude out-

p u t siream i s d i r e c t l y i n j e c t e d .

Alternatively,

closed photobiore-

a c t o r w h i c h e x c l u d e o u t s i d e a i r c o u l d be u s e d f o r p r o d u c t i o n o f h y drogen,

ammonium and o t h e r compounds.

Such a l g a l s y s t e m s w o u l d r e -

q u i r e o n l y s i m p l e g r o w t h medium ( b r a c k i s h w a t e r be u s e d i n many c a s e s ) ,

o r sea w a t e r c o u l d

need l i t t l e or no n i t r o g e n f e r t i l i s e r

c o u l d b e s e t up on d e s e r t o r s t e r i l e l a n d ,

and

w h e r e t h e y w o u l d n o t com-

pete with agriculture. I f t h e a i m were t o p e r m a n e n t l y s t o r e c a r b o n , c o u l d be f i x e d w i t h a l d e h y d e s , r e d d r y i n d e s e r t areas.

Section o f a l g a l s t r a i n s ( a research area

w h i c h shows c o n s i d e r a b l e p o t e n t i a l ) C02-scavenging

t h e a l g a l biomass

f i x e d as o r g a n i c c a r b o n a t e s o r s t o -

w o u l d be r e q u i r e d f o r maximum

and p h o t o s y n t h e t i c e f f i c i e n c y ,

o f carbon content a f t e r harvesting. r r i e r s w o u l d n e e d t o be o v e r c o m e ,

as w e l l as s t a b i l i t y

A l t h o u g h more t e c h n o l o g i c a l ba-

a l g a l s y s t e m s o f f e r c e r t a i n ad-

v a n t a g e s o v e r c a r b o n s t o r a g e a s wood.

S t r a i n s e l e c t i o n and g e n e t i c

m a n i p u l a t i o n w o u l d be g r e a t l y e n h a n c e d by t h e s h o r t g e n e r a t i o n t i m e , o f f e r i n g t h e p o t e n t i a l o f a wide range o f p r o d u c t s and c h a r a c t tics.

E f f i c i e n c y o f l i g h t e n e r g y c o n v e r s i o n ( a n d hence C O P

rate)

is a l s o p o t e n t i a l l y much h i g h e r t h a n f o r f o r e s t s ,

ris-

fixation

which would

reduce the requirement f o r land. Another p o s s i b l e response t o t h e b u i l d - u p w o u l d be t h e l a r g e - s c a l e

o f a t m o s p h e r i c COP

reforestation (or other reveqetation)

of

TABLE 2 C a r b o n f l u x e s and sinks-a s y n t h e s i s o f d a t a f r o m B a r n o l a e t e l . ( 1 9 8 7 1 , B o l i n e t a l . (19861, G i f f o r d (1980), Houghton e t a l . (19871, Kohlmaier e t a l . (1987), T r a b a l k a (1985), L i e t h (1985), S i e g e n t h a l e r 8 Oeschger ( 1 9 8 7 ) a n d M i n t z e r ( 1 9 8 7 ) 1311. ~

~~

CO

CO CO

2 2 2

i n atmosphere ( p r e s e n t a n n u a l i n c r e a s e )

i n atmosphere

(preindustrial)

i n atmosphere (change from p r e i n d u s t r i a l t o 1986)

1.5

ppm ( = 3. 2 Pg c a r b o n )

272 ppm ( = 575 Pg c a r b o n ) 7 4 ppm ( = 27 X; 155 Pg c a r b o n r e m a i n e d i n atmosphere

COi! f o s s i l - f u e l e m i s s i o n s ( 1 9 8 6 )

5.3

Pg c a r b o n p e r y e a r

Cog fossil-fuel

183

2 15 2 0.9

emissions (cumulative t o date)

Pg c a r b o n (1.0

C02 b i o s p h e r i c e m i s s i o n s ( p r e s e n t )

1.8

C02 b i o s p h e r i c e m i s s i o n ( c u m u l a t i v e t o d a t e )

150

F o s s i l f u e l s carbon content

1 7 0 Pg c a r b o n

(reserves)

F o s s i l f u e l s carbon content (resources) Biomass ( t e r r e s t r i a l ;

80 X i n t r e e s )

-

2.6)

6500 Pg c a r b o n 560 Pg c a r b o n

Gross p r i m a r y p r o d u c t i o n ( t e r r e s t r i a l )

120 Pg c a r b o n p e r y e a r

Net p r i m a r y p r o d u c t i o n ( t e r r e s t r i a l )

60 Pg c a r b o n p e r y e a r

Net p r i m a r y p r o d u c t i o n ( a q u a t i c )

46 Pg c a r b o n p e r y e a r

Net p r i m a r y p r o d u c t i o n ( t o t a l )

1 0 6 Pg c a r b o n p e r y e a r

S o i l carbon content

1 5 1 5 Pg c a r b o n

(75 m )

carbon content

725 Pg c a r b o n

O c e a n - i n t e r m e d i a t e a n d deep c a r b o n c o n t e n t

38000 Pg c a r b o n

C02 u p t a k e b y o c e a n

1.9

Pg c a r b o n p e r y e a r

C o g uptake by freshwater

0.8

Pg c a r b o n p e r y e a r

1 Pg = 10

15 g

Pg c a r b o n p e r yea1

5 0 Pg c a r b o n

3 Pg c a r b o n

Biomass ( a q u a t i c )

Ocean-surface

~~

346 ppm ( = 730 Pg c a r b o n )

C02 i n a t m o s p h e r e ( 1 9 8 6 )

I N

4 N I

p r e v i o u s l y degraded lands.

273

-

A t present,

i t i s e s t i m a t e d t h a t about

1 . 8 b i l l i o n t o n n e s o f c a r b o n a r e r e l e a s e d a n n u a l l y b y f o r e s t des t r u c t i o n and a l s o l a r g e amounts f r o m o t h e r f o r m s o f d e v e g e t a t i o n s u c h as g r a s s l a n d b u r n i n g s ,

c o m p a r e d w i t h a b o u t 5.2

p e r annum f r o m f o s s i l f u e l b u r n i n g .

b i l l i o n tonnes

The f i r s t s t e p w o u l d t h e r e f o r e

be t o h a l t t h e n e t r e m o v a l o f t r e e s and o t h e r p l a n t c o v e r ,

and t h e n

t o r e v e r s e i t by t r e e p l s n t i n g c o m b i n e d w i t h g o o d f o r e s t management.

T h i s o b v i o u s l y m u s t be c o m b i n e d on a g l o b a l s c a l e w i t h i n c r e -

ased energy use e f f i c i e n c y and o t h e r measures t o decrease t h e emis s i o n s o f greenhouse gases.

ELECTRICITY GENERATION V I A

7

IMMOBILIZED P H O T O S Y S T E M S

Whole c e l l s o f c y a n o b a c t e r i a ,

isolated thylakoids,

individual

p h o t o s y s t e m s a n d e v e n c h l o r o p h y l l d e p o s i t e d on s e m i c o n d u c t o r e l e c t r o d e s c a n be u s e d f o r t h e g e n e r a t i o n o f p h o t o c u r r e n t s a n d p h o t o voltages.

M a s t i q o c l a d u s l a m i n o s u s i m m o b i l i z e d i n Ca a l g i n a t e a n d

d e p o s i t e d on t h e s u r f a c e o f an o p t i c a l l y t r a n s p a r e n t Sn02 e l e c t r o d e when

illuminated

photocurrent, 132

I.

i n an e l e c t r o c h e m i c a l c e l l n o t o n l y g e n e r a t e d a

b u t a l s o c o n t i n u e d t o grow on t h e e l e c t r o d e s u r f a c e

The " l i v i n g a l g a l e l e c t r o d e "

generating a steady photocurrent.

f u n c t i o n e d f o r more t h a n 2 0 d a y s ,

I n a much m o r e s i m p l i f i e d s y s t e m ,

P h o t o s y s t e m I 1 membrane p a r t i c l e s i m m o b i l i z e d o n a d y e - d e r i v a t i s e d T i 0 2 e l e c t r o d e g e n e r a t e d p h o t o c u r r e n t s o f t h e o r d e r o f 35p A cm-' o f e l e c t r o d e s u r f a c e and 10 m A mg-l photon-to-current

chlorophyll.

Maximum i n c i d e n t

c o n v e r s i o n e f f i c i e n c y was 1 2 % [331. B i o s o l a r

b a t t e r i e s w i t h p h o t o s y n t h e t i c p i g m e n t s sandwiched between e l e c t r o d e s u r f a c e s may s o o n b e a v a i l a b l e .

8

CONCLUSION We h a v e o u t l i n e d some p r o c e s s e s w h e r e b y i m m o b i l i z e d p h o t o s y n t h e -

t i c systems c a n be a p p l i e d f o r

t h e p r o d u c t i o n o f f u e l s and c h e m i c a l s

and f o r t h e a l l e v i a t i o n o f e n v i r o n m e n t a l p o l l u t i o n .

Most o f t h e s e

t e c h n i q u e s a r e s t i l l i n a l a b o r a t o r y s t a g e and need a c a l i n g up before extensive f i e l d t r i a l s . conversion technology wide f l u c t u a t i o n s

The p r o b l e m i n h e r e n t i n s o l a r e n e r g y

is t h a t t h e i n c i d e n t e n e r g y i s d i f f u s e w i t h

i n i n s o l a t i o n o v e r b o t h d a i l y and annual c y c l e s ,

t h i s d i s a d v a n t a g e i n s o l a r e n e r g y u t i l i s a t i o n whereby l a r g e c o l l e c t i n g s u r f a c e s a r e r e q u i r e d s h o u l d be w e i g h e d a g a i n s t i t s u n i q u e advantage t h a t s o l a r energy i s b o t h c o s t and p o l l u t i o n f r e e .

We d o

n o t v e n t u r e a t p r e s e n t t o compare t h e e c o n o m i c s o f s o l a r ' e n e r g y

-

274

-

technology a g a i n s t those u s i n g c o n v e n t i a l energy d e r i v e d from fos s i l f u e l s and n u c l e a r f i s s i o n . terest,

a l l over the world,

t h e p r o d u c t i o n o f H2 from water. cesses,

we h o p e ,

However,

t h e r e i s now a r e n e w e d i n -

i n s o l a r conversion,

particularly in

I n t e r e s t and s u p p o r t

for other pro-

would f o l l o w .

REFERENCES

1

2 3

4

5

6 7

8 9

10

11 12

13 14 15 16

Summaries o f I n v i t e d P r e s e n t a t i o n s , ' S o l a r P h o t o c o n v e r s i o n P r o c e s s e s For R e c y c l i n g C O from t h e atmosphere', Colorado S p r i n g s , U S A , M a r c h 1990. g E R I , G o l d e n , C o l o r a d o . A.W. R u t h e r f o r d , ( 1 9 8 9 ) ' P h o t o s y s t e m 11, t h e W a t e r - s p l i t t i n q enzyme', T I E S , 1 4 , 227-232. D. G u s t a n d T.A,Moore (1989) ' M i m i c k i n q P h o t o s y n t h e s i s ' , S c i e n c e , 244, 3 5 - 4 1 , S e e a l s o : New S c i e n t i s t , L o n d o n , 26 May 1990, p. 34. J.W. V e r h o e v e n , J. K r o o n , M.N. Paddon-Row and A.M. Oliver (1989) ' E l e c t r o n and energy t r a n s f e r a c r o s s r i g i d , s a t u r a t e d h y d r o c a r b o n b r i d g e s ' : m o l e c u l a r e n g i n e e r i n g o f t h e b r i d g e conH a l l a n d G. G r a s s i ( e d s . ) P h o t o c o n v e r s i o n f i g u r a t i o n ' i n D.O. Processes f o r Energy and Chemicals, E l s e v i e r A p p l i e d Science, L o n d o n pp. 100-108. L. H a u s s l i n g , C. M e r t e s d o r f and H. R i n g s d o r f (1989) 'Photochem i c a l l y i n d u c e d s t r u c t u r a l changes i n m u l t i l a y e r s and columnar pp. 5 3 - 6 7 . systems' I.W i l l n e r , D. M a n d l e r a n d R . Maidaw ( 1 9 8 7 ) ' B i o - m o d e l s a n d a r t i f i c i a l m o d e l s f o r p h o t o s y n t h e s i s ' . N e w J. o f C h e m i s t r y , ll, 109-122. D.O. H a l l and K.K. Rao ( 1 9 8 8 ) ' I m m o b i l i z e d p h o t o s y n t h e t i c memb r a n e s and c e l l s f o r t h e p r o d u c t i o n o f f u e l s and c h e m i c a l s ' , Schmur, J . M . a n d Chapman, D. ( e d s . ) B i o t e c h n o i n G r a b e r , B.P., l o g i c a l A p p l i c a t i o n s o f L i p i d M i c r o - s t r u c t u r e s , Plenum Press, New Y o r k pp. 2 2 5 - 2 4 9 . K . Mosbach, ed. ( 1 9 8 7 ) M e t h o d s i n E n z y m o l o g y . V o l . Immobil i z e d Enzymes and C e l l s . A c a d e m i c P r e s s , New Y o r k . M. B r o u e r s , D - J - S h i a n d D.O. H a l l , ( 1 9 8 8 ) " I m m o b i l i z a t i o n methods f o r c y a n o b a c t e r i a i n s o l i d m a t r i c e s " , Methods i n Enzymolog y , 167, 6 2 9 - 6 3 6 . P.E. G i s b y and D.O. H a l l ( 1 9 8 0 ) ' B i o p h o t o l y t i c H 2 p r o d u c t i o n u s i n g a l g i n a t e - i m m o b i l i z e d c h l o r o p l a s t s , enzymes a n d s y n t h e t i c c a t a l y s t s ' , N a t u r e 287, 251-253. A.W. B u n c h ( 1 9 8 8 ) The u s e s a n d f u t u r e p o t e n t i a l o f m i c r o b i a l h o l l o w - f i b r e b i o r e a c t o r s , J . M i c r o b . M e t h o d s , E, 1 0 3 - 1 1 9 . M. C h e r y a n and M.A. M e h a i a ( 1 9 8 6 ) 'Membrane b i o r e a c t o r s ' i n W.C. McGregor ( e d . ) Membrane S e p a r a t i o n s i n B i o t e c h n o l o g y , Marc e l D e k k e r , New Y o r k pp. 2 5 2 - 3 0 1 . D.O. H a l l and K . K . Rao ( 1 9 8 9 ) ' I m m o b i l i z e d p h o t o s y n t h e t i c membranes and c e l l s f o r t h e p r o d u c t i o n o f f u e l s and c h e m i c a l s ' C h i m i c a o g g i , 1, ( 3 ) 41-47. M.A. B o r o w i t z k a a n d L . J . B o r o w i t z k a e d s . ( 1 9 8 8 ) M i c r o a l g a l B 0 t e c h n o l o g y . Cambridge U n i v e r s i t y Press. R.C. Cresswell, 1.A.V Rees a n d N. Shah e d s . ( 1 9 8 9 ) A l g a l a n d C y a n o b a c t e r i a l B i o t e c h n o l o g y . Longman S c i e n t i f i c B T e c h n i c a l London. M.D. T r e v a n a n d A.L. Mak ( 1 9 8 8 ) ' I m m o b i l i z e d a l g a e a n d t h e i r P O t e n t i a l f o r u s e a s b i o c a t a l y s t s ' . TIBTECH, 6 , 6 8 - 7 3

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P.M. V i g n a i s , A . C o l b e a u , J.C. W i l s o n a n d Y . J o u a n n e a u ( 1 9 8 5 ) ’Hydrogenase, n i t r o g e n a s e and hydrogen m e t a b o l i s m i n t h e p h o t o s y n t h e t i c b a c t e r i a ’ , A d v a n c e s M i c r o b . P h y s i o l . 26, 156-232. T . M a t s u n a g a a n d A. M i t s u i ( 1 9 8 2 ) ’ S e a w a t e r b a s e d h y d r o g e n p r o d u c t i o n by i m m o b i l i z e d marine p h o t o s y n t h e t i c b a c t e r i a ’ B i o t e c h . B i o e n g . Symp. l2, 4 4 1 - 4 5 0 . D . J . . S h i , M. B r o u e r s , D.O. H a l l a n d R.J. R o b i n s ( 1 9 8 7 ) ’The e f f e c t s o f i m m o b i l i z a t i o n on t h e b i o c h e m i c a l , p h y s i o l o g i c a l a n d m o r p h o l o g i c a l f e a t u r e s o f Anabaena a z o l l a e ’ , P l a n t a , 293-308. E . C . Greenbaum ( 1 9 8 5 ) ’ P l a t i n i z e d c h l o r o p l a s t s : a n o v e l p h o t o 1373-1375. c a t a l y t i c m a t e r i a l ’ , Science C.H. F o y e r a n d D.O. H a l l ( 1 9 8 0 ) ’ O x y g e n m e t a b o l i s m i n t h e a c t i v e c h l o r o p l a s t ’ TIBS 5 , 1 8 8 - 1 9 1 . M.A. de l a Rosa, K . K . Rao a n d D.O. H a l l ( 1 9 8 6 ) ’ H y d r o g e n p e r o x i d e p h o t o p r o d u c t i o n by f r e e and i m m o b i l i z e d s p i n a c h t h y l a k o i d s , P h o t o b i o c h e m . P h o t o b i o p h y s . ll, 1 7 3 - 1 8 7 . F.F. d e l a Rosa a n d I. M o r a l e s ( 1 9 8 9 ) ’ P h o t o b i o l o g i c a l p r o d u c t i o n 373-377. o f h y d r o g e n p e r o x i d e : A m o d e l s y s t e m ’ . S o l a r E n e r g y 9, M-F Coquempot a n d 0 . Thomas ( 1 9 8 4 ) ’ I m m o b i l i z e d c h l o r o p l a s t memb r a n e s : K i n e t i c s s p e c t s o f t h e i r c o n t i n u o u s use i n b a t c h and c h e m o s t a t ’ , Enzyme M i c r o b . T e c h n o l . 6 , 3 2 1 - 3 2 4 . A . M u a l l e m , D . B r u c e a n d D.O. H a l l ( i 9 8 3 ) ’ P h o t o p r o d u c t i o n o f H 2 a n d NADPH b y p o l y u r e t h a n e - i m m o b i l i z e d c y a n o b a c t e r i a ’ , B i o t e c h . L e t $ 2 , 365-368. P. C u e n d e t a n d M. G r a e t z e l ( 1 9 8 4 ) ’ P h o t o r e d u c t i o n o f NAD t o NADH i n semiconductor d i s p e r s i o n s ’ , Photochem. P h o t o b i o l . 9 , 6 0 9 - 6 1 2. D.J. Sh i and 0.0. H a l l (1988) ’ A z o l l a and i m m o b i l i z e d cysnobact e r i a : from t r a d i t i o n a l a g r i c u l t u r e t o biotechnology, Plants 5-12. Today, J. F r e n z , C . L a r g e a u and E . C a s a d e v a l l ( 1 9 8 9 ) ’ H y d r o c a r b o n r e covery by e x t r a c t i o n w i t h a b i o c o m p a t i b l e s o l v e n t f r o m f r e e and i m m o b i l i z e d c u l t u r e s o f B o t r y o c o c c u s b r a u n i i , Enzyme M i c r o b . T e c h n o l . 11, 717-724. M . O r e n - S G r n i r , M. A v r o n , a n d H . D e g a n i ( 1 9 8 8 ) FEBS L e t t . , 2 , 124-128, J. de l a Noue a n d D. P r o u l x ( 1 9 8 8 ) ’ B i o l o g i c a l t e r t i a r y t r e a t ment o f u r b a n waste w a t e r s w i t h c h i t o s a n - i m m o b i l i z e d Phormidium’ A p p l . M i c r o b i o l . B i o t e c h n o l . 29, 292-297 D.O. H a l l ( 1 9 8 9 ) ’ C a r b o n f l o w s i n t h e b i o s p h e r e : p r e s e n t a n d f u t u r e ’ , J. G e o l . S O C . London. 146, 1 7 5 - 1 8 1 . H. O c h i a i , A. Tanska and S. F u k u i ( 1 9 8 3 ) ’ I m m o b i l i z e d O r g a n e l l e s ’ , A p p l . B i o c h e m . B i o e n g . 4, 1 5 3 - 1 8 7 . K . K . Rao, D . O . H a l l , N. V l a c h o p o u l o s , M . G r a t z e l , M . C . W . Evans a n d M. S e i b e r t ( 1 9 9 0 ) ’ P h o t o e l e c t r o c h e m i c a l r e s p o n s e s o f p h o t o s y s t e m I 1 p a r t i c l e s i m m o b i l i z e d on d y e - d e r i v a t i s e d T i 0 2 f i l m s ’ J. Photochem. P h o t o b i o l . 8 . B i o l . 2 , 3 7 9 - 3 8 9 .

172,

20 21 22 23 24

25

26 27

=,

1,

28

29 30

,

31 32 33

This Page Intentionally Left TBlank

ANAEROBIC T R E A T M E N T OF E X C R E M E N T S FROM LARGE-SCALE ANIMAL FARMS

J. K E R E K R E T Y , B. P E T R O V I C O V A ,

K.

BODA a n d 0.

ADAMEC

S l o v a k Academy o f S c i e n c e , I n s t i t u t e o f Animal B i o c h e m i s t r y and G e n e t i c s , Ivanka p r i Dunaji, Czechoslovakian Federative Republic

ABSTHACT The m e t h a n o g e n i c d i g e s t i o n o f p o u l t r y m a n u r e i s u s e d a s a moresulting i n large-scale

d e l [if w a s t e management

f e r m e n t a t i o n p r o c e s s t a k e s away a b o u t 40

animal

L o f the

COO,

farms.

The

removes t h e

a g g r e s s i v e o d o r , r e d u c e s b o t h t h e number o f p a t h o g e n s a n d w i l d o a t g r a s s seeds,

produces gas f u e l ( a b o u t 1 m3 o f b i o g a s f r o m 1 k g o f

degraded o r g a n i c m a t t e r ) t o smmonia.

and changes t h e o r g a n i c a l l y bound n i t r o g e n

The m a t e r i a l a n d e n e r g y b a l a n c e o f a f a r m w i t h 100 0 0 0

l a y e r s i s counted.

The b a l a n c e d a n a e r o b i c t r e a t m e n t a n d d r y i n g o f

e x c r e m e n t s i s s u g g e s t e d a s a mode w h i c h may s o l v e t h e e n v i r o n m e n t a l

problems o f l a r g e - s c a l e

animal farms l u c r a t i v e l y .

INTRODUCTION The p r e v i o u s e c o n o m i c a l l y

o r i e n t e d development i n t h e meat,

m i l k a n d e g g p r o d u c t i o n r e s u l t e d i n a g r e a t number o f i n t e n s i v e l i t t e r l e s s farms. p i g production,

T h i s was t r u e e s p e c i a l l y i n t h e c a s e o f p o u l t r y o r where t h e p r o d u c t i o n u n i t s a c h i e v e d t h e h i g h e s t c o n -

c e n t r a t i o n o f animals.

The number o f f a r m s i n C z e c h o s l o v a k i a c o n s i -

1. F o r e x a m p l e , t h e r e s r e about 5 8 egg p r o d u c i n g farms h o u s i n g o v e r 100 000 hens i n

d e r e d a s h i g h c a p a c i t y u n i t s a r e shown i n Tab.

cages,

t h a t i s 45

L o f t h e t o t a l number o f l s y e r s .

The c o n c e n t r a t i o n o f a l a r g e number o f a n i m a l s i n a r e l a t i v e l y s m a l l space g e n e r a t e s s e r i o u s e c o l o g i c a l problems, immense v o l u m e o f e x c r e m e n t s .

because o f t h e

M a n i p u l a t i o n and s t o r a g e problems o f

t h i s c o n t i n u a l l y g r o w i n g mass may a c h i e v e t h e c r i t i c a l p o i n t , t i n g t h e s i z e o f l i v e s t o c k number. the ground water or r i v e r s ,

limi-

The p o t e n t i a l c o n t a m i n a t i o n o f

and t h e e m i s s i o n o f a g g r e s s i v e odor a r e

the primary environmental

278

r i s k s connected w i t h t h e occurrence o f

t h i s hazardous m a t e r i a l . TABLE 1 Large-scale

A n i m a l Farms,

U s i n g L i t t e r l e s s Technology,

Category o f Farm Animals more t h a n

I1

Number of Farms

Number of Animals

I1

Average of Animals i n one Farm

(1)

x

of Total Number of Animals

450

164

1 0 2 500

6 25

450

93

98 9 5 2

1 064

3.1

3 000

166

1 053 7 6 8

6 348

17 .O

100 0 0 0

58

1 0 352 1 3 0

1 7 8 485

44.7

I

Swine L a y i n g Hens

II

i n CSFR

According t o the data i n Table 2., one o f t h e m o s t p o l l u t i n g a n i m a l w a s t e s .

5.5

p o u l t r y manure seems t o b e The t h e o r e t i c a l BOD v a l u e

o f e x c r e m e n t s p r o d u c e d b y 1 0 0 0 k g o f l a y i n g h e n s i s a b o u t 2.5 higher than i n other animal species.

times

And r e p r e s e n t s a p o l l u t i o n

e q u i v a l e n t o f about 100 i n h a b i t a n t s . The a n n u a l BOO v a l u e o f t h e w h o l e amount o f l i v e s t o c k w a s t e s produced i n l a r g e - s c a l e

l i t t e r l e s s a n i m a l farms r e p r e s e n t s 164 m i l l i -

on k g O 2 i n Czechoslovakia. o f 8.3

T h i s e q u a l s t o t h e e n v i r o n m e n t a l demand

million o f inhabitants,

t h i s country’s

r e p r e s e n t i n g m o r e t h a n one h a l f o f

population.

TABLE 2 E x c r e m e n t P r o d u c t i o n o f 1 0 0 0 k g L i v e W e i g h t o f Farm A n i m a l s a n d t h e i r Pollution Equivalents

P r o d u c t i o n o f Excr e m e n t s k g TS/d

Dairy Cattle

Beef

11.08 1.90

Pigs

Laying Hens

10.08

7.10

24.26

2.12

2.44

5.82

BOD kg 02/d Number o f E q u i v a l e n t Inhabitants

THE PRINCIPLE

35.2

39.2

45.0

107.8

OF M E T H A N O G E N E S I S

The s t u d y o f m e t h a n o g e n i c a l l y

o r i e n t e d a n a e r o b i c t r e a t m e n t and

i t s a p p l i c a t i o n t o a n i m a l wastes were m o t i v a t e d b y t h e l a t e s t w o r l d

-

279

-

energy c r i s i s i n t h e m i d d l e o f t h e s e v e n t i e s .

T h i s was t h e r e a s o n

why t h e p r e v i o u s t r e n d o f d r y i n g t h e p o u l t r y manure was changed. The r e c e n t e m p h a s i s on e n v i r o n m e n t a l p r o t e c t i o n a n d p o l l u t i o n c o n t r o l has i n c r e a s e d t h e e f f o r t f o r f u r t h e r development o f methanogen i c fermentation.

The r e s e a r c h r e s u l t s a n d t h e t e c h n o l o g i c a l e x p e -

r i e n c e s p r o m i s e t h a t a n a e r o b i c d i g e s t i o n may b e one o f t h e p o s s i b l e ways o f a n e f f e c t i v e f a r m w a s t e management. The p r i n c i p l e o f m e t h a n o g e n e s i s i s b a s e d o n t h e t r e a t m e n t o f e x c r e m e n t s b y a m i x e d c u l t u r e o f m i c r o o r g a n i s m s i n sn o x y g e n f r e e atmosphere under m e s o p h i l i c t e m p e r a t u r e c o n d i t i o n s a t about 40

i n a s t i r r e d digester.

OC

The r e t e n t i o n t i m e f o r s p e c i f i c a n i m a l w s s t e

s u b s t r s t e r a n g e s b e t w e e n 15 t o 4 0 d a y s .

The p r o d u c t s o f t h e m i c r o -

b i a l t r a n s f o r m a t i o n o f o r g a n i c m a t t e r a r e b i o g a s a n d f e r m e n t e d ma-

1).

t e r i a l (Fig.

I

I

AGRONOMY L

w4

I-IX

%

&'

I

----- -

- -I I

I

ANIMAL

I

FARM

I

u) W

I

t-

cn

9

Z W

Fig.

1

A

1

u) W Iu)

nJ

I I

OUTPUTS MEAT, MILK, EGGS

INPUTS FORAGE ENERGY

I I

5, M E T H A N O G E N E S I S

I I

- - _ -METHANE -_-___

I

-1

1. The p l a c e o f m e t h a n o g e n e s i s i n t h e a g r i c u l t u r a l e c o s y s t e m .

Biogas

-

t h e main p r o d u c t o f anaerobic f e r m e n t a t i o n ,

o f methane and c a r b o n d i o x i d e .

consists

The h i g h methane c o n c e n t r a t i o n i n

b i o g a s p e r m i t s i t s d i r e c t use as a f u e l .

I n s p e c i a l cases i f needed

t h e r e i s a l s o t h e p o s s i b i l i t y t o p u r i f y i t t o t h e n a t u r a l gas q u a l i ty. The s e c o n d p r o d u c t o f t h e p r o c e s s i s f e r m e n t e d s l u d g e .

It i s a

w a t e r s u s p e n s i o n o f t h e s o l i d manure p s r t i c l e s and t h e s o l u b l e organ i c and i n o r g a n i c substances.

Under a n a e r o b i c c o n d i t i o n s t h e g r e a t e r

p a r t o f t h e o r g a n i c m a t e r i a l i s t r a n s f o r m e d t o an i n o r g a n i c o r more

simple orgsnic matter. nitrogen,

280

-

The e s s e n t i a l f e r t i l i z e r n u t r i e n t s ,

such as

p h o s p h o r u s a s w e l l ss h u m u s o g e n i c s u b s t a n c e s a r e b e i n g s a -

v e d a n d may b e r e c y c l e d .

M E T H A N O G E N I C FERMENTATION OF POULTRY MANURE The m e t h a n o g e n i c t r e a t m e n t o f p o u l t r y m a n u r e , ged l a y e r s ,

e s p e c i a l l y o f cs-

i s p r e s e n t e d s s a m o d e l f o r w a s t e management.

The r e s e s r c h was a i m e d s t s o l v i n g t h e f o l l o w i n g p r o b l e m s :

1. The a p p l i c s t i o n o f m e t h a n o g e n e s i s t o a s u b s t r a t e w i t h a high TS content.

2.

The e f f e c t i v e u s e o f p r o d u c e d e n e r g y .

3 . The management o f t h e f e r m e n t e d s l u d g e . Ad 1. R e c e n t l y t h e a p p l i c s t i o n o f a n a e r o b i c d i g e s t i o n t o p o u l t r y m a n u r e p r o c e s s i n g wss c o n n e c t e d w i t h some s e r i o u s p r o b l e m s

. The

main

X

o b s t s c l e wss t h e n e c e s s i t y t o d i l u t e e x c r e m e n t s t o s l e v e l o f 4-6

i n o r d e r t o a v o i d p o t e n t i a l i n h i b i t i o n b y h i g h ammonia c o n c e n t r a t i o n s .

However,

w a t e r a d d i t i o n i n c r e a s e d t h e s l u d g e volume end enhanced e-

n e r g y demand f o r h e a t i n g i t t o t h e f e r m e n t a t i o n t e m p e r a t u r e . The r e s u l t s o f t h e e x p e r i m e n t s o f o u r l s b o r s t o r y show t h a t ,

the

m i x e d a n a e r o b i c c o n s o r t i u m may b e a d a p t e d t o a r e l a t i v e l y h i g h smmon i a nitrogen concentration.

As i t i s s e e n i n F i g .

2 t h e methane p r o -

d u c t i o n i n s t i m e p e r i o d o f s b o u t 80 weeks s l i g h t l y i n c r e a s e s . i n c r e m e n t i s s b o u t 0.0216

MJ/kg/week.

The f e r m e n t a t i o n p r o c e s s was

n o t i n h i b i t e d a t l e v e l s r a n g i n g b e t w e e n 4.1-5.9

7.5

g N/1

The

g N/1,

extremely

(2).

-

cn

ff E K R GY PRCI)UCTIDN

-TREK,

0'

10

20

3b

io

50

$0

I

70

80

WEEKS

F i g . 2. The d a i l y e n e r g y p r o d u c t i o n f r o m 1 k g o f t o t a l s o l i d s i n p u t o f p o u l t r y manure.

-

281

-

The l i q u i d f o r m o f t h e s u b s t r a t e i s e s s e n t i a l n o t b e c a u s e o f t h e proper fermentation process, b y pumps.

a b o u t 1 5 76, liquid.

b u t because o f i t s t r a n s p o r t a t i o n

F o r t h i s r e a s o n t h e s o l i d s c o n t e n t o f t h e i n p u t may be w h i c h i s t h e c o n c e n t r a t i o n a t w h i c h t h e m a n u r e becomes

T h i s T S c o n t e n t does n o t i n t e r f e r e w i t h t h e methanogenic d i -

gestion. The c a l c u l a t i o n s b a s e d o n t h e l a b o r a t o r y s c a l e m e t h a n e p r o d u c t i o n l e a d t o t h e c o n c l u s i o n t h a t s t a r t i n g from 4

X o f TS c o n t e n t ,

t h e l a r g e s c a l e t e c h n o l o g y may a l s o b e p e r f o r m e d a s an e n e r g e t i c a l l y e f f i c i e n t o p e r a t i o n (see Fig.

3 ) . The m e t h a n o g e n e s i s o f p o u l t r y ma-

n u r e w i t h a 12 % T S c o n c e n t r a t i o n u n d e r m e s o p h i l i c c o n d i t i o n s may p r o d u c e up t o 10 M J o f n e t e n e r g y p e r 1 k g o f t h e t o t a l s o l i d s i n put.

-

90

TOTAL SOLIDS CONCENTRATION ['//,I F i g . 3 . The t h e o r e t i c a l n e t e n e r g y y i e l d and t h e t o t a l s o l i d s c o n c e n t r a t i o n o f p o u l t r y manure.

Ad 2 .

I n t h e s p e c i f i c c a s e o f p o u l t r y manure t h e p r o d u c e d e n e r g y may

b e e f f e c t i v e l y u t i l i z e d by u s i n g a model o f b a l a n c e d a n a e r o b i c t r e atment and d r y i n g .

I n the fermentation process only t h a t p a r t o f

t h e e x c r e m e n t i s u s e d w h i c h p r o d u c e s a s u f f i c i e n t amount o f e n e r g y

f o r d r y i n g t h e r e s t o f i t . The r a t i o b e t w e e n f e r m e n t e d manure a n d t h e m a t e r i a l w h i c h i s d r i e d i s a b o u t 3:2 o f b o t h o p t i o n s a r e shown i n T a b l e 3 .

(Fig.

4).

The m a t e r i a l b a l a n c e

The f e r m e n t e d s l u d g e a s w e l l

a s t h e d r i e d e x c r e m e n t s may b e u s e d a s f e r t i l i z e r s i n a g r i c u l t u r e . Such a f l o w s h e e t s i g n i f i c a n t l y material, rial.

reduces t h e t o t a l volume o f waste

and s i m p l i f i e s t h e m a n i p u l a t i o n o f t h e r e s t o f t h e mate-

-

Fig.

4.

282

-

B a l a n c e d methanogenesia and d r y i n g o f p o u l t r y manure.

TABLE 3 The M a t e r i a l B a l a n c e o f B o t h V a r i a n t s o f t h e M e t h a n o g e n e s i s A p p l i c a t i o n i n a F a r m w i t h 100 000 L a y e r Hens Methanogenesis and D r y i n g

Methanogenesis P h y s i o l o g i c a l Manure 15 400

T e c h n o l o g i c a l Water

Ik g / d 1 1kg/d 1

F e r m e n t e r I n p u t T S 12 %

Ikg/dl

32 0 8 0

Fermented Sludge

[ kg/d

P r o d u c t i o n T S 25 %

B i o g a s Volume 62,5 D r i e d Excrementa

16 680

1

30 1 8 0

X CH4 [ m 3 / d l [ kg/d 1

1 338

-

1 5 400 I

,

10 180 1 9 580 18 410

-

1 700

This organization o f t h e technology i s supported a l s o by the e c o l o g i c a l aspect.

As i t i s s e e n i n F i g .

5 the application o f the

b a l a n c e d d i g e s t i o n a n d d r y i n g r e m o v e s a b o u t 6 5 X o f t h e w a s t e COD, w h i l e t h e s i n g l e methanogenic f e r m e n t a t i o n e l i m i n a t e s o n l y 40 % o f

-

-

283

i t . The v a l u e o f COD o f p h y s i o l o g i c a l m a n u r e i s a b o u t 3 3 7 . 6 1 2 7 1 . 9 1 and a f t e r a n a e r o b i c t r e a t m e n t i t i s a b o u t 98.67 2 45.15

METHANOGENESIS AND DRYING

METHANOGENESIS

5000

&OOO 3000

2000 1000 0 EXCREMENTS F i g . 5. drying.

FERMENTED SLU)GE

EXCREMENTS 'ERMENTED SLU~GE

The r e m o v e d COD u s i n g m e t h a n o g e n e s i s a n d m e t h a n a g e g i s a n d

T h i s s u b s t a n t i a l l y r e d u c e s t h e p r o b l e m o f waste m a n i p u l a t i o n

a s well a s t h e p o l l u t i o n r i s k . M o r e o v e r , i t o f f e r s a c o m p l e m e n t a r y energy source f o r d i r e c t u t i l i z a t i o n . Ad 3 .

The f e r t i l i z i n g q u a l i t y o f t h e d i g e s t e d s u d g e c o n t r i b u t e s t o

economical and environmental p r o t e c t i o n e f f e c t .

The a m m o n i a c a l f o r m

o f n i t r o g e n may a u p p o r t t h e n u t r i e n t r e c y c l i n g , o w i n g t o i t s b e t t e r u t i l i z a t i o n by p l a n t s .

The f e r m e n t a t i o n p r o c e s s d o e s n o t s i g n i f i -

c a n t l y d e c r e a s e t h e n i t r o g e n c o n t e n t o f manure

ig.

6.

T H E ADVANTAGES OF A N A E R O B I C T R E A T M E N T The b e n e f i t s o f t h e p r e s e n t e d mode o f a n i m a l w a s t e m a n a g e m e n t may b e i l l u s t r a t e d i n c o m p a r i s o n t o t h e g e n e r a l l y known a e r o b i c p r o -

cess ( T a b . 4 ) . (e.i.

The l a t t e r e l i m i n a t e s p r a c t i c a l l y t h e t o t a l p o l l u t i o n

COD o r BOD) o f t h e l i q u i d m a n u r e a n d e n a b l e s i t s d i r e c t a p p l i -

c a t i o n t o t h e water r e c i p i e n t a .

I t s u b s t a n t i a l l y reduces t h e troub-

l e s o f t h e waste m a n i p u l a t i o n m i n i m i z i n g i t s volume. On t h e o t h e r hand, t h e a e r o b i c t r e a t m e n t r e q u i r e s i n a d d i t i o n t o i n v e s t m e n t s a l s o enormous p r o c e s s e n e r g y i n p u t s .

The a p p r o x i m a t e a s s e s s m e n t o f t h e

-

284

-

d a i l y e n e r g y c o n s u m p t i o n o f t h e a e r o b i c t r e a t m e n t o f p o u l t r y msnu-

r e o n a 100 0 0 0 l a y e r f a r m r e p r e s e n t s 1 8 . 7 1

9

GJ.

8

E 22

6 z

2D

18 1:6 14 1:2

l,o

0.8

0

10

30

40

50

60

70

80

30

40

50

60

70

80

32.5 - -

2 -. 1S -I--

0.51 0

I

10

20

WEEKS F i g 6. The n i t r o g e n c o n t e n t o f p o u l t r y m a n u r e b e f o r e a n d s f t e r methanogenesis.

Treatment

Var.

IGJ 1

Process Energy

Anaerobic 1 Var.

6.11

2

Aerobic

3.73

18.71

Energy Requirement f o r F e r t i l e Nutrient Substitution

[GJ1

-

-

12.60

P r o d u c t i o n o f Energy

[GJI

35.74

21.80

-

Sum E n e r g y

lGJl

*

+ 29.63

+

18.07*

-

31.31

The e n e r g y i s u s e d f o r e x c r e m e n t d r y i n g The a e r o b i c e l i m i n a t i o n o f a n n u a l p o l l u t i o n c a u s e d b y p o u l t r y

farms u s i n g caged l a y e r t e c h n o l o g y i n C z e c h o s l o v a k i a r e p r e s e n t s a t o t a l p r o c e s s e n e r g y i n p u t o f a b o u t 1 1 8 0 TJ. Moreover,

t h e a e r o b i c method o r t h e s t o r a g e u n d e r n o r m a l con-

d i t i o n s d e s t r o y s t h e e s s e n t i a l f e r t i l i z e r n u t r i e n t s and e l i m i n a t e s them from n a t u r a l r e c y c l i n g . industrial fertilizers is,

The n e c e s s i t y o f t h e i r s u b s t i t u t i o n b y

o w i n g t o a h i g h e n e r g y demand f o r t h e i r

production,

285

-

a g a i n c o n n e c t e d w i t h an a d d i t i o n a l e n e r g y c o n s u m p t i o n .

The s u b s t i t u t i o n o f a n a e r o b i c a l l y

saved n i t r o g e n r e q u i r e a o n l y i n

t h e case o f a e r o b i c t r e a t m e n t o f p o u l t r y manure an energy i n p u t o f

000 l a y e r s o f 2376 T J / y e a r

12.6[1 GJ/100

i n Czechoslovakia.

T a b l e 5 shows t h e h y p o t h e t i c a l e n e r g y s a v i n g i n C z e c h o s l o v a k i a o n l y i n t h e case o f

a t methanogenic f e r m e n t s t i o n o f a n i m a l waste, l a y i n g hens.

i t may s a v e a b o u t 1 7 %

According t o t h i s c a l c u l a t i o n s

o f a n n u a l n a t u r a l g a s p r o d u c t i o n o f CSFR

(3).

TABLE 5 C a l c u l a t i o n s o f Annual Energy Saving a t Anserobic Treatment o f P o u l t r y Manure i n t h e CSFR

I

810 5 5 0

GJ

Saved e n e r g y i n f e r t i l e nutrients

2 3 7 6 000

GJ

Saved Eneinqy by A n a e r o b i c Treatment S u b s t i t u t i o n

1 1 8 0 000

GJ

The h y p o t h e t i c e n e r g y s a v i n g

4 266 5 5 0

GJ

Net Energy P r o d u c t i o n

The e n e r g y p r o d u c e d b y t h e a n a e r o b i c p r o c e s s comes f r o m r e n e w able resources,

which i s an a l t e r n a t i v e o f l i m i t e d q u a n t i t i e s o f

f o s s i l f u e l s or n u c l e a r energy c a r r i e r s n o t o n l y from t h e e n e r g e t i c p o i n t o f view

b u t a l s o from t h e e n v i r o n m e n t a l p o i n t o f view.

The

energy produced by t h i s method does n o t cause such a s e v e r e p o l l u t i o n o f t h e atmosphere a s t h e f o s s i l s o r t h e n u c l e a r power p l a n t s do.

I n a d d i t i o n t o the energetic aspect, ce o f t h e anaerobic treatment: fermented sludge.

there i s another preferen-

the better f e r t i l e quality o f the

The b e n e f i t o f t h e t r a n s f o r m a t i o n o f o r g a n i c a l l y

b o u n d n i t r o g e n t o ammonia h a s b e e n m e n t i o n e d a b o v e . me a u t h o r s ,

g e n s a n d t h e number o f w i l d o a t - g r a s s s e e d s p o s i t i v e h y g i e n i c consequences i . e . requirement.

A c c o r d i n g t o so-

t h e a n a e r o b i c t r e a t m e n t a l s o r e d u c e s t h e number o f p a t h o -

(4). T h i s l e s d s t o some

the reduction o f the herbicide

The f e r m e n t a t i o n p r o c e s s a l s o e l i m i n a t e s t h e a g g r e s a i -

ve o d o r o f e x c r e m e n t s a n d t h e i r t o x i c compounds o c c u r r i n g d u r i n g spontaneous

degradation o f organic matter.

I n some d e v e l o p e d c o u n t r i e s (Denmark,

H o l l a n d and I t a l y ) t h e

s o i l a p p l i c a t i o n o f n o n f e r m e n t e d manure i s p r o h i b i t e d .

-

286

-

CONCLUSION A s s u m i n g t h e p r e s e n t k n o w l e d g e , o n e may s t a t e t h a t ,

t h e metha-

nogenic fermentation p r o c e s s can be a p p l i e d t o l a r g e s c a l e animal f a r m s , e s p e c i a l l y p o u l t r y f a r m s , a s an e c o l o g i c a l l y and economi-

cally e f f e c t i v e technology.

T h i s c a n b e a c h i e v e d b o t h by t h e o r g a -

n i z a t i o n o f t e c h n o l o g y a s w e l l a s by u s i n g h i g h l y e f f i c i e n t m i x e d c u l t u r e s o f r e s i s t a n t m i c r o o r g a n i s m s a d a p t e d t o a h i g h ammonia n i trogen concentration.

LITERATURE 1 2

3

4

K . Boda a n d M . C e r n f , M o f n o s t i p o u f i t i a m e t a n o g e n e z y p r e s p r a c o v a n i e exkrementov hospoderskych z v i e r a t v podmienkach 2 i v o E i S n e j v f r o b y S R , V e s t n f k CSAZ E . 1 0 9 , SZN P r a h a 1 9 8 7 . 2. P e c h a n , 0 . K n a p p o v a , B . P e t r o v i E o v A a n d 0 . Adamec, A n a e r o b i c d i g e s t i o n o f p o u l t r y m a n u r e a t h i g h ammonium n i t r o g e n c o n c e n t r a t i o n s . Biol. Wastes 2 0 , 1987, 117-131. S t a t i s t i c k 6 r o E e n k a ESSR 1 9 8 8 , s . 6 6 1 , ALFA/SNTL 1 9 8 8 Bigadan Danmark, 1989. 0 . Adsmec,

INTENSIFICATION AND ECOLOGICAL ASPECTS OF M E T H A N E FERMENTATION OF AGRICULTURAL W A S T E S

A.P.

M.J.

BEKER,

GRINBERGS,

J.E.

BLUMBERGS a n d M.K.

V.E.

DAVIDS,

L.J.

LABANE,

MARAUSKA

I n s t i t u t e o f Microbiology, 226067 R i g a , L a t v i a

L a t v i a n Academy o f S c i e n c e s ,

Methane f e r m e n t a t i o n o f a g r i c u l t u r a l wastes,

including l i q u i d

f a r m w a s t e s c a n b e recommended as a good m e t h o d i n e n s u r i n g e n v i ronment p r o t e c t i o n . tages:

Methane f e r m e n t a t i o n possesses s e v e r a l advan-

substrate detoxication,

r a e n d weed s e e d s , More o v e r ,

i n a c t i v a t i o n o f pathogenic m i c r o f l o -

dehelminthization

and s u b s t r a t e d e o d o r a t i o n .

methane f e r m e n t a t i o n a l l o w s a v a l u a b l e f e r t i l i z e r and a

s o u r c e o f humus f o r m i n g s u b s t a n c e s , nergy source,

as w e l l as b i o g a s

-

a l o c a l e-

t o be o b t a i n e d .

T h r e e m e t h o d s a r e m a i n l y u s e d i n o r d e r t o i n t e n s i f y any b i o t e c h n o l o g i c a l process:

s e l e c t i o n o f a more p r o d u c t i v e s t r a i n ,

i t s

physiologico-biochemical a c t i v a t i o n and v a r i o u s t e c h n o l o g i c a l meth o d s . The r e a l i z a t i o n o f i s a v e r y s i m p l e one.

spontaneous methane f e r m e n t a t i o n p r o c e s s

You n e e d a h e r m e t i z e d r e s e r v o i r w i t h o r g a n i c

s u b s t r a t e a n d a n a e r o b i c c o n d i t i o n s h e a t e d up t o 3 0 - 5 0

C.

I n a few

weeks methane f o r m a t i o n t a k e s p l a c e f o l l o w e d b y b i o g a s e x c r e t i o n . I n t h e p r o c e s s o f methane f e r m e n t a t i o n t h e r e p a r t i c i p a t e s a v e r y complex a s s o c i a t i o n o f microorganisms w h i c h r e a l i z e s a b i o d e g r a d a t i o n o f o r g a n i c a u b s t a n c e s i n a c e r t a i n sequence i n t h e f o l l o wing processes:

1

h y d r o l y s i s and a c i d f o r m a t i o n ;

2

a c e t o g e n e s i s and d e h y d r o g e n i z a t i o n ;

3

methanogeneais. I n t e r r e l a t i o n s o f microorganisms r e a l i z i n g these conversions

i n a s u b s t r a t e s r e v e r y complex.

For instance,

a c i d s i n h i b i t methanogenic b a c t e r i a . robes,

t h e formed o r g a n i c

The l a t t e r a r e o b l i g a t e anae-

a n d d u e t o t h i s t h e y depend a l s o on t h e m i c r o o r g a n i s m s r o n -

auming t h e oxygen f r o m s u b s t r a t e .

S u b s t r a t e s f o r methanogenic bacte-

r i a are acetate,

formiate,

-

288

carbon dioxide,

m e t h a n o l and m e t h y l a m i n e

w h i c h a r e f o r m e d due t o t h e a c t i v i t i e s o f c e r t a i n o t h e r m i c r o o r o g a nisms.

An i n t e s i f i c a t i o n o f s u c h a c o m p l e x p r o c e s s a s m e t h a n e f e r -

mentation,

p o s s e s s e s i t s own s p e c i f i c a t i o n s as c o m p a r e d t o b i o t e c h -

n o l o g i c a l processes which are r e a l i z e d w i t h monocultures.

The s e -

l e c t i o n o f p r o d u c e r s and t h e f o r m a t i o n o f a r t i f i c i a l a s s o c i a t i o n are not i n practice yet, b e i n g done.

however,

investigations i n t h i s f i e l d are

Physiologico-biochemical

activation,

natural associations t o a given substrate,

the adaptation o f

t h e r e g u l a t i o n o f medium

c o m p o s i t i o n a n d t h e o p t i m i z a t i o n o f pH a n d medium t e m p e r a t u r e a r e giving a positive result. The i n s t i t u t e o f M i c r o b i o l o g y o f t h e L a t v i a n Academy o f S c i e n ces,

is c a r r y i n g o u t t h e m e t h a n e f e r m e n t a t i o n o f p i g b r e e d i n g f a r m

l i q u i d w a s t e s as w e l l a s w a s t e s o f a g r i c u l t u r a l p l a n t s . t a b l i s h e d t h a t a t h e r m o p h i l i c regime (53O-55OC)

I t was e s -

significantly in-

a s c o m p a r e d t o a m e s o p h y l i c one ( 3 5 ' -

creases t h e methanogenesis, 4OoC),

which s t i m u l a t e s an i n a c t i v a t i o n o f t h e pathogenous m i c r o -

flora,

h e l m i n t h s a n d weed s e e d s .

T a b l e 1 shows t h e i n f l u e n c e o f t h e t h e r m o p h i l i c t o t a l amount o f b a c t e r i a , and E .

regime on t h e

as w e l l a s on some p h y s i o l o g i c a l g r o u p s

c o l i i n the substrate

( l i q u i d p i g manure)

a f t e r 3 days and

a f t e r 1 t o 3 months o f methane f e r m e n t a t i o n ( c o u n t o f m i c r o o r g a n i s m s i n 1 m l o f native substrate).

TABLE 1 D y n a m i c s o f some g r o u p s o f m i c r o f l o r a g r o w t h i n p i g s l u r r y u p o n t h e r m o p h i l i c a n d m e s o p h y l i c methane d i g e s t i o n

DM

Sample

Proteolytic ucing bacteria

[%I

Coli bacteri

Mesophylic digestion

1.1

6.8

5.10

4.106

A f t e r 1 month.

1.2

7.7

2.106

6.10 4

A f t e r 2 month.

0.4

8.7

7.10~

3.10~

Fresh s l u r r y .

0.8

7.3

8.10

4.106

A f t e r 3 days.

0.8

7.5

4.10~

3.10~

A f t e r 5 days.

0.6

7.5

2.10~

0.5

7.5

2.10

Fresh

slurry.

Thermophilic

After

27 d a y s .

I

3.10~

lo4 lo3

4.10'

digestion

lo5 2.10

lo4

4.10 5.

4.10~ I

4.10'

lo3

1

6.10'

3

0

2

0

lo3

0 I

t

-

289

-

Upon m e s o p h y l i c f e r m e n t a t i o n d u r i n g 2 m o n t h s , o f E.

c o l i b a c t e r i a were o b s e r v e d i n t h e s u b s t r a t e .

r e g i m e i n a c t i v a t e d a l l c e l l s o f E.

c o l i i n 3 days.

1000 l i v i n g c e l l s The t h e r m o p h i l i c Methane fermen-

t a t i o n o f wastes a l l o w s t h e d e g r a d a t i o n n o t o n l y o f carbohydrates, p r o t e i n s and l i p i d s ,

b u t a l s o o f a r o m a t i c a n d h e t e r o c y c l i c compounds.

D u r i n g t h e methane f e r m e n t a t i o n ,

p r a c t i c a l l y a l l petrochemicals are

metabolized. The i n f l u e n c e o f t h e t h e r m o p h i l i c m e t h a n e f e r m e n t a t i o n on t h e d e g r e e o f a d e n i n e and c y t o s i n e b i o d e g r a d a t i o n was i n v e s t i g a t e d .

I t was f o u n d t h a t a t 55OC a b o u t 65 compounds w e r e d e g r a d e d , t h a t the thermophilic c y c l i c compounds.

o f t h e t o t a l amount o f t h e s e

and o n l y 40-45

X

a t 35OC.

This indicates

regime increases the degradation o f hetero-

These e x a m p l e show,

m e n t a t i o n by p h y s i o l o g i c a l methods Fig.

X

-

how t o i n t e n s i f y m e t h a n e f e r by r e g u l a t i o n t e m p e r a t u r e .

1 shows s u g g e s t e d d e c o m p o s i t i o n o f some a r o m a t i c a n d h e t e r o -

c y c l i c compounds d u r i n g m e t h a n e f e r m e n t a t i o n (Sahm, c i a l e x p e r i m e n t s i t was d e m o n s t r a t e d ( T a b l e 2 ) a s sodium t r i c h l o r a c e t a t e , s i m a s i n e ( a n a c t i v e

bis-ethylamino-simm-triazine)

1984).

I n spe-

t h a t such p e s t i c i d e s reagent 2-chlor-4,6

a n d t i l t 2 5 0 ( S w i s s p r e p a r a t i o n ) , were

b i o d e g r a d e d d u r i n g t h e t h e r m o p h i l i c methane f e r m e n t a t i o n . Methane f e r m e n t a t i o n can be used a l s o f o r t h e e f f e c t i v e u t i l i z a t i o n o f wastes from f e e d p r o d u c t i o n i n farms.

TABLE 2 A d e g r e e o f some added p e s t i c i d e d e s t r u c t i o n u p o n 1 9 - d a y a n a e r o b i c thermophilic

Pesticide

fermentation o f p i g s l u r r y

Concentration o f pesticide Initial

[mg/ll

D e s t r u c t i o n degree

Postfermentation

Simasine

100

2.4

97.6

Simasine

150

4.0

97.4

Simasine

200

13.7

93.2

Na-trichloracetate

8

0

100

Na-trichloracetate

24

0

100

Na-trichloracetate

40

0

100

Tilt-250

4.2

0.16

T i I t - 250

10.5

0.20

98.1

T i I t - 250

21

1.80

91.4

96.2

[X

-

290

BENZOIC ACID

-

CYCLOHEXANECARBOXYLIC

ACID

!

2-OXOCYCCOHEXANE

'

CARBOXYLIC ACID

'

I

I

' 3 H 2 \H20

I

//

I

/

&:H2cmH PHENYL ACETIC

I

6:b:H ADlPlC ACID

ACID

F i g . 1. S u g g e s t e d m o d e l f o r d e c o m p o s i t i o n o f some a r o m a t i c compounds t o methane (Sahm, 1 9 8 4 ) . L a t v i a n s p e c i a l i s t s d e v e l o p e d a t e c h n o l o g y o f wet g r e e n c r o p f r a c t i o n a t i o n and l i q u i d l e a f p r o t e i n c o a g u l a t e p r o d u c t i o n from alfalfa,

c l o v e r and o t h e r g r a s s e s .

f i r m "Uzvara"

i n Latvia.

The p r o d u c t i v i t y o f t h e e q u i p m e n t i s 5 - 7

t o n s o f green crop p e r hour. presented i n Fig.

T h i s t e c h n o l o g y i s b a s e d on a g r o -

The f l o w s h e e t o f t h e p r o c e s s T P F - 1 i s

2.

M o i s t and p r o t e i n r i c h g r e e n c r o p i s t r a n s p o r t e d b y a f e e d i n g l i n e i n t o a f r a c t i o n i n g device

-

lacetrator,

t h e n t o a screw press.

The j u i c e i s f e d b a t c h w i s e o r c o n t i n u o u s l y i n t o a f e r m e n t a t o r - c o a gulator.

D u r i n g f e r m e n t a t i o n t h e p r o t e i n c o a g u l a t e s and s e d i m e n t s

i n the form o f coagulate.

Coagulate,

c o n t a i n i n g 3-5

% protein,

i s

-

291

b a t c h w i s e pumped i n t o a c o l l e c t o r .

-

The s t a b i l i z e d j u i c e f r o m t h e

top p a r t o f the fermentator-coagulator daily).

i s used t o feed p i g s ( 2 - 3

1

F r e s h o r s i l a g e d p r e s s cakes a r e f e d t o ruminants. JUICE WHEY

BIOGAS

A JUICE

GREEN MASS 3

-

A

FRACTIONING

1 - FEEDER

5 - JUICE 8 11

I I L I COAGU-l

BLOCK

- GREEN

FERMENTATION

PUMP JUICE

, ,

F i g . 2. F l o w - s h e e t fermented j u i c e .

I'

BLOCK

ELEVATOR , 2 - CONVEYER , 3 - SHREDDER

MASS

COLLECTOR , 6 - PUMP , 7 - SIEVE

- COAGULATE - TANK FOR

Y

9 -COAGULATE

- SEPARATOR

O F FIBROUS

COLLECTOR , 10 - FERMENTOR

,

4 - PRESS ,

FRACTION,

- COAGULATOR ,

1 2 - BIOREACTOR ;

f o r t h e p r o d u c t i o n o f p r o t e i n c o a g u l a t e and

Comparative e x p e r i m e n t s have d e m o n s t r a t e d t h a t t h e T P F - 1 c e s s consumes, technology.

a l m o s t b y an o r d e r ,

The c o s t o f a 5

w i t h a s t a f f o f 1 - 2 people.

-

7 t/h

e q u i p m e n t i s 50 t h o u s a n d r o u b l e s ,

With s i n g l e s h i f t work,

o f herbage d u r i n g 5 months every y e a r , sand r o u b l e s ,

pro-

l e s s energy t h a n t h e t r a d i t i o n a l t r e a t i n g 5600 t

t h e e x p e n d i t u r e was 53 t h o u -

as compared w i t h t h e p r o d u c t i o n o f g r a s s f l o u r

from

herbage. We s t u d i e d t h e p o s s i b i l i t y o f a m o r e r a t i o n a l u s e o f p r o t e i n f r e e f e r m e n t e d brown j u i c e by s u b j e c t i n g i t t o methane f e r m e n t a t i o n . Spontaneous a n a e r o b i c f e r m e n t a t i o n o f l e a f j u i c e a c c o r d i n g t o t h e TPF-1 p r o c e s s c a n b e d e f i n i t e , tation, ce.

as t h e f i r s t s t a g e o f m e t h a n e f e r m e n -

i n which development o f a c i d p r o d u c i n g m i c r o f l o r a t a k e s . p l a -

Table 3 p r e s e n t s m i c r o b i o l o g i c a l d a t a o f a l f a l f a j u i c e

t a t i o n d u r i n g 4 8 h a t room t e m p e r a t u r e .

fermen-

93 % a c i d producing bacte-

r i a f r o m t o t a l amount t a k e s p a r t i n t h e f e r m e n t a t i o n p r o c e s s u n d e r anaerobic conditions. acid,

0.2-0.4

Fermented j u i c e c o n t a i n s 0.7-1.2

X of lactic

?6 o f a c e t i c a c i d a n d pH14.0-4.5.

A novel e f f i c i e n t process f o r anaerobic thermophilic bioconv e r s i o n o f b r o w n j u i c e was r e a l i z e d i n a l a b o r a t o r y s c a l e f e r m e n t e r

w i t h a b o t t o m l a y e r o f a c t i v e biomass and a c a r r i e r o f a b i o l o g i c a l

-

292

-

o r i q i n with immobilized microorganisms i n i t s t o p s e c t i o n (Fig. The c h e m i c a l c o m p o s i t i o n o f

3).

t h e i n i t i a l and f e r m e n t e d brown j u i c e

( r e t e n t i o n t i m e 5.6 l a y s a n d l o a d 11 g COD/day)

i s p r e s e n t e d i n Tab-

l e 4. '4BLE 3 Crowth o f m i c r o f l o r a upon a s p o n t a n e o u s a n a e r o b i c f e r m e n t a t i o n o f 5lfalfa juice

I Group

of microorganisms

I

N[mill./mll

I

[XI

I

I ~ ~ c t e r i at o, t a l ~

Acid producing b a c t e r i a

1 Proteolytic

1 Yeasts

bacteria

~

'

0.001 0.001

-

3 . An e q u i p m e n t fo: a n a e r o b i c f e r m e n t a t i o n o f s i l a g e e f f l u e n t a n d bror:n j u i c e . 1 - s u b s t r a t e s t o r a g e t a n k ; 2 - p e r i s t a l t i c pump; - r e a c t o r , 3 . 1 - l a y e r o f t h e s e d i m e n t , 3 . 2 - h e a t e r , 3 . 3 - micr o o r g h n i s m c a r r i e r ; 4 - d i s c h a r g e p h a s e s e p a r a t o r ; 5 - g a s meter. Fig.

A 50 m 3

b i o r e a c t o r was i n s t a l l e d o n t h e a q r o f i r m " U z v a r a " f o r

t h e r e a l i z a t i o n o f m e t h a n e f e r m e n t a t i o n . One o f t h e c o m p a r t m e n t s i n t h e r e a c t o r i s equipped w i t h capron b r u s h e s t o i n t e n s i f y methanoge-

nesis. I n s p e c i a l e x p e r i m e n t s i t w a s d e m o n s t r a t e d t h a t a d h e s i o n o f m e t h a n o g e n i c b a c t e r i a a t c a p r o n s u r f a c e s i s m o s t e f f e c t i v e a s compareo with t h e g i a s s one (Fig. 4).

The t o t a l a m o u n t o f m e t h a n o g e n i c

b a c t e r i a c a l c u l a t e d o n 1 g d r y w e i g h t o n c a p r o n b r u s h e s is 10 bu!

12 ,

i n l i q c i d i s l o l o . We f o u n d t h a t 6 0 X t o 7 0 X o f m e t h a n o g e n i c

-

293

-

b a c t e r i a a r e i n an i m m o b i l i z e d s t a t e .

1 m3 o f whey o r b r o w n j u i c e

y i e l d s 1 5 - 2 0 m 3 o f b i o g a s w i t h 55-60

?A m e t h a n e . D u r i n g m e t h a n e f e r -

X reduction o f juice

m e n t a t i o n a 70-80 takes place,

COO and i t s n e u t r a l i z a t i o n

h e n c e i t c a n be w e l l u s e d f o r t h e w a t e r i n g o f f i e i d s ,

t h u s i n c r e a s i n g h e r b a g e y i e l d by 1 0 - 2 5 76. TABLE 4 Chemical c o m p o s i t i o n o f t h e i n i t i a l and f e r m e n t e d brown j u i c e Initial juice

Parame t e r Dry

matter content.

[XI

Fermented j u i c e

4.55

2.5 39.4

44.4

COO [ g / l l

18.3

NH4 1 9 / 1 1

0.70

0.77

PH

4.0

7.1

Acetic acid

[%I [#I

Propionic acid Lactic acid [ X I n-butyric

acid [ % I

n-valeric

acid

[%I

0.044

0.107

0.009

0.170

1.617

0.0

0.0

0.002

0.010

0.013

CAPRON

* t 4

-f

I V

/.-

2.0

0.5

"

/

----

/ /

f, 1

2

3

CONTROL

, , ,

I

4

7

WEEKS

5

6

OF FERMENTATION

F i g . 4. An e f f e c t o f i m m o b i l i z a t i o n s u r f a c e ( c a p r o n a n d g l a s s ) on t h e process o f methanogenesis. C o n t r o l - w i t h o u t i m m o b i l i z a t i o n surface.

Good r e s u l t s w e r e o b t a i n e d d u r i n g m e t h a n e f e r m e n t a t i o n o f b r o w n j u i c e t o g e t h e r w i t h a n i m a l manure ( F i g .

5).

T h i s means t h a t wet

g r e e n c r o p f r a c t i o n a t i o n a c c o r d i n g t o t h e TPF-1 p r o c e s s m u s t be o r -

-

294 -

g a n i z e d d i r e c t l y on t h e a n i m a l f a r m s t o u t i l i z e i n one u n i t o f b i o r e a c t o r , both t h e w a s t e s o f f e e d t r e a t m e n t and farming. A very s i g n i f i c a n t f a c t o r i n a n i n t e n s i f i c a t i o n of methane fer-

m e n t a t i o n is an o p t i m i z a t i o n o f t h e C/N rich with nitrogen,

r e l a t i o n . Farm w a s t e s a r e

and due t o t h e given example demonstrated above,

a n a d d i t i o n o f p l a n t mass brown j u i c e c o n s i d e r a b l y r a i s e d t h e b i o gas yield.

: :I 0.8

0.4

I

I

I

I

I

I

l

l

1

2

3

4

5

6

7

FERMENTATION DAYS

F i g . 5 . An e f f e c t o f b r o w n j u i c e a d d i t i o n o n t h e p r o c e s s o f p i g s l u r r y methanogenesis. E x p e r i m e n t a l l y t h e e f f i c a c y was t e s t e d o f w h e a t s t r a w a d d i t i o n t o p i g s l u r r y . As o n e s e e s f r o m F i g . 6 t h e b i o g a s y i e l d c a n b e r a i s e d by a d d i n g up t o 8 % c h o p p e d straw ( i n d r y w e i g h t ) .

Thus, a straw

u t i l i z a t i o n i s p o s s i b l e i f t h e r e i s a b i o r e a c t o r b y t h e f a r m . An addition of straw t o slurry improves t h e s u b s t r a t e surface, bettering properties a s the fermentation microflora practically does n o t d e s t r o y l i g n i n , b u t h e m i c e l l u l o s e and c e l l u l o s e a t a s h o r t e n e d r e t e n t i o n time c a n b e t o a g r e a t e x t e n t p r e s e r v e d a s humus f o r m i n g factors

.

-E

: 30 E

8 '/o

-

STRAW - BIOGAS

I

8 '10 STRAW- CHq

10

20

30

40

50 DAYS

F i g . 6 . E f f e c t o f a n 8 % a d d i t i o n o f s t r a w on m e t h a n o g e n e s i s of p i g farm wastes.

-

295

The a g r i c u l t u r a l f i r m " O g r e " r e a c t o r s o f 100 m 3 c a p a c i t y f o r i n a thermophilic

regime.

-

has been u s i n g f o r s i x y e a r s b i o -

f e r m e n t i n g methane from p i g s l u r r y

Mean t e c h n i c a l and e c o n o m i c d a t a a r e g i -

ven i n Table 5. TABLE 5 Average p a r a m e t e r s o f a 1 2 month work o f t h e e x p e r i m e n t a l b i o e n e r g e t i c e q u i p m e n t on t h e s t a t e f a r m " O q r e " 3 per m I 3 o f sludge i n b i o r e a c t o r , [ m /m rdayl. D a i l y y i e l d o f biogas

[m3

3

D a i l y y i e l d o f biogas [m3

2.6

per kg 1

3

o f d r y o r g a n i c m a t t e r , [ m /kg*day 1. C o n t e n t o r methane i n b i o g a s ,

[XI.

D e s t r u c t i o n o f d r y o r g a n i c m a t t e r , [ 861.

0.5 65 33

A t present t h i s farm i s organizing a complete b i o t e c h n o l o g i c a l system o f waste t r e a t m e n t

( a p p r o x i m a t e l y f o r 20,000

pigs).

t e m i n c l u d e s methane f e r m e n t a t i o n o f p i g f a r m wastes,

The s y s -

t h e usage o f

f e r m e n t e d manure f o r t h e w a t e r i n g o f g r e e n c r o p f i e l d s and t h e e quipment f o r pigs.

f r a c t i o n a t i o n o f grass t o obtain p r o t e i n coagulate f o r

The c a l c u l a t e d i n c o m e p e r y e a r e q u a l s 244,000

r o u b l e s and t h e

t o t a l investment c o s t o f t h i s system i s about 1 . 6 m i l l i o n r o u b l e s . We c a l c u l a t e d t h e m e n t i o n e d i n v e s t m e n t c o s t b y c o v e r i n g t h e i n c o m e d u r i n g t h e 6-7

year e x p l o i t a t i o n o f t h e system.

A f t e r t h e methane f e r m e n t a t i o n , was p a r t l y e l i m i n a t e d . i s

t h e s p e c i f i c o d o r o f manure

The e c o l o g i c a l e f f e c t i s m o s t i m p o r t a n t ,

as

t h e e c o n o m i c a l one f r o m s u c h a w a s t l e s s t e c h n o l o g y .

O u r t o t s 1 c o n c l u s i o n i s ; m e t h a n e f e r m e n t a t i o n c a n b e recommend e d a s a g o o d and s i m p l e m e t h o d f o r e n v i r o n m e n t a l p r o t e c t i o n i n agriculture.

LITERATURE

1 2

3

H . Sahm A n a e r o b i c W a s t e w a t e r T r e a t m e n t , A d v a n c e s i n B i o c h e m i c a l E n g i n e e r i n g , B i o t e c h n o l o g y , S p r i n g e r , B e r l i n , H e i d e l b e r g , New Y o r k , T o k i o , 1984, p. 8 5 - 1 1 5 . M.J. B e k e r , A.P. G r i n b e r g s , L . J . Labane, C a r b o n h y d r a t e b i o c o n v e r s i o n i n methane f e r m e n t a t i o n . S e v e n t h A u s t r a l i a n B i o t e c h n o l o g y conference. U n i v e r s i t y o f Melbourne, 2 5 t h - 2 8 t h August, Proceed i n g s , 1986, p. 3 5 2 - 3 5 5 . C . Cooney and D. W i s e , T h e r m o p h i l i c a n a e r o b i c d i g e s t i o n o f s o l i d w a s t e f o r f u e l g a s p r o d u c t i o n . B i o t e c h . B i o e n g . , 1 9 7 5 , v o l . 17, p. 1119.

4

5

6 7 8

J.G.

296

-

Z e i k u s , The b i o l o g y o f m e t h a n o g e n i c b a c t e r i a . B a c t e r i o l . Rev., 1 9 7 7 . , v o l . 4 1 , No 2, p . 514-541. M A . Beker, A.A. K l i n t s a r e , A.A. U p i t i s , A.P. G r i n b e r g s , A.P. Vilc a n s and M . K . M a r a u s k a . E x p e r i m e n t a l s t u d y a n d i n d u s t r i a l u s e o f anaerobic f e r m e n t a t i o n f o r o b t a i n i n g feeds and b i o g a s . World B i o t e c h . R e p o r t . 1985, v o l . 3: A s i a p. 427-430. V.S. D u b r o v s k i i a n d U.E. V i e s t u r s , Metanovoe s b r a z i v a n i e s e l s k o h o z a i s t v e n i h otxodov, Riga, Z i n a t n e , 1988. ( i n r u s s i a n ) B i o t e c h n o l o g i a k o r m o p r o i z v i d s t v a i pererabotka othodov. ( e d i t e d by M . J . B e k e r ) , R i g a , Z i n a t n e , 1 9 8 7 , ( i n r u s s i a n ) . T r a n s f o r m a c i a p r o d u k t o v f o t o s i n t e z a . ( e d i t e d by M . J . B e k e r ) , Riga, Z i n a t n e , 1984. ( i n r u s s i a n ) .

E C O N O M I C A N D B I O E N E R G E T I C A S P E C T S OF M E T H A N O G E N E S I S

-

QUANTITATIVE INVESTIGATIONS

S.

VASSILEVA,

M.

ROBEVA'

and S .

MUTAFOV'

C e n t r a l L a b o r a t o r y B i o i n s t r u m e n t a t i o n and A u t o m a t i o n 1 I n s t i t u t e o f Microbiology B u l g a r i a n Academy o f S c i e n c e , S o f i a , B u l g a r i a

INTRODUCTION

One o f t h e b a s i c c o n t e m p o r a r y t a s k s i s k e e p i n g t h e e c o l o g i c a l balance i n nature.

Increasing accumulation o f i n d u s t r i a l ,

agricul-

t u r a l and m u n i c i p a l w a s t e s h a v e a s e r i o u s i m p a c t on t h e e c o l o g i c a l equilibrium.

These w a s t e s a r e a v a l u a b l e r a w m a t e r i a l f o r o b t a i n i n g

e n e r g y r i c h compounds,

s u c h as v a r i o u s a l c o h o l s a n d m e t h a n e ,

t h e i r s t o r a g e more o f t e n t h a n n o t i s v e r y d i f f i c u l t .

yet

T h a t i s why

t h e c r e a t i o n o f t e c h n o l o g i e s i n c l u d i n g r e c y c l i n g o f waste i n i n d u s t r y and a g r i c u l t u r e

i s a primary task.

The p r e s e n t work d e a l s w i t h some b i o e n e r g e t i c f a c t o r s S o ,

6, r

,

Y T 111 and t h e i r i m p a c t on b i o g a s a n d e t h a n o l o u t p u t i n l a b o r a t o r y conditions.

The d e s i g n o f m a t h e m a t i c a l m o d e l s s i m u l a t i n g t h e b e h a -

v i o u r on r e a l s y s t e m s o n a c o m p u t e r i s an i m p o r t a n t a s p e c t o f t h e m e t h o d o l o g y o f p r e s e n t day e n g i n e e r i n g a n a l y s i s .

The m o d e l s p r e s e n -

t e d i n t h i s w o r k a r e f o r m u l a t e d by u s i n g e x p e r i m e n t a l d a t a s u m m a r i z i n g t h e b i o c h e m i c a l as w e l l as p h y s i o l o g i c a l a s p e c t s o f t h e m e t h a nogenesis.

O u r m a i n g o a l was t o c r e a t e a m a t h e m a t i c a l m o d e l w h i c h

c o u l d be used f o r p r e d i c t i n g t h e p r o c e s s o f p r o d u c t i v i t y and r e n t a b ilit.y

.

M A T E R I A L AND METHODS The q u a n t i t a t i v e a n d q u a l i t a t i v e a n a l y s i s o f t h e i n p u t and o u t p u t s u b s t a n c e s was b a s e d on t h e m a s s - e n e r g y thermodynamic e f f i c i e n c y ,

b a l a n c e m e t h o d 111. The

t h e d i s t r i b u t i o n o f c a r b o n and energy o f

t h e s u b s t r a t e w i t h d e f i n e d c h e m i c a l c o m p o s i t i o n was e s t a b l i s h e d b y t h e amrnount o f C H 4 a n d C 0 2 p r o d u c e d .

The k i n e t i c s o f d e g r a d a t i o n o f

these s u b s t r a t e s from t h e mixed m i c r o b i a l p o p u l a t i o n t a k e n from t h e

-

298

-

a c t i v a t e d s l u d g e was e s t a b l i s h e d o n t h e b a s i s o f t h e c h a n g e s i n t h e amount and c o m p o s i t i o n o f t h e o u t p u t g a s m i x t u r e . The m e t h o d o l o g i c a l b a s i s f o r a l l t h e s e e x p e r i m e n t s was e l a b o r a t e d i n t h e Biotechnology Department o f t h e M i c r o b i o l o g i c a l I n s t i t u t e i n Prague 121. As a m o d e l p r o c e s s f o r a n a e r o b i c d e g r a d a t i o n i n w h i c h t h e p r o t h e e x p e r i m e n t s w i t h a c u l t u r e o f 5.

d u c t i s i n l i q u i d phase,

cepe-

v i s i a e s t r a i n R X I I was u s e d f r o m t h e c o l l e c t i o n o f t h e M i c r o b i o l o g i c a l I n s t i t u t e i n Prague. ethanol,

carbon dioxide,

The t i m e c o u r s e o f t h e b i o m a s s ,

glucose,

i n o r g a n i c n i t r o g e n a n d pH was m e a s u r e d .

The c h a n g e s i n t h e c o n t e n t o f p r o t e i n a n d t o t a l R N A were c l o s e l y ob-

I n t h e second

served.

and t h i r d experiment o f a s e t o f t h r e e e x p e r i -

X o f e t h a n o l was a d d e d i n t h e r e a c t i o n

m e n t s an amount o f 2 a n d 5 mixture.

The q u a n t i t y o f medium and i n n o c u l u m was e q u a l

[2,

31.

MODELLING M E T H A N E YIELD A S A FUNCTION OF BIOENERGETIC FACTOR OF THE S U B S T R A T E

Based o n 3 2 t h r i c e r e p e a t e d e x p e r i m e n t s c a r r i e d o u t w i t h 7 s u b strates, time, exp.

t h e f o l l o w i n g models f o r b i o g a s p r o d u c t i o n as a f u n c t i o n o f

have been o b t a i n e d : No 1 P(t)

(1)

209

=

s t a t i s t i c a l c r i t e r i u m RL exp.

=

exp.

0,9556;

exp.

No 4

(4)

= 502 -

107.102/t

+

398.102/t2;

P(t)

924

+

178.102/t

-

0,76.1O6/t’;

P(t)

= 83,5 +

P(t)

=

2.37.t

+

0.0495.t’;

-

290.103/t

5

(5)

=

P(t)

0,9502;

exp.No

RL

0,7598;

No 3

R2

I

5980/t2;

0,9253;

(3)

RL

+

No 2

(2) RL

2240/t

0,3951;

406

+

74,60/t

2

;

exp.No

2

=

-

6

(6) R

299

p(t)

=

5888

P(t)

=

575

-

+ 324.lo3/t2;

0,26.105/t

0,7931;

e x p o No 7 (7)

+

-

620/t

233.10

3

2

/t

,

R 2 = 0,9435. With t h e e x c e p t i o n o f m o d e l No 4 ,

t h e s t r u c t u r e s o f models a r e

The c o e f f i c i e n t o f t h e m o d e l P ( t ) = a

similar.

+

b/t

+ c/t2 (a, b

and c ) c o u l d be p r e s e n t e d as a f u n c t i o n o f b i o e n e r g e t i c T 6 , r, Y ( s e e T a b l e 1).

factors So,

YT

so

6

r

1.

0,66

1

0,17

0,028

exp.

No

2.

4,95

2

0,18

0,060

3.

4,95

3

0,32

0,160

4.

0,66

4

0,40

0,260

5.

393

4

0,18

0,120

6.

0,99

4,67

0,48

0,370

7.

0,66

5

0,55

0,460

T h i s dependence c o u l d be e x p r e s s e d i n t h e f o l l o w i n g models:

(8)

a

=

statistical

(9)

b

=

(10)

F

=

c

=

0,56413;

1,15735.So

0,49499.Y

criteria F 7,1865

T

=

R

-

0,68232.r

R

=

59,645,

+

-

-

0,9997;

3,57902. r

+ 3,83303. 6

-

0,74596;

-

-9,15623

12,98107.So T + 14,15378.Y ;

R

+ 1,96685.6

;

3,5058.So T 5,63393.Y

F = 0,31364;

+

0,06746

=

+

10,90564.r

-

17,63561.6

+

0,83242.

The m o d e l s (8)

-

(lo),

s u b s t i t u t e d i n t h e equations (1)

-

(7),

show q u a n t i t a t i v e d e p e n d e n c e b e t w e e n m e t h a n e p r o d u c t i v i t y a n d b i o e nergetic factors o f t h e substrate.

For convenience t h e b i o e n e r g e t i c

-

300 -

f a c t o r s and t h e c o e f f i c i e n t s were s c a l e d up a s f o l l o w s : 6 -2 , b - b.10-3, c - c . ~ O - ~ . a - a.10

-

6

.lo,

yT

-

YT.102,

Fig.

1 shows e x p e r i m e n t a l d a t a f o r m e t h a n e p r o d u c t i v i t y d e p e n -

d i n g on b i o e n e r g e t i c

factors o f d i f f e r e n t substrates.

A pH1 1 - 1 0 pH3 1 - 1

D H ~1 - 1

,

25 t Ihl

0

Fig.

1.

INFLUENCE OF SOME A B I O T I C FACTORS ON ALCOHOL FERMENTATION As a m o d e l p r o c e s s f o r a n a e r o b i c d e g r a d a t i o n i n w h i c h t h e p r o three experiments w i t h a c u l t u r e o f

d u c t i s i n a l i q u i d phase, S.

c e r e v i s i a e were c a r r i e d o u t .

Changes i n t h e p r o t e i n c o n t e n t w e r e

m e a s u r e d i n t h e t i m e c o u r s e o f t h e b i o m a s s X, c a r b o n d i o x i d e C02,

nitrogen

glucose S , ethanol E,

N , pH. The ammount o f 2 a n d 5 X e t h a -

n o l was added i n t h e s e c o n d a n d t h i r d e x p e r i m e n t . E x p e r i m e n t a l d a t a were e l a b o r a t e d w i t h t h e aim o f smooth s p l i nes,

b u t t h e d e r i v a t i v e s were d e t e r m i n e d by t h e method o f p o l y n o m i a l

approximation w i t h parabol passing through every three neighbouring points. A p p l y i n g t h e method o f n o n l i n e a r r e g r e s s i o n t h e f o l l o w i n g models were o b t a i n e d . exp.No (1)

1 dX/dt

=

dS/dt

= 1,42 + 9,02/X

E

u.X

-

0,879/X2

= 2599 - 0,2335.5 + 0,006.S 2

-

301

(4)

d(C02)/dt

=

(5)

u

+0,0879/pH

I

0,366

6,72

8,39/X

dX/dt

=

u.X

dS/dt

=

1/[9,ll(dX/dt

E

=

-

59,O

d(COp)/dt

u

-

-

0,00296/pHL

-

0,2331’

- 3 , 8 + 5,31.X

= 0,0738 -

2,37/X2

-

0,5361

+ 0,0033.5 2

0,782.5 I

+

-

0,905.X 2

+ 0,136.(dpH/dt)

0,24l.(dpH/dt)

exp.No 3

=

(1)

dX/dt

(2)

dS/dt

(3)

E

(4)

d(C02)/dt

(5)

u ‘= 0,0639

=

u.X -1,69

79,6

-

- 64,2.dX/dt

+ 237.(dX/dt) 2

0,885.S 0,636

I

-

+ O,OO19/X

0,075.(dpH/dt)

-

O,lll.(dpH/dt)’

D i s c r i m i n a t i o n o f t h e m o d e l s f o r t h r e e e x p e r i m e n t s was b a s e d on a s t a t i s t i c c r i t e r i a . The f o r m u l a e f o r s p e c i f i c g r o w t h r a t e ,

d e p e n d i n g on pH,

c a n be

u s e d f o r o p t i m a l c o n t r o l [ 4 , 51. Changes o f pH f o r t h r e e e x p e r i m e n t s a r e shown i n F i g .

2.

CONCLUSION The e x p e r i m e n t a l d a t a were p r e s e n t e d i n m a t h e m a t i c a l m o d e l s , d e s c r i p t i n g p r o c e s s p r o d u c t i v i t y as a f u n c t i o n o f b i o e n e r g e t i c f a c tors. The m o d e l s c o n c e r n i n g b i o g a s p r o d u c t i o n shown above c a n s e r v e f o r p r e d i c t i o n process p r o d u c t i v i t y f o r a given time a t t h e determined s t a r t i n g c o n d i t i o n . Two i m p o r t a n t e c o n o m i c a l t a s k s c o u l d b e s o l v e d w i t h t h e m o d e l s T f ( S o , 6 , r , Y 1:

P ( t ) and P =

1. p r e d i c t i o n o f b i o g a s y i e l d b a s e d on t h e i n p u t b i o e n e r g e t i c p a rameters o f the substrate; 2.

maximizing o f the P ( t ) a t the condition o f the biodegradation o f o r g a n i c wastes for m i n i m a l t i m e course.

-

302

-

0 20

70 t Ihl

Fig.

2.

The c o m p l e t e s o l v i n g o f t h e s e t w o t a s k s a n d t h e c h o i c e o f a s u i t a b l e method c o u l d be t h e aim o f f u r t h e r work.

L I S T OF SYMBOLS

s

-

substrate concentration

6

-

mass f r a c t i o n o f c a r b o n

Y

- degree

YT

-

P

-

o f reductance

theoretical yield product (biogas) product

f r o m ammonium o x a l i c u m

product from n a t r i u m formicum p3

p4 p5 '6

p7

-

product from g l y c i n product from l a c t i c a c i d product from natrium aceticum product from propionic acid product from b u t y r i c a c i d

LITERATURE

1

2

M . S o b o t k a , J . V o t r u b a , I. H a v l i k a n d I . G . M i n k e v i c h , The M a s s Energy Balance o f Anaerobic Methane P r o d u c t i o n . F o g l i a M i c r o b i o l o g i c a 2 8 , 1983, p . 1 9 5 - 2 0 4 . M. R o b e v a , Fh. T h e s i s o f D i s s e r t a t i o n : A n a e r o b i c p r o d u c t i o n o f E n e r g y - R i c h Compounds: B i o t e c h n o l o g i c a l a s p e c t s , 1988.

3

4

5

S.H. Z i n d e r and R . A .

303

-

Man, I s o l a t i o n a n d c h a r a c t e r i z a t i o n o f a T h e r m o p h i l i c s t r a i n o f M e t h a n o s a r c i n a u n a b l e t o u q e H2C02 f o r M e c h a n o g e n e s i s . A p p l . Env. M i c r o b i o l . 3 8 , 1 9 7 9 , p . 996 - 1008. K . S i g l e r , Knotkova and A. K o t y k , F a c t o r s g o v e r n i n g s u b s t r a t e i n d u c e d g e n e r a t i o n a n d e x t e n t i o n o f p r o t o n s i n t h e y e a s t 5. c e r e v i s i a e . Biochem. B i o p h y s . A c t a 6 4 3 : 1 9 8 1 , p. 5 7 2 - 5 8 2 . L.N. A n d r e y e v a and V . V . B i r y u k o v , A n a l y s i s o f m a t h e m a t i c a l mod e l s o f t h e e f f e c t o f pH on t h e f e r m e n t a t i o n p r o c e s s a n d t h e i r use f o r c a l c u l a t i n g o p t i m a l f e r m e n t a t i o n c o n d i t i o n s . B i o t e c h n o l . B i o e n g . Symp. No 4 , 1 9 7 4 , p. 6 1 - 7 6 .

This Page Intentionally Left TBlank

R E C O V E R Y OF ENERGY FROM MUNICIPAL SOLID WASTE

I N FABRICATED

DIGESTERS

J. COOMBS a n d Y . R .

COOMBS

CPL S c i e n t i f i c T O , S c i e n c e House, B e r k s h i r e , RG14 5 P X , UK.

Winchcombe Road,

Newbury,

SUMMARY The u s e o f a n a e r o b i c d i g e s t i o n (AD) o f t h e o r g a n i c f r a c t i o n (OF) o f m u n i c i p a l s o l i d w a s t e (MSW) i n f a b r i c a t e d d i g e s t e r s w i t h t h e o b j e c t i v e o f b o t h waste d i s p o s a l and e n e r g y g e n e r a t i o n i s r e viewed. V a r i o u s t y p e s o f feeds a r e d i s c u s s e d i n terms o f composit i o n , energy c o n t e n t and b i o d e g r a d a b i l i t y i n r e l a t i o n t o p o t e n t i a l gas e v o l u t i o n . P o s s i b i l i t i e s o f i n c r e a s i n g gas p r o d u c t i o n b y p r e t r e a t m e n t o f t h e OF/MSW o r b y u s e o f m u l t i - s t a g e r e a c t o r s a r e d i s cussed and r e s u l t s from v a r i o u s p i l o t and c o m m e r c i a l s c a l e f a c i l i t i e s u s e d t o i l l u s t r a t e t h e most i m p o r t a n t f a c t o r s w h i c h d e t e r m i n e gas p r o d u c t i o n r a t e s and economic v i a b i l i t y . Advantages o f thermop h i l i c d i g e s t i o n o f s o u r c e - s o r t e d m a t e r i a l , admixed w i t h o r g a n i c sludges, a t h i g h s o l i d s concentration i n simple d i g e s t e r s w i t h low c a p i t a l cost are stressed.

INTRODUCTION A l t h o u g h an i n c r e a s i n g amount o f e n e r g y i s b e i n g r e c o v e r e d i n t h e f o r m o f l a n d f i l l g a s f r o m MSW t h i s is a c h i e v e d o n l y o v e r a l o n g perioo or

Lime

-

o r r e n e s r i m a r e u a r arouriu

IU

years ur

SU.

LYIIUII-

l l i n g a l s o has t h e d i s a d v a n t a g e t h a t m a t e r i a l s such as m e t a l s , a n d p l a s t i c s a r e n o t r e c o v e r e d u n l e s s s o r t e d a t s o u r c e and, more,

a s i g n i f i c a n t amount o f t h e e n e r g y c o n t e n t ,

n i c materials r e s i s t a n t t o biodegradation,

glass

further-

p r e s e n t i n orga-

i s not recovered.

The a-

mount o f e n e r g y t h a t i s r e c o v e r e d f r o m MSW c a n be i n c r e a s e d by a d o p t i n g thermal or thermochemical techniques ration,

pyrolysis,

g a s i f i c a t i o n or

including direct incine-

m a n u f a c t u r i n g RDF.

Sorting plants

have been d e s i g n e d t o r e c o v e r f e r r o u s and o t h e r m e t a l s , and/or

glass.

r i a l s a s r e f u s e d e r i v e d f u e l s (RDF lets)

plastics

I n g e n e r a l such p l a n t a l s o r e c o v e r t h e o r g a n i c mate-

-

i n t h e form o f f l u f f or p e l -

o r as s e r o b i c c o m p o s t . I r r e a p e c t i v e o f t h e range o f p r o d u c t s g e n e r a t e d waste s o r t i n g

p l a n t a r e c a p i t a l i n t e n a i v e and have s i g n i f i c a n t r u n n i n g c o s t s . s e c o s t s c a n be o f f s e t ,

The-

a n d i n some c a s e a t h e o v e r a l l c o s t p e r t o n n e

- 306

-

o f MSW t r e a t e d r e d u c e d b e l o w t h a t o f l a n d f i l l i n g ,

i f t h e waste t r e -

atment p l a n t g e n e r a t e s income f r o m t h e s a l e o f r e c o v e r e d s o l i d mater i a l s (metal,

plastic,

glass,

t h e s a l e o f energy o r f u e l s .

paper,

card,

b o a r d o r wood)

and/or

I f t h e income f r o m s a l e o f e n e r g y and

m a t e r i a l s i s n o t i n excess o f c a p i t a l and r u n n i n g c o s t s t h e d i f f e r e n c e can be c o n v e r t e d i n t o a t i p p i n g f e e ,

charged f o r waste d i s p o -

s a l through the p l a n t . P l a n t p r o d u c i n g s o l i d f u e l s f r o m MSW u s e t h e d r i e r f r a c t i o n , i n c l u d i n g paper,

card,

wood,

p l a s t i c s and l a m i n a t e s ,

which form t h e

b u l k o f t h e m a t e r i a l r e c o v e r e d as f l u f f o r p e l l e t e d RDF.

The c a l o r i -

f i c v a l u e o f t h e s o l i d f u e l i n c r e a s e s w i t h r e m o v a l o f b o t h i n e r t mat e r i a l and m o i s t o r g a n i c s d e r i v e d f r o m f o o d wastes, e t c (Fig.

1). I t i s t h i s e a s i l y biodegradable,

garden c l i p p i n g s ,

or putrescible,

1 CV(net)=7.06MJ k g - 1

,9

H20 ASH

c

~ 3 3 % =29%

Raw r e f u s e Removal o f m e t a Is Glass and noncombs.

2 CV(net)=9.ZMJ k g - l HzO ~ 3 9 % ASH =12.62 3 CV(net)=10.5MJkgH20 ~ 3 9 % ASH =8.3?6

1

4 CV(net)-12.9MJ k g - l H20 =28% ASH ~8.5% 1 5 CV(net)=26.OMJ kg-

=20x

YlELEDlWElGHT OF FUEL AS

'/o

Removal

of

e t c and

-

meta I s 1 0 mm

Removal o f m e t a e t c - 10 mm p u t rescibles Only

plastic

remaining

OF INPU'I

F i g . 1. E f f e c t o f s o r t i n g on t h e p o t e n t i a l f o r r e c o v e r i n g e n e r g y f r o m r a w w a s t e by c o m b u s t i o n . f r a c t i o n which c o n t r i b u t e s l a r g e l y t o the formation o f biogas i n l a n d f i l l s and i s t h e most o b v i o u s s u b s t r a t e f o r A D i n f a b r i c a t e d d i gesters.

The i n c o r p o r a t i o n o f

wet p u t r e s c i b l e m a t e r i a l s i n w a s t e

f r a c t i o n s w h i c h a r e t o b e u s e d as d i r e c t c o m b u s t i o n f u e l s d e c r e a s e s t h e c a l o r i f i c value.

I t i s c l e a r t h a t e n e r g y p r o d u c t i o n f r o m MSW c a n

be o p t i m i s e d by i n c l u d i n g A D i n i n t e g r a t e d w a s t e s o r t i n g a n d m a t e r i a l s r e c o v e r y systems.

I n t h e s i m p l e s t c o n c e p t AD o f t h e p u t r e s c i b -

l e f r a c t i o n w i l l r e c o v e r e n e r g y f r o m a component o f t h e w a s t e w h i c h i s not otherwise o f value,

d e t r a c t s f r o m t h e c a l o r i f i c v a l u e o f RDF

a n d ifs e p a r a t e d c u r r e n t l y

-

307

requires disposal t o l a n d f i l l .

The amount

o f gas e n e r g y a n d i t s m a r k e t v a l u e d e p e n d s on t h e c o m p o s i t i o n o f wast e and p l a n t d e s i g n s a s w e l l a s c u r r e n t g a s ,

o i l and e l e c t r i c i t y p r i -

ces.

A l l waste s e p a r a t i o n p l a n t use s i g n i f i c a n t amounts o f p r o c e s s energy;

f r o m 1 5 t o 75 kWh p e r t o n n e o f MSW t r e a t e d r e s u l t i n g i n t o -

t a l a n n u a l energy b i l l s e x c e e d i n g E 300.000 ce,

i n some i n s t a n c e s .

Hen-

t h e main b e n e f i t o f i n t e g r a t i n g a d i g e s t e r i n t o such p l a n t i s

r e p l a c i n g bought i n energy. intangible benefits b e t t e r separation,

I n a d d i t i o n t h e r e may b e m e a s u r a b l e

from other aspects,

or

such as improved s o l i d f u e l s ,

decreased l a n d f i l l i n g c o s t s ,

decreased problems

o f LFG c o n t r o l a n d l e s s l e a c h a t e g e n e r a t i o n . However,

s o f a r t h e number o f A D p l a n t t r e a t i n g w a s t e w h i c h h a -

ve b e e n b u i l t i s l i m i t e d t o a s m a l l number o f p i l o t , and c o m m e r c i a l p l a n t i n EC c o u n t r i e s , and i n t h e US.

i n Japan

The l i m i t a t i o n s shown b y s i m p l e s y s t e m s r e f l e c t de-

g r a d a t i o n o f l i g n o c e l l u l o s i c as a r a t e l i m i t i n g s t e p . exist for increasing digestibility, using multi-stage merization.

demonstration

elsewhere i n Europe,

Possibilities

and hence t o t a l gas p r o d u c t i o n ,

processes i n c l u d i n g chemical or b i o l o g i c a l depoly-

I n c r e a s e d gas p r o d u c t i o n would f a v o u r t h e g e n e r a t i o n o f

e l e c t r i c i t y f o r s u p p l y t o t h e g r i d w h i c h may become more a t t r a c t i v e f r o m an e c o n o m i c v i e w p o i n t i n t h e f u t u r e .

R e s u l t s from such p r o j e c t s

a r e used t o e v a l u a t e o p t i m a 1 p r o c e s s o p t i o n s . AVAILABILITY,

C O M P O S I T I O N AND ENERGY OF MSW

The n a t u r e o f MSW a v a i l a b l e i n a g i v e n l o c a l i t y i s t h e m o s t i m portant

f a c t o r i n t h e s i z i n g and d e s i g n o f a waste t r e a t m e n t p l a n t .

Composition w i l l i n f l u e n c e the separation flowsheet, gy c o n t e n t ,

t h e t o t a l ener-

t h e p r o p o r t i o n o f energy t h a t can be r e c o v e r e d and t h e

c h o i c e o f technology used t o r e c o v e r t h i s energy. t e w i l l determine the s i z e o f the p l a n t .

The amount o f was-

There a r e two c l e a r a l t e r -

n a t i v e s i n a p p l y i n g t h e AD p r o c e s s t o t r e a t m e n t o f OF/MSW.

The f i r s t

i s t o u s e AD a a a means o f r e m o v i n g a f r a c t i o n o f t h e w a s t e w h i c h h a s l i t t l e v a l u e a n d may d e t r a c t

f r o m t h e e f f i c i e n c y and c o s t e f f e c -

tiveness o f other energy of m a t e r i a l recovery processes. i s t o d e s i g n an e n e r g y r e c o v e r y p r o c e s s b a s e d on AD,

The s e c o n d

which uses f r a c -

t i o n o f MSW a s f e e d s t o c k a n d i s o p t i m i s e d f o r e n e r g y r e c o v e r y . The amount o f g a s w h i c h c a n b e g e n e r a t e d f r o m a g i v e n w e i g h t o f u n s o r t e d MSW as c o l l e c t e d w i l l b e p r o p o r t i o n a l t o t h e c o n t e n t o f e a s i l y biodegradable material.

I n o r d e r t o i n c r e a s e gas p r o d u c t i o n i t

-

308

-

may b e n e c e s s a r y t o s e p a r s t e t h e MSW t o i n c r e a s e t h e c o n t e n t o f b i o d e g r a d a b l e v o l a t i l e s o l i d s (bVS) se d i g e s t i b i l i t y

pretreate i t t o increa-

content,

o r add o t h e r o r g a n i c m a t e r i a l .

Additions could i n -

c l u d e s u i t a b l e i n d u s t r i a l or sewage s l u d g e s a s w e l l as o t h e r s o l i d ( i n d u s t r i a l or a g r i c u l t u r a l ) energy crops.

r e s i d u e s o r even purpose grown biomass

T h i s may b e p a r t i c u l a r l y t r u e i n c o u n t r i e s w h e r e t h e

r e l a t i v e p r o p o r t i o n o f p u t r e s c i b l e m a t t e r is l o w ( F i g .

I2O 100

2).

1

80

60 40

20 0 5 6 7 8 9 10 11 TOWN OR COUNTRY El WTRESCBLES 63 PAPER ETC PLASTIC DMETAL BGLASS OTHER 2

1

3

4

f3l

F i g . 2 . C o m p o s i t i o n o f MSW f r o m v a r i o u s s o u r c e s : 1. F r a n c e , 2 . U K , 3 J a p a n , 4. Canada, 5.Sweden, 6 . U S A , 7 . F r a n c e , 8 . I t a l y , 9 . Swed en, 1 0 . I t a l y , 11. G r e e c e .

.

E N E R G Y POTENTIAL I f o r g a n i c m a t t e r i s combusted i n a b o i l e r o r f u r n a c e t h e a v s i l a b l e h e a t w i l l d e c r e a s e a s t h e m o i s t u r e c o n t e n t i n c r e a s e s due t o t h e f a c t t h a t p a r t o f t h e e n e r g y i s u s e d i n t h e e v a p o r a t i o n o f water.

However,

t h e energy w h i c h can be r e c o v e r e d by a n a e r o b i c d i g e s -

t i o n i s n o t a f f e c t e d by t h e m o i s t u r e c o n t e n t s i n c e t h e p r o c e s s t e k e s p l a c e i n an aqueous e n v i r o n m e n t .

I n general,

vegetable matter

w i t h a h i g h e r w a t e r c o n t e n t i s more e a s i l y d i g e s t e d t h a n m a t e r i a l s such as paper,

n a t u r a l f i b r e s and t e x t i l e s ,

c a r d a n d wood.

Hence,

e n e r g y r e c o v e r y f r o m w a s t e c a n b e m a x i m i s e d by s e p a r a t i n g t h e o r g a n i c material i n t o a putrescible fraction for and a n o n - d i g e s t i b l e

f r a c t i o n f o r combustion.

anaerobic d i g e s t i o n

-

310 -

GAS PRODUCTION H i s t o r i c a l l y , i n p r a c t i c a l systems, o r i n r e s e a r c h s t u d i e s , t h e r e h a s b e e n a t e n d e n c y t o o p t i m i s e s p e c i f i c g a s p r o d u c t i o n ( c u m/kg

t h e r e t e n t i o n time ( d e c r e a s i n g t h e l o a d i n g o r

increasing

VS) b y

dilution rate).

However, a s t h e r e t e n t i o n time i s i n c r e a s e d t h e r e i s

a corresponding increase i n t h e s i z e o f the vessel required t o deal w i t h a g i v e n d a i l y i n p u t o f raw m a t e r i a l .

As

per volume o f g a s p r o d u c e d w i l l i n c r e a s e .

Gas p r o d u c t i o n a l s o ref-

l e c t s the loading regime,

a result,

capital cost

composition of feed and temperature.

w i l l p r e f e r e n t i a l l y d i g e s t s o l u b l e components and l o w - l i g n i f i e d

ganic solids.

I n OF/M‘;W

l e f t as n o n - d i g e s t e d may br, d i g e s t e d

AD

or-

d i g e s t i o n o v e r 5 0 L o f t h e m a t e r i a l may b e

s o l i d s w h e r e a s 95-98 % o f pure v e g e t a b l e m a t t e r

An a p p r o x i m a t i o n o f t h e p o t e n t i a l g a s p r o d u c t i o n

c a n b e c a l c u l a t e d b y m u l t i p l y i n g t h e VS b y a n e x p e r i m e n t a l t h e o r e t i c a l s p e c i f i c g a s y i e l d , w h i c h f o r MSW may b e i n t h e r a n g e o f 0 . 3 5 0.75

c u m/kg

to

VS a d d e d ( F i g . 4 a ) .

-

b

[L

v , >

kJ ’01

a

I

,.=l

,”lf

..

?;

\.

~

.35 .3 0

0

CSTR

5

10

105 10 15 20 25 LOAOIG RATE IKG VSlrn31DAY I

8662-

I 0

*.

AND SIMILAR PLANT

,

,

,

15

20

25

-/ -. HIGH DIGESTERS THERMOPHILIC

’ SOLIDS

* MESOPHlLlC 1

5 10 15 20 25 LOADING RATE IKG VS/m3/DAY 1

F i g . 4 . a - S p e c i f i c b i o a a s y i e l d s a s FI f t i n c t i o n o f o r g a n i c l o a d i n g r a t e . b -Volumetric b i o g a s y i e l d s as a f u n c t i o n o f o r g a n i c l o a d i n g rate.

-

309

-

The d r y b a s i s HV o f o r g a n i c w a s t e s o f n a t u r a l o r i g i n w i t h a h i g h c a r b o h y d r a t e c o n t e n t i s a r o u n d 16-18

MJ/kg,

2 3 MJ/kg a s p r o t e i n o r o i l c o n t e n t i n c r e a s e s . c a r d e d m a t e r i a l d r o p s due t o w a t e r c o n t e n t .

i n c r e a s i n g towards

The a c t u a l HV o f d i s -

The HV o f MSW c a n t h u s

be d e t e r m i n e d by summation o f t h e a c t u a l H V s o f components c o r r e c t e d f o r p e r c e n t a g e c o m p o s i t i o n a n d w a t e r c o n t e n t a s shown i n F i g u -

r e 3 . I n t h e UK i t h a s b e e n e s t i m a t e d t h a t t h e HV f o r t y p i c a l r e f u s e i s between 7.5

and 9 GJ/tonne

MSW.

Assuming t h a t p l a n t t r i m -

mings and f o o d wastes are t h e main s u b s t r a t e s f o r a n a e r o b i c d i g e s t i o n and l a r g e l y c a r b o h y d r a t e ,

t h e n t h e r e a l i s a b l e energy p e r tonne

o f MSW w i l l r e f l e c t t h e d r y w e i g h t c o m p o s i t i o n r a t h e r t h a n w e t w e i g h t g i v i n g an e n e r g y c o n t r i b u t i o n f r o m p u t r e s c i b l e m a t e r i a l o b t a i n e d t h r o u g h AD o f

a r o u n d 2 G J p e r t o n n e MSW.

T h i s can be compared w i t h

t h e h e a t g e n e r a t e d b y i n c i n e r a t i o n o f a r o u n d 6 G J p e r t o n n e MSW,

or

t h a t r e c o v e r e d i n R O F p e l l e t s o f a r o u n d 7 GH p e r t o n n e MSW.

a

b

F i g . 3 . F r a c t i o n a l composition, energy c o n t e n t , m o i s t u r e c o n t e n t a n d d i s t r i b u t i o n o f e n e r g y b e t w e e n f r a c t i o n s o f MSW. a - P e r c e n t c o m p o s i t i o n o f MSW; b - S p e c i f i c e n e r g y c o n t e n t o f f r a c t i o n s ; c- Percentage moisture; d - Actual recoverable energy content o f e a c h f r a c t i o n ( L H V ) . From l e f t t o r i g h t i n e a c h f i g u r e 1. Food w a s t e s , 2 . P a p e r , 3 . C a r d , 4 . P l a s t i c s , 5. T e x t i l e s , 6 . R u b b e r , 7 . L e a t h e r , 8. P l a n t d e b r i s , 9. Wood.

- 311

I n general,

-

r a t e s o f gas p r o d u c t i o n a r e i n c r e a s e d as r e a c t o r

t e m p e r a t u r e i s i n c r e a s e d f r o m t h e m e s o p h i l i c r a n g e ( a r o u n d 3 5 C) t o the thermophilic

range (around 55 C).

The s o l i d s c o n t e n t o f t h e ma-

t e r i a l as l o a d e d i n t o t h e d i g e s t e r h a s a v e r y s i g n i f i c a n t e f f e c t on t h e volume o f lids;

t h e d i g e s t e r r e q u i r e d t o t r e a t a g i v e n w e i g h t o f so-

the higher the s o l i d s content o f feed t h e lower t h e d i g e s t i o n

volume r e q u i r e d .

A t t h e same t i m e t h e volume o f l i q u i d e f f l u e n t ge-

n e r a t e d i s s i m i l a r l y decreased.

Hence,

d e s i g n systems which can accept, l o g i c a l view,

i t i s clearly preferable t o

f r o m b o t h a t e c h n i c a l and m i c r o b i o -

a s h i g h an i n p u t c o n c e n t r a t i o n o f MSW a s p o s s i b l e .

W i t h i n t h e ranges o f V S c o n c e n t r a t i o n which do n o t i n h i b i t m i c r o b i a l a c t i v i t y t h e v o l u m e t r i c gas p r o d u c t i o n r a t e ( c u m methane/cu m d i g e s t e r volume)

w i l l also increase w i t h increase i n s o l i d s concentra-

tion. The o n l y way t o i n c r e a s e s o l i d s c o n t e n t w h i l s t m a i n t a i n i n g a constant

-

r e t e n t i o n t i m e i s t o increase the organic l o a d i n g r a t e (ORL

t h e q u a n t i t y o f o r g a n i c m a t t e r f e d p e r u n i t volume o f d i g e s t e r p e r

u n i t time;

k g VS/cu

m/day).

The ORL a n d t h e f e e d c o n c e n t r a t i o n d e -

f i n e t h e d e t e n t i o n t i m e f o r a g i v e n volume.

The d e t e n t i o n t i m e i s

c h a n g e d o n l y by c h a n g i n g t h e f e e d c o n c e n t r a t i o n .

However,

f o r any

p a r t i c u l a r r e t e n t i o n t i m e t h e r e w i l l be a l i m i t t o t h e l o a d i n g r a t e that i s practicable.

With c o n v e n t i o n a l pumps t h e maximum s o l i d s c o n -

t e n t i s l i m i t e d t o a r o u n d 1 0 t o 1 2 % .However, f e e d o f up t o 30 % s o l i d s may be l o a d e d .

u s i n g s l u r r y pumps,

Increase i n feed s o l i d s a t

a q i v e n H R T i n c r e a s e s t h e l o a d i n g r a t e u n t i l a t some u p p e r l i m i t a d d i t i o n a l biomass s u b s t r a t e w i l l

r e m a i n u n c r e a c t e d due t o d i f f u s i o n

o r m o t a b o l i c l i m i t a t i o n s . M e t h a n e p r o d u c t i o n p r o b a b l y becomes l i m i ted at solids

0.f

o v e r 3 0 7;.

However,

up t o t h i s l e v e l v o l u m e t r i c g a s

p r o d ~ c t i o nw i l l i n c r e a s e p r o p o r t i o n a l l y t o l o a d i n g r a t e a s shown i n F i g u r e 4b.

E N G I I E E R I N G AND S Y S T E M S D E S I G N T h r e e d i s t i n c t a p p r o a c h e s t o t r e a t i n g t h e OF/MSW c a n b e t a k e n . The f i r s t

i s t o use a d i l u t e s l u r r y o f t h e m a t e r i a l ,

w i t h sewage s l u d g e , red tank reactor

-

p o s s i b l y mixed

i n a conventional tank reactor (continuous s t i r CSTR).

The s e c o n d a p p r o a c h i s t o d i g e s t t h e ma-

t e r i a l a t a higher s o l i d s content,

possibly using a larger

o f t h e w a s t e i n c l u d i n g more p a p e r e t c .

fraction

The t h i r d o p t i o n i s t o p r e -

t r e a t t h e w a s t e i n o r d e r t o i n c r e a s e t h e amount o f s o l u b l e m a t e r i a l p r i o r t o d i g e s t i o n i n a second s t a g e r e a c t o r .

D e p e n d i n g on t h e e x -

-

-

312

t e n t o f s o l u b i l i s a t i o n t h i s second

r e a c t o r c o u l d b e any o f t h e

r e c o g n i s e d h i g h r a t e systems i n c l u d i n g CSTR, UASB,

anaerobic f i l t e r s ,

c o n t a c t systems o r f l u i d i s e d bed r e a c t o r s .

A l l t h r e e o p t i o n s have been i n v e s t i g a t e d i n e x t e n s i v e p i l o t p l a n t s t u d i e s or commercial p l a n t .

These i n c l u d e a ) C o n v e n t i o n a l

f u l l y m i x e d d i g e s t e r s R E F O R M (US); ( B e l g i u m ) and V a l o r g a ( F r a n c e ) ; (Japan).

b ) H i g h s o l i d s d i g e s t e r s Dranco c)

Two p h a s e d i g e s t e r s H i t a c h i

C o n v e n t i o n a l C S T R r e a c t o r s may be u s e d w i t h d i l u t e d s l u r -

r i e s p r o d u c e d u s i n g w a t e r o r sewage s l u d g e i n m e c h a n i c a l l y m i x e d d i -

X s o l i d s . O p e r a t e d a s a one = MRT (where HRT h y d r a u l i c re-

g e s t e r s o p e r a t i n g a t between 3 and 8 pass f u l l y mixed r e a c t o r HRT = SRT tention time,

SRT

=

t i o n t i m e ) . However,

s o l i d s r e t e n t i o n t i m e and MRT = m i c r o b i a l r e t e n l a r g e t a n k s may b e r e q u i r e d t o a c h i e v e t h e m i -

nimum SRTs o f b e t w e e n 1 5 a n d 30 d a y s i n t h e m e s o p h i l i c r a n g e a t a t e m p e r a t u r e o f 35 C.

Such p r o c e s s e s c a n o n l y b e u s e d w i t h OF/MSW i f

the material i s d i l u t e d f i r s t .

T h i s means t h a t f r o m 6 t o 1 0 c u m o f

w a t e r w i l l have t o be added p e r t o n n e o f o r g a n i c m a t e r i a l i n o r d e r t o reduce t h e T S c o n t e n t t o around 5 t o 10

X. T h i s w a t e r

w i l l have

t o be h e a t e d a n d w i l l i n t u r n f o r m an e f f l u e n t w h i c h w i l l r e q u i r e post-digestion

treatment.

The t e r m h i g h s o l i d s d i g e s t i o n , as i t i s s o m e t i m e s c a l l e d ,

or dry anaerobic fermentation

i s used t o d e s c r i b e systems i n which t h e

t o t a l s o l i d s c o n c e n t r a t i o n w i t h i n t h e d i g e s t e r i s more t h a n 20 t o

25 X .

Such d r y f e r m e n t a t i o n s h a v e a n a d v a n t a g e w h e r e t h e f e e d s t o c k ,

s u c h as MSW,

i s a v a i l a b l e w i t h m o i s t u r e c o n t e n t s o f l e s s t h a n 60 t o

7 0 X s i n c e t h e n e e d t o trdd w a t e r i s r e d u c e d . sed p o s t - d i g e s t e r

treatment costs,

This r e s u l t s i n decrea-

d e c r e a s e d r e a c t o r v o l u m e and t h e

p o s s i b i l i t y o f h i g h e r OLRs a t l o n g e r r e t e n t i o n t i m e s .

These i n c l u d e

s y s t e m s i n w h i c h t h e m a t e r i a l i s p a r t l y m i x e d as w e l l a s s y s t e m s w h i c h e s s e n t i a l l y work a s p l u g f l o w r e a c t o r s u n d e r t h e i n f l u e n c e o f gravity.

P l u g f l o w d i g e s t e r s t y p i c a l l y r e c e i v e f e e d a t one e n d

w h i l s t e f f l u e n t i s removed from t h e o t h e r .

MRT.

However,

and e f f l u e n t

Hence a g a i n H R T

SRT

=

such systems d i f f e r f r o m t h e CSTR i n t h a t i n f l u e n t a r e n o t mixed and hence u n d i g e s t e d m a t e r i a l i s n o t l o s t

a l t h o u g h t h e r e c a n b e some v e r t i c a l m i x i n g d u e t o g a s p r o d u c t i o n and some s o l i d s s e p a r a t i o n d u e t o g r a v i t y .

Consequently,

t h e SRT can

e x c e e d t h e HRT.

I t i s a l s o q u i t e common f o r t h e r e a c t o r t o become i n e f f e c t two stage,

w i t h h y d r o l y s i s and a c i d p r o d u c t i o n d c c u r i n g a t t h e

in-

-

313

-

f l u e n t end a n d m e t h a n o g e n e s i s a t t h e o u t f l o w .

T h i s can l e a d t o prob-

lems w i t h f e e d s t o c k s such as MSW s i n c e u n l e s s microorganisms a r e c o n t i n u a l l y s e e d e d b a c k i n t o t h e r e a c t o r m e t h a n o g e n s may wash o u t preferentially

causing decreased s t a b i l i t y .

A d d i t i o n o f sewage s l u d -

ge o r r e c y c l e o f t h e e f f l u e n t c a n h e l p overcome

such problems.

High

s o l i d s s y s t e m s c a n show v e r y h i g h r a t e s o f g a s p r o d u c t i o n r e f l e c t i n g t h e h i g h l o a d i n g r a t e s a n d a r e more r o b u s t due t o t h e f a c t t h a t c r u s t f o r m a t i o n a n d a c c u m u l a t i o n o f i n e r t s o l i d s d o e s n o t o c c u r a s i n mo-

re d i l u t e s t i r r e d tank reactors.

Systems have a l s o been r e s e a r c h e d

or designed i n which the o v e r a l l process o f conversion o f i n s o l u b l e p o l y m e r i c m a t e r i a l t o methane i s c a r r i e d o u t i n two o r more r e a c t o r s a r r a n g e d i n s e r i e s i n s u c h a way t h a t t h e p r o c e s s t a k e s p l a c e i n t w o microbiologically

independent phases.

Conditions i n the f i r s t

reactor are adjusted i n order t o o p t i -

m i s e t h e g r o w t h o f o r g a n i s m s w h i c h a r e c a p a b l e o f b r e a k i n g down b i o polymers with the release o f s h o r t chain f a t t y acids.

Since t h i s re-

s u l t s i n l i q u e f a c t i o n and a c i d o g e n e s i s t h e s e r e a c t o r s a r e sometimes r e f f e r e d t o as t h e liquefaction-acidfication

(LA)

phase.

The s o l u -

t i o n o f VFAs p r o d u c e d i s t h e n p a s s e d t o t h e s e c o n d p h a s e w h i c h may b e one o f any o f t h e s o - c a l l e d

h i g h r a t e second g e n e r a t i o n d i g e s t i o n

s y s t e m s where m e t h a n o g e n e s i s o c c u r s .

The a d v a n t a g e s o f two phase

systems r e l a t e t o i n c r e a s e d c o n v e r s i o n o f p o l y m e r i c m a t e r i a l and t h e f a c t t h a t t h e f i r s t s t a g e may be o f s i m p l e c o n s t r u c t i o n a n d l a r g e size,

o f t e n c o n s i s t i n g o f a simple container through which l i q u i d

i s p e r c o l a t e d b e f o r e p a s s i n g t o t h e second r e a c t o r .

A t one e x t r e m e

proposals t o recycle leachate through l i n e d l a n d f i l l s i t e s with the o u t f l o w p a s s i n g t h r o u g h a d i g e s t e r c a n be r e g a r d e d a s a l a r g e t w o phase p e r c o l a t i o n system. c u s s e d above.

The f i r s t p h a s e i s r a t e l i m i t i n g ,

I t i s possible t o pretreat the s o l i d material i n order

t o increase d i g e s t i b i l i t y , of

as d i s -

cellulolytic

or t o incubate the material with cultures

or l i g n o l y t i c f u n g i o r b a c t e r i a .

The r a n g e o f p r e -

t r e a t m e n t s w h i c h have been t r i e d w i t h v a r i o u s s t a r t i n g m a t e r i a l s , i n c l u d i n g a g r i c u l t u r a l r e s i d u e s and i n d u s t r i a l wastes, se w h i c h have been used i n t h e t r e a t m e n t o f s i m i l a r

order t o produce paper pulp,

resemble tho-

feedstocks i n

increase d i g e s t i b i l i t y o f animal

feeds

or generate a fermentation feedstock f o r f u e l a l c o h o l production These i n c l u d e ateam t r e a t m e n t s o f v a r i o u s t y p e s , a c i d and a l k a l i t r e a t m e n t s , etc.

steam e x p l o s i o n ,

t h e use o f hydrogen p e r o x i d e ,

solvents,

I n g e n e r a l t h e s e a p p r o a c h e s r e m a i n more o f a c a d e m i c i n t e r e s t

t h a n r e a l p r a c t i c a l a p p l i c a b i l i t y f o r two reasons.

The f i r s t r e l a t e s

-

314

-

t o c o s t s o f such t r e a t m e n t and t h e second t o p r o b l e m s o f e f f l u e n t treatment,

chemical n e u t r a l i s a t i o n etc.

ECONOMICS A t t e m p t s t o answer

t h e q u e s t i o n o f economic b e n e f i t s o f adding

an A D p l a n t t o a w a s t e d i s p o s a l f a c i l i t y h a v e b e e n made b y c o n s i d e r i n g t h e n e t cash b a l a n c e p e r c u b i c m e t r e o f d i g e s t e r volume, systems o p e r a t e d under v a r i o u s regimes,

for

which r e s u l t from various

energy s a l e p r i c e s w i t h v a r y i n g c a p i t a l c o s t s expressed i n terms o f p e r c u b i c m e t r e d i g e s t e r volume,

a s s u m i n g a m a r k e t v a l u e o f gas

o f b e t w e e n 1 0 and 3 0 p p e r t h e r m a n d a v a l u e f o r e l e c t r i c i t y o f b e t ween 2 a n d 6 p p e r KWh

(Fig.

5).

zoo] 1004

/

-100 I I 10 20 30 I PENCElTHERM 1

ELECTRICITY PRKE [plKWhl

401

z -40

- 80 -120

-60 -80 200 300 400 500

200 300

200

400 500

DIGESTER COST [ E l m 3

300 400 500

1

F i g . 5.. Changes i n t h e n e t a n n u a l c a s h f l o w , e x p r e s s e d i n t e r m s o f L/cubi m e t r e d i g e s t e r v o l u m e p e r annum, f o r s y s t e m s d e s i g n e d f o r the anaerobic digestion o f t h e organic f r a c t i o n o f municipal s o l i d waste. A ( t o p row) n e t cash f l o w as a f u n c t i o n o f f u e l p r i c e f o r s y s t e m s i n w h i c h an i n c l u s i v e c o s t o f f, 3 0 0 p e r c u b i c m e t r e o f d i g e s t e r c a p a c i t y i s assumed. B ( b o t t o m r o w ) n e t c a s h f l o w as a f u n c t i o n o f c a p i t a l c o s t s a t f i x e d gas p r i c e o f 15p p e r t h e r m o r e l e c t r i c i t y p r i c e o f 2 , 5 p/KWh. L e f t a n d r i g h t v e r t i c a l c o l u m n s a r e c a l c u l a t e d on t h e a s s u m p t i o n t h a t t h e s y s t e m i s h e a t e d f r o m an e x t e r n a l b o i l e r u s i n g non- d i g e s t i b l e m a t e r i a l o r w a s t e h e a t f r o m a C H P u n i t . C e n t r a l column g i v e s r e s u l t s f o r systems h e a t e d by b u r n i n g p a r t o f 1 = CSTR digester; 2 = mesophilic high s o l i d s digester t h e gas. and 3 = t h e r m o p h i l i c h i g h s o l i d s d i g e s t e r .

- 315 -

I n t h e s e c a l c u l a t i o n s g a s p r o d u c t i o n is e s t i m a t e d from t h e aver a g e v a l u e s u n d e r e a c h c o n d i t i o n d e r i v e d from d a t a shown i n f i g u r e 3 and 4.

For c o n v e n i e n c e a n i n t e r e s t r a t e o f 10 L o v e r a 1 5 y e a r li-

f e i s u s e d t o compute t h e a n n u a l c a p i t a l c h a r g e a s s o c i a t e d w i t h varying digester costs.

A d d i t i o n a l e s t i m a t e s were m a d e f o r o p e r a t i n g

c o s t s a n d t h e sum s u b t r a c t e d f r o m t h e r e v e n u e f r o m s a l e s o f n e t g a s

or e l e c t r i c i t y f o r d i f f e r i n g d i q e s t e r t y p e s , a s shown i n f i g u r e 5 , t o g i v e a n e t c o s t . C a l c u l a t i o n s assumed t h a t t h e d i g e s t e r producing e l e c t r i c i t y w a s h e a t e d u s i n g h e a t f r o m a CHP u n i t .

D i g e s t e r s produ-

c i n g g a s were a s s u m e d e i t h e r t o b e h e a t e d by g a s c o m b u s t i o n o r b y h e a t from i n c i n e r a t i o n i n a b o i l e r , ded.

t h e c o s t o f which was n o t i n c l u -

Obviously t h e d e t a i l e d f i g u r e s a r e site s p e c i f i c .

However,

this

g e n e r a l a n a l y s i s s u p p o r t s t h e c o n c l u s i o n s o f V e a l t h a t CSTR s y s t e m s

are u n l i k e l y t o be economic, b u t a t t h e same t i m e shows t h e p o t e n t i a l o f h i g h s o l i d s t h e r m o p h i l i c d i g e s t e r s . However, systems,

even with such

u n l e s s e n e r g y p r i c e s r i s e a b o v e t h e lower limits o f 1 5 p

p e r t h e r m o r 2 , 5 p p e r KWh, e c o n o m i c s a r e d e p e n d e n t o n a n a l l - i n

ca-

p i t a l c o s t o f d i g e s t i o n o f a r o u n d € 2 0 0 t o € 250 p e r c u b i c metre. CONCLUSIONS T h e g e n e r a l c o n c l u s i o n i s t h a t t h e d i g e s t i o n o f s l u r r i e d MSW w i t h sewage s l u d g e i s b a s i c a l l y an e s t a b l i s h e d technology.

However,

a t p r e s e n t t h e r e i s l e s s e x p e r i e n c e w i t h A D s y s t e m s b a s e d on h i g h e r s o l i d s and l o a d i n g r a t e s .

J u s t i f i c a t i o n f o r AD systems d e s i g n e d t o

p r o d u c e g a s or power f o r s a l e , r a t i n g energy f o r in-house

r a t h e r t h a n j u s t a s a means o f gene-

use i n a sorting/RDF

p l a n t , d e p e n d s on

assumptions r e l a t i n g t o t h e f u t u r e p r i c e o f energy a s well a s f u t u re methods o f d i s p o s a l o f wastes and/or

identification of additio-

n a l b e n e f i t s a s s o c i a t e d w i t h t h e r o u t e t h r o u g h f a b r i c a t e d AD systems. I n t h e s h o r t term p r o b a b l y t h e m o r e i m p o r t a n t f a c t o r s w h i c h a r e a l r e a d y p r o m p t i n g many w a s t e d i s p o s a l a u t h o r i t i e s t o c o n s i d e r t h i s o p t i o n r e l a t e t o waste disposal.

These i n c l u d e d e c r e a s i n g a v a i l a b i l i -

t y o f l a n d f o r w a s t e d e p o s i t , more s t r i n g e n t l e g i s l a t i o n o n c o n t r o l o f l e a c h a t e and g a s l e a k s from l a n d f i l l s i t e s l e a d i n g t o h i g h e r c o s t s , s i m i l a r i n c r e a s e d c o s t s of i n c i n e r a t i o n due t o t h e need t o i n s t a l l stack-gas

c l e a n u p , c h a n g e s i n l e g i s l a t i o n on r e l e a s e of

s l u d g e i n t o t h e s e a or t o l a n d and environmental c o n s i d e r a t i o n s i n general.

I n some c o u n t r i e s t h e e c o n o m i c c l i m a t e f o r s m a l l s c a l e po-

wer g e n e r a t i o n f r o m w a s t e s i s i m p r o v i n g , a s s o c i a t e d w i t h t h e c o n c e r n a b o u t n u c l e a r power on t h e o n e hand a n d c a r b o n d i o x i d e l e v e l s a n d

a c i d r a i n on t h e o t h e r .

316

-

A t t h e same t i m e i n c r e a s e d r e c y c l i n g o f com-

p o n e n t s o f MSW w i l l r e v e r s e t h e t r e n d w h i c h h a s b e e n s e e n o v e r t h e l a s t 20 y e a r s o r so,

w h e r e t h e amount o f b i o d e g r a d a b l e m a t e r i a l de-

c l i n e d as p l a s t i c s r e p l a c e d paper as t h e major packaging m a t e r i a l a n d p e o p l e w e n t f r o m p r e p a r i n g t h e i r own f o o d t o b u y i n g p r o c e s s e d food.

The c u r r e n t t r e n d t o w a r d s h e a l t h i e r e a t i n g ,

more v e g e t a b l e s

and b i o d e g r a d a b l e p a c k a g i n g w i l l a l l i n c r e a s e t h e p u t r e s c i b l e f r a c tion,

f a v o u r i n g AD.

I n t h e UK s u c h c o n s i d e r a t i o n s h a v e p r o m p t e d a

number o f c i t y c o u n s i l s ,

whilst the

UK

including Cardiff,

t o look at t h i s option

Department o f Energy i s f u n d i n g r e s e a r c h and d e v e l o p -

ment s t u d i e s a n d p l a n t .

E l s e w h e r e t h e E C E n e r g y Programme ( D G X V I I )

h a s f u n d e d a number o f d e m o n s t r a t i o n s a n d i n F r a n c e s e v e r a l m o r e

f u l l s c a l e p l a n t a r e planned.

LANDFILL GAS FUEL AN0 ECOLOGICAL PROBLEMS

2.

PIETRZYK

T e c h n i c a l U n i v e r s i t y o f Cracow,

1

POLAND.

INTRODUCTION Annual i n c r e m e n t o f r e f u s e g e n e r a t i o n and a i r and w a t e r p o l l u -

t i o n i s r e a l l y h i g h e r t h a n f o o d o r i n d u s t r i a l p r o d u c t i o n and g r o w t h o f the population i t s e l f (Fig.

1).

~~

WASTE

Fig.

PRODUCTION

INCREASE PROD

1. G r o w t h o f p r o d u c t i o n . A c i t i z e n i n a w e l l d e v e l o p e d c o u n t r y p r o d u c e s 1.5

o f m u n i c i p a l waste y e a r l y .

with time.

cubic meters

T h i s amount h a s a t e n d e n c y t o i n c r e a s e

Because o f t h e h i g h d e n s i t y o f t h e p o p u l a t i o n ,

municipal

w a s t e becomes a s e r i o u s p r o b l e m n o t o n l y f o r m u n i c i p a l i t i e s b u t a l s o f o r c i t i z e n s themselves. Waste w a t e r t r e a t m e n t i s i m p o r t a n t t o e n s u r e c l e a n r i v e r s a n d

water resources.

318

-

The p u r i f i c a t i o n p r o c e s s i s r e a l i z e d a l s o w i t h p o l -

l u t e d f l u e gases i n t h e industry. Solid municipal wastes a r e f a i r l y o f t e n not disposed of immediately.

They a r e m a i n l y l a n d f i l l e d . S t o c k s

o f t h e m g r o w p e r m a n e n t l y a n d we a r e t h r e a t e n e d w i t h a t i m e bomb.

Up

t o now t h e r e h a s b e e n a l a c k o f s u c c e s s f u l w a y s t o c o m p l e t e d e g r a d a t i o n , destroying or u t i l i z a t i o n of a l l t h e ingredients of t h e municip a l waste. The p r i n c i p a l ways o f d e a l i n g w i t h m u n i c i p a l w a s t e a r e : ling,

recyc-

i n c i n e r a t i o n , c o m p o s t i n g and l a n d f i l l i n g . Most o f t h e w a s t e

( U S - 9 5 % , UK-89 % ,F R G - 7 1 % ) i s l a n d f i l l e d . T h e r e m a i n i n g p a r t i s i n c i n e r a t e d ( U S - 5 X , UK-10 3

% ,F R G - 2 5

X). P a r t s o f t h e r e f u s e ( b e l o w 2 0

X) o r c o m p o s t e d ( U S - 1 X , %) l i k e g l a s s ,

rubber,

FRG-

plastics,

c e l l u l o s e and e s p e c i a l l y non f e r r o u s m e t a l s c a n be r e c y c l e d .

I t is

t h e most e f i c i e n t and e c o n o m i c a l way f o r s e c o n d a r y m a t e r i a l r e c o very

[ll.

he w a s t e i n c i n e r a t i o n p r o c e s s i s r e a l i z e d i n s p e c i a l f u r -

naces with heat recovery

f o r t h e s t e a m o r warm w a t e r p r o d u c t i o n .

Combustion is o f t e n supported with n a t u r a l f u e l s ( o i l ,

natural gas)

b e c a u s e o f h i g h h u m i d i t y and low c a l o r i f i c v a l u e o f r e f u s e . compounds

Waste

i k e PVC a n d f r e o n s ( f r o m s p r a y c a n s ) c r e a t e d i o x i n e s a n d

f u r a n e s i n combustion chambers. nents of f l u e gases.

T h e s e a r e t h e h i g h l y t o x i c compo-

Some y e a r s a g o e v e r y m a j o r t o w n h a d o n e o b j e c t

- t o b u i l d an i n c i n e r a t i o n p l a n t f o r municipal r e f u s e . S u i t a b l e l o c a t i o n s a r e commonly d i s c u s s e d . As

t h e b i o d e g r a d a t i o n methods a r e used:

aerobic composting i n

h e a t e d and a e r a t e d r e a c t o r s , a n a e r o b i c biodecomposition o f hydrocarb o n s i n dump c o n d i t i o n s .

The main p r o d u c t o f t h e a n a e r o b i c p r o c e s s

l a n d f i l l g a s c o n t a i n s methane and carbon d i o x i d e . nical progress,

I n s p i t e of tech-

t h e century old p r a c t i c e of l a n d f i l l i n g remains t h e

p r i n c i p l e means o f w a s t e d i s p o s a l .

The p o p u l a r i t y o f l a n d f i l l i n g i s

d u e t o low t e c h n o l o g i c a l r e q u i r e m e n t s , c o s t s o f t h i s method.

s i m p l i c i t y and low i n v e s t m e n t

The m a i n d r a w b a c k i s t h a t much l a n d i s n e e d e d

and i t i s g e t t i n g more a n d more d i f f i c u l t t o o b t a i n i t i n t h e v i c i nity of large cities.

L a n d f i l l i n g a s a means o f s o l i d w a s t e d i s p o s a l

is generally a nuisance t o the surrounding area a s is the recently

criticized incinerators. One o f t h e r e a s o n s why l a n d f i l l s a r e a p r o b l e m i s b e c a u s e o f t h e generation of landfill gas. 55-33

T h i s i s a m i x t u r e o f 44-66

?i C 0 2 a n d s o m e o d o r i n g r e d i e n t s .

% methane and

Due t o t h e h i g h c o n t e n t o f

m e t h a n e , when l a n d f i l l g a s m i x e s w i t h a i r t h e r e i s a d a n g e r o f f i r e and p o s s i b l e explosions.

The h a z a r d s o f e x p l o s i o n s e x t e n d o u t s i d e t h e

-

319

-

l a n d f i l l limits b e c a u s e t h e g a s c a n m i g r a t e u n d e r t h e s u r f a c e o f t h e e a r t h f o r hundreds

o f meters.

From e a c h k i l o g r a m o f w a s t e c a l c u l a t e d , t h e c a r b o n b a l a n c e t h e m3 o f g a s 1 2 1 . I n p r a c t i c e t h i s vam3 g a s / k g w a s t e e x t e n d e d f o r 20 y e a r s .

o r e t i c a l l y o b t a i n e d i s 0.26-0.41 l u e i s r e d u c e d t o 0.10-0.20

The c h a n g e o f g a s g e n e r a t i o n i n e x p l o i t a t i o n time i s shown i n [ 3 1 Fig.

2.

The s p e e d o f t h e g a s g e n e r a t i o n i s p r o p o r t i o n a l

ber o f b a c t e r i a and t h e i r a c t i v i t y , (humidity,

contents of antibiotics).

t o t h e num-

due t o the external conditions Absence o f oxygen i n biomass is

a very important factor f o r successful running of t h e decomposition process.

100

ma

I I

I

SUPPLY

STORAGE

TIME

0

Fig.

2.

Production of t h e l a n d f i l l g a s .

L a n d f i l l g a s is generated i n t h r e e s e q u e n t i a l s t a g e s and t h e p r o d u c t s o f e a c h one a r e u s e d a s a raw m a t e r i a l i n t h e n e x t . o f t h e c u r v e s f o r main g a s e o u s c o m p o n e n t s a r e shown i n F i g .

2

Trends 3.

U T I L I Z A T I O N OF T H E LANDFILL G A S To r e d u c e t h e r i s k o f e x p l o s i o n s t h e g a s i s e x t r a c t e d by a s y s -

tem o f w e l l s d r i l l e d i n t h e l a n d f i l l , w h i c h i s t h e n f i l l e d w i t h t h e 4 ) . The p i p e s c o n n e c t t h e wells w h i c h extract t h e g a s [ 4 1 . T h e w e l l s a r e 1 0 0 meters a p a r t ( 1 p e r h e c t a r e )

pebbles and a i r sealed (Fig.

-

320

-

I

f

Y

I

1

3 0

>

& z

0 t In 0 a

z

0 V

in

cn

a

a _J

4 LL

n z

a _I

TIME Fig.

3.

L a n d f i l l gas p r o d u c t i o n p a t t e r n .

DESTRUCTION TORCH Fig.

4.

UTILIZATION FUEL

FURNACE

OVEN

BOILER

CHP

L a n d f i l l gas p r o c e e d i n g

3

a n d e a c h y i e l d s an a v e r a g e 0 . 4

m / m i n o f gas.

ned i n a t o r c h o r i n f u r n a c e s .

The g a s a f t e r p u r i f i c a t i o n i s i n c i n e -

The r a w g a s c a n b e b u r -

r a t e d w i t h h e a t r e c o v e r y i n a b o i l e r or gas e n g i n e ( F i g . lers,

5).

I n boi-

p u r e g a s i s u s e d f o r warm w a t e r p r o d u c t i o n ( t e m p e r a t u r e

o r hot water (90 h i g h C02 c o n t e n t , c a l o r i c value 10

OC)

f o r central heating.

55

OC)

R e d u c i n g t o a minimum t h e

l a n d f i l l gas can b e q u a l i f i e d between gas L ( l o w

X C o g ) a n d g a s H ( h i g h c a l o r i c v a l u e 5 7;

i n t r o d u c e d i n t o n a t u r a l gas n e t w o r k .

C02)

and

-

321

-

Q

MUNICIPAL

( W A S T E WATER BARRIER) Fig.

5.

L a n d f i l l gas r e c o v e r y w e l l .

The p u r e g a s h a s a d v a n t a g e s a s a f u e l i n t h e i n t e r n a l c o m b u s t i on engine o r i n t h e gas t u r b i n e . o n l y w i t h gas, of

The c a r b u r e t o r e n g i n e c a n b e f e d

w h i l e a d i e s e l u n i t r e q u i r e s an a d d i t i o n o f 20-30

o i l f o r gas-air

and Power) system,

mixture ignition. o n l y 30

X

I n CHP ( C o g e n e r a t i o n o f H e a t

X o f t h e c h e m i c a l energy o f t h e gas i s b u t 40-60 X may b e r e c o v e d f r o m

u t i l i z e d f o r generating e l e c t r i c i t y ;

waste h e a t ( c o o l i n g water and e x h a u s t gases) and used f o r domestic purposes ( F i g .

6).

U t i l i z i n g t h e l a n d f i l l g a s i n an e n g i n e e n o u g h e n e r g y i s gener a t e d t o c o v e r e n e r g y demand a t t h e l a n d f i l l s i t e ( p r o p u l s i o n o f pumps a n d f a n s )

and e n a b l e s h o t w a t e r p r o d u c t i o n f o r c l e a n i n g t r u c k s

and c o n t a i n e r s .

From 1 h a o f l a n d f i l l i t i s p o s s i b l e t o o b t a i n

30 kWatta i n e l e c t r i c i t y

and a d d i t i o n a l l y 50 k W a t t s o f h e a t .

Elec-

t r i c i t y g e n e r a t i n g f r o m t h e l a n d f i l l g a s b y means o f e n g i n e a n d e l e c t r i c g e n e r a t o r i s e c o n o m i c a l when t h e a r e a o f l a n d f i l l i s a minimum o f 10 h a a n d a d e p t h 8-12 m e t e r s .

I f t h e gas i s b u r n e d i n water b o i l e r s , n i c i t y of approx.

t h e l a n d f i l l can be covered.

h e a t demand i n t h e v i -

From 1 h a (1 w e l l )

one o b t a i n s

100 k W a t t s o f h e a t w h i c h i s enough t o s u p p l y h e a t f o r c e n t r a l

h e a t i n g a n d warm w a t e r p r o d u c t i o n t o 6 s i n g l e f a m i l y h o u s e s .

Such b e -

-

322

-

n e n e f i t s i n c l u d e reduced p r i m a r y energy consumption f o r a g i v e n l e v e l o f economic o u t p u t ( F i g .

7).

EL.

EN.

CHP

Fig.

6 . L a n d f i l l gas i n s t a l l a t i o n w i t h e n e r g y r e c o v e r y .

Nevertheless,

u t i l i z a t i o n o f energy from t h e l a n d f i l l gas has

a m a r g i n a l i m p a c t on t h e e n e r g y b a l a n c e i n t h e c o m m u n i t y a n d t h e l o c a t i o n o f t h e l a n d f i l l n a t u r a l l y l i m i t s t h e number o f t h e p o t e n t i a l users.

The e c o l o g i c a l i m p a c t o f t h e p r o b l e m i s f a r m o r e s i g n i -

ficant.

L a n d f i l l gas

-

ecological problem

L a n d f i l l gas d e n s i t y i s s i m i l a r t o a i r , i s quite different.

but i t s optical density

T h i s causes t h e r e f r a c t i o n o f l i g h t and t h e r e f -

l e c t i o n o f r a y s on a i r - l a n d f i l l

gas i n t e r f a c i a l a u r f a c e .

I t i s possi-

b l e t h a t u n e x p l a i n a b l e c a r a c c i d e n t s d u r i n g f u l l d a y l i g h t may b e c a u s e d b y e n t e r i n g an a r e a w i t h a l i g h t c o n c e n t r a t i o n o f l a n d f i l l

gas.

-

323

-

300 (30 O/o 1

ELECTRICAL ENERGY 200

STATION I L8 % ) 100 SYSTEM GAS

I 0

1000

2000 V

FIG:

7.

(85%)

( 8 5 O/Ol

BOILER [ H E A T )

C o s t o f energy g e n e r a t i o n from

The a p p r o a c h i n g c a r c a n b e i n v i s i b l e ,

[nm3/h1

l a n d f i l l gas. as i f i t was u n d e r w a t e r .

This

phen0meno.n i n a d d i t i o n t o t h e l a c k o f c o n c e n t r a t i o n c a n l e a d t o f a t a l r e s u l t s (Fig.

6).

A

Fig.

8.

L a n d f i l l gas l a y e r a s a m i r r o r .

L a n d f i l l w i t h a i r c r e a t e s an e x p l o s i v e m i x t u r e .

For hydrogen-

a i r m i x t u r e t h e i g n i t i o n range i s between 4 and 7 5 % o f H 2 and t h e same f o r m e t h a n e i s 5 . 3

t o 16 X.

The s t o i c h i o m e t r i c d e t o n a t i o n r a n g e

f o r methane i s f i v e t i m e s s m a l l e r t h a n t h a t

for

hydrogen,

b u t the

i n c r e a s e o f p r e s s u r e i n t h e d e t o n a t i o n wave o f m e t h a n e i s 5 0 X h i g p2 h e r t h a n t h a t f o r hydrogen ( p r e s s u r e i n c r e a s e - f o r CH4 i s e q u a l 31

P1

and f o r H 2 i s o n l y 20).

Assuming t h a t l a n d f i l l gas c o n s i s t s o n l y o f q a r d i n g t h e o t h e r components,

CH4 a n d C02 d i s r e -

t h e f l u e gases a f t e r c o m b u s t i o n o f 1 m

o f g a s w i l l a l w a y s i n c l u d e 1 m3 o f C02. When t h e c o n c e n t r a t i o n o f m e t h a n e c h a n g e s f r o m 1 0 0 % t o 40

X i n t h e l a n d f i l l gas t h e maximal

3

-

324

-

C02 c o n c e n t r a t i o n i n d r y f l u e g a s e s i n c r e a s e s f r o m 1 1 . 7

t o 2 5 76.

T h a t i s much h i g h e r C02 c o n t e n t s t h a n f o r p u r e c a r b o n i n c i n e r a t i n g

o r f o r e n e r g e t i c c o a l ( 1 8 . 4 + 1 8 . 8 X).

( 2 1 7;)

T h i s i s h a r m f u l because o f t h e green house e f f e c t . s i n c e l a n d f i l l g a s w i t h o n l y 5 0 7; r i c value o f n a t u r a l gas.

Especially

o f methane h a s h a l f o f t h e c a l o -

I n o t h e r words,

one m u s t u s e t w i c e t h e

l a n d f i l l g a s t o a c h i e v e t h e same amount o f h e a t a n d t h e same i s p r o d u c e d t w i c e more C02 w h i c h s h o u l d b e l a u n c h e d i n t o t h e a t m o s p h e r e . Combustion o f l a n d f i l l gas i s l e s s h a r m f u l t o t h e e n v i r o n m e n t t h a n i n c i n e r a t i o n o f g o l i d m u n i c i p a l waste i t s e l f . contain PVC,

nor polyurethanes.

r i n e compounds a n d i n t h e c a s e o f drogen.

S u l f u r hydrogen,

The g a s d o e s n o t

The i n c i n e r a t i o n o f P V C y i e l d s c h l o p o l y u r e t h a n e s forms t h e cyano hy-

f r e o n s and f l u o r i n e hydrocarbons a r e t h e

most h a r m f u l s u b s t a n c e s i n l a n d f i l l gas. When t h e i n c i n e r a t i o n p r o c e s s i s n o t r e a l i z e d a t t o o h i g h temperatures, ted (Fig.

s t r o n g l y t o x i c b e n s o d i o x i n e and b e n s o f u r a n e s a r e genera-

9).

[ 5 1 These p r o d u c t s a r e v e r y s t a b l e a n d c a n b e decom-

posed o n l y t h r o u g h c o m b u s t i o n i n f u r n a c e s a t t e m p e r a t u r e s above 1300 'C.

T h i s i s why t h i s i s t h e o n l y way o f c o m b u s t i o n p e r m i t t e d i n

some c o u n t r i e s . cumstances,

L a n d f i l l gas c o n t a i n s ,

d e p e n d i n g on t h e l o c a l c i r -

1 5 t 2 0 0 mgs o f c h l o r i n e p e r m3 a n d t h e c h l o r i n e h y d r o -

c a r b o n compounds ( S e v e s o t o x i n e ) a r e t h e p r o m i n e n t e n v i r o n m e n t a l p r o b lems i n l a n d f i l l gas combustion,

a n d i s f a r m o r e h a z a r d o u s t h a n com-

m o n l y r e c o g n i z e d s u l f u r compounds.

DlBENZODlOXlNE

2 3.7.0

TCDD

( TE TRACHLORINEDIBENZODIOXINE

Fig.

DIBENZOFURANE

2 3 7 . 0 TCDF

1

9. D i o x i n e a n d f u r a n e t o x i n .

( TETRACHLORINEDIBENZO FURANE 1

-

-

325

When i n l a n d f i l l g a s t h e c o n t e n t s o f C02 i n c r e a s e s , t h e

amount

o f w a t e r s t e a m i n f l u e g a s e s d e c r e a s e s a n d t h e i r dew p o i n t w i l l b e lower.

I n flue

g a s e s f r o m p u r e m e t h a n e H20 m o l f r a c t i o n i s 0 . 1 9

t h e dew p o i n t 56 t o 0.4

-

OC,

and

when m e t h a n e c o n t e n t s i n l a n d f i l l g a s d e c r e a s e s

t h e dew p o i n t i s a t 53

OC.

I t makes e x p l o i t a t i o n o f c o n -

densing the appliance r a t h e r d i f f i c u l t b u t r e a l l y possible t o j e t . The c o n d e n s i n g a p p l i a n c e a s s u r e s h i g h e r t h e r m a l e f f i c i e n c y t h a n c l a s s i c gas u n i t .

T h e s e a r e u s e d a s d o m e s t i c and communal b o i l e r s

a n d warm w a t e r s h e a t e r s .

Gas c o n s u m p t i o n f o r t h e s e i s 1 0

-

15

X lo-

wer t h a n i t i s f o r c l a s s i c g a s u n i t s . Unfortunately

the sulfur,

c h l o r i n e a n d f l u o r i n e compounds f r o m

l a n d f i l l g a s go i n t o c o m b u s t i o n p r o d u c t s a n d i n t h e p r e s e n c e o f c o n densate they c r e a t e a very hazardous water s o l u t i o n . corrosive e f f e c t s o f the ingredients, l o w e r t h a n 25 mg/m

3

,

the total

Considering the

f l u o r i n e should be

c h l o r i n e l o w e r t h a n 5 0 mg/m3 a n d s u l f u r h y d r o -

g e n l o w e r t h a n 1 5 0 0 mg/m3

o f l a n d f i l l gas.

P r o b l e m number one f o r l a n d f i l l g a s i s e c o n o m i c a n d t h e r e l i a b i l i t y o f t h e c l e a n i n g and e n r i c h i n g process. means o f :

-

dry

I n t h e a d s o r p t i o n method,

I t can be r e a l i z e d by

or wet-absorption treatment.

a d s o r p t i o n way,

[ 6 1 u s i n g t h e a c t i v a t e d carbon and

t h e z e o l i n e o r t h e m o l e c u l a r membrane u n d e r p r e s s u r e 2 - 3 MPa a r e c a p t u r e d and removed under pressure

2-3

-

s u l f u r hydrogen,

p i p e l i n e s o f a gas s y s t e m .

C a r b o n a c t i v e i s r e g e n e r a t e d b y means o f

h i g h t e m p e r a t u r e w a t e r steam. using zeolite,

f r e o n s and carbon d i o x i d e

1 0 ) . Gas a f t e r p u r i f i c a t i o n f l o w s i n t o

MPa ( F i g .

Carbon d i o x i d e i s removed from t h e gas

and a f t e r two s t a g e s t h e a d s o r b e r t o t a l methane i n -

c r e a s e s t o 95 %. I n t h e a b s o r p t i o n p r o c e s s [ 6 1 c a r b o n d i o x i d e i s washed o u t f r o m l a n d f i l l g a s u s i n g p u r e w a t e r i n t h e p r e s s u r e s w i n g mode, c a l l y - i n amine b a t h ( F i g .

11).

o r chemi-

I n t h e p r e s s u r e swing method

-

car-

bon d i o x i d e i s t r e a t e d by water i n t h e a b s o r p t i o n tower under p r e -

s s u r e 1 MPa, a n d t h e n t h e s a t u r a t e d w a t e r i s d e c o m p r e s s e d i n t h e s t r i p p e r w h e r e C02 i s r e l e a s e d . t h e gas i n a c h e m i c a l b a t h .

O t h e r i n g r e d i e n t s a r e removed from

Compressed m e t h a n e f l i e s i n t o t h e c o l l e c -

tor. I n t h e system ( f o r l o w 5 mg/m3, 0.5

mg/m

3

communal u s e ) g a s H 2 S m u s t b e r e d u c e d t o b e -

C02 b e l o w 5 % ( m i n H gas)

. For

a n d c h l o r i n e compounds b e l o w

gas c l e a n i n g and e n r i c h i n g

method i s p r e f e r r e d .

-

the dry (adsorption)

-

STEAM

ZEO-

C ACT

LlTE

ADS

ADS

ADS

+

Fig.

Fig.

-

CACT

Fe

11

326

+

I

1 0 . Gas c l e a n i n g p l a n t ( d r y m e t h o d ) .

11. Gas c l e a n i n g p l a n t ( w e t m e t h o d ) .

-

327

-

CONCLUSIONS

L a n d f i l l gas i s t h e f u e l r e c e i v e d from s o l i d waste i n a v e r y s i m p l e and e c o n o m i c way i n a n a t u r a l r e a c t o r

-

landfill.

C o m b u s t i o n and u t i l i z a t i o n o f l a n d f i l l g a s i s , hazardous t h a n s o l i d waste i t s e l f , med i n t o g a s w i t h 4 0 t 6 0

as a r u l e ,

less

I n the bioprocess i t i s transfor-

X o f refuses hydrocarbons.

The p r o b l e m i s c o n n e c t e d w i t h g a s e o u s i n g r e d i e n t s ( c h l o r i n e , f l u o r i n e compounds) w h i c h a t l o w t e m p e r a t u r e s o f t h e c o m b u s t i o n p r o cess ( b e l o w 1300 furane.

OC)

cause p r o d u c t i o n o f h i g h l y t o x i c d i o x i n e and

By r e m o v i n g t h o s e ,

k i n g gas c l e a n i n g p l a n t ,

the a p p l i c a t i o n o f the economically

i s very important.

wor-

I n t h e l a n d f i l l g a s com-

b u s t i o n p r o c e s s more C02 i s p r o d u c e d p e r 1 kWh t h a n i n p u r e c a r b o n burning.

BIBLIOGRAPHY

1 2

3 4

5 6

7

R . P o r t e r a n d 1. R o b e r t s - E n e r Q y s a v i n q- b .y w a s t e s r e c y c l i n g , _. London 1985. W . Dahm - T e c h n i s c h e u n d w i r t s c h a f t l i c h e A s p e k t e d e r D e p o n i e gasnutzung. W i s s e n s c h a f t u. Umwelt 1986 - n r 3-4. Z. P i e t r z y k , T. S t y p k a Gaz w y s y p i s k o w y n i e k o n w e n c j o n a l n e ;r;dlo e n e r g i i . Gas Woda T e c h n i k a S a n i t a r n a 1 9 8 7 n r 6. R . L o o c k - A s p e k t e d e r D e p o n i e g a s n u t z u n g . Gas E r d g a s 1 9 8 8 n r 6. T . Weidenbach, I. K e r n e r a n d D . Radek D i o x i n d i e chemise Zeit-bombe Kiepenhauer W i t s c h K b l n 1984. K . Hedden - A u f b e r e i t u n g u n d N u t z u n g v o n D e p o n i e g e s . Gas E r d g a s 1987 n r 1 1 / 1 2 . K.H. L a u e r K l a r u n d D e p o n i e g a s a l s Ausgang p r o d u k t e z u r H e r s t e l l u n g v o n E r g a s - A u s t a u s c h g a s e n . Gas E r d g a s 1 9 8 7 n r 8.

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-

-

-

This Page Intentionally Left TBlank

BIOMASS AND THE PROBLEMS OF E C O L O G Y ,

E.S.

A G R O C H E M I S T R Y AND E N E R G Y

PANTSKHAVA

A.N. Each I n s t i t u t e o f B i o c h e m i s t r y , U S S R Academy o f S c i e n c e s , L e n i n s k y p r . 3 3 , Moscow, 1 1 7 0 7 1 , U S S R About 95 % o f modern f u e l

s o u r c e s f o r m e d a s a r e s u l t o f an-

c i e n t p h o t o s y n t h e s i s , and r e p r e s e n t t h e a n c i e n t biomass t h a t has been c o n v e r t e d t o f o s s i l f u e l energy t h r o u g h a v e r y complex geophysical,

g e o c h e m i c a l and b i o l o g i c a l p r o c e s s e s .

Thus, biomass

-

t h e r e a r e a number o f p o s s i b l e a p p l i c a t i o n s o f m o d e r n t h e m a j o r a c c u m u l a t o r o f s o l a r e n e r g y on t h e E a r t h

-

as

an a d d i t i o n a l f u e l s o u r c e . The n e c e s s i t y o f t h e d e v e l o p m e n t o f t h i s f u e l s o u r c e i s d i c t a t e d n o t o n l y b y e c o l o g i c a l p r o b l e m s s u c h as a t m o s p h e r i c p o l l u t i o n w i t h e x c e s s i v e C02 a n d h e a t e n e r g y r e s u l t i n g f r o m t h e c o m b u s t i o n o f organic fuels, fuels.

b u t m a i n l y by t h e i n c r e a s i n g d e f i c i e n c y o f f o s s i l

A new b r a n c h o f modern e n g i n e e r i g n g aimed a t s o l v i n g t h e two

problems,

i.e.

f u e l s u p p l y and e x t e r n a l e n v i r o n m e n t a l p r o t e c t i o n ,

called "bioenergetics".

I t is b o t h f u n d a m e n t a l

a r i s e n from modern b i o t e c h n o l o g y , gineering.

i s

and a p p l i e d s c i e n c e

chemical technology,

and power en-

B i o e n e r g e t i c s i s t h e s t u d y o f s o l a r energy b i o c o n v e r s i o n

i n t o b i o m a s s w h i c h d e v e l o p s ways o f b i o l o g i c a l a n d t h e r m o c h e m i c a l t r a n s f o r m a t i o n o f biomass t o f u e l and energy

(1).

The i n t r o d u c t i o n o f a c h i e v e m e n t s i n b i o e n e r g e t i c s i n t o t h e n a t i o n a l economy d e p e n d s ,

f ir st of all,

on t h e s o l u t i o n o f t h e t a s k s

connected with i n t e n s i f i c a t i o n o f o r g a n i c m a t e r i a l c o n v e r s i o n t o f u e l and w i t h l a r g e - s c a l e

p r o d u c t i o n o f biomass.

O n l y i n t e n s i v e and f u n d a -

mental research i n t o the problem w i l l enable a h i g h l y - e f f i c i e n t b r a n c h o f t h e n a t i o n a l economy t o meet t h e r e q u i r e m e n t s o f s c i e n t i f i c and t e c h n i c a l p r o g r e s s t h a t w i l l be e s t a b l i s h e d .

The s o l u t i o n

o f p r o b l e m s o f b i o e n e r g e t i c s d e p e n d s on t h e f o l l o w i n g :

-

t h e p r o s p e c t f o r and development o f h i g h l y - p r o d u c t i v e o f biomass,

e.g.

photosynthesis,

dustrial cultivation o f plants

-

sources

p r o d u c t i o n o f woody b i o m a s s , production of

hydrocarbons,

inpro-

I O

/B

Liquid hydrocarbons, plant oils, latex

i

A

55

Industrial processing

F oa on du sf at uc ft uf sr e ad n gd o o d s m Liquid

Agricultural processing

-

Astuffs

\

olid

\

Therrnochemical

1. L i q u i d f u e l : e t h a n o l , b u t a n o l , 2,3-butandiol, acetone, organic acids 2. Gaseous f u e l :

biogas, hydrogen

3 . Heat e n e r g y r e l e a s e d d u r i n g biooxidation

1. D i r e c t c o m b u s t i o n e l e c t r i c power

-

heat energy

conversion

-

w

2 . P y r o l y s i s - s o l i d f u e l ( c o a l ) , l i q u i d and gaseous f u e l (synthesis gas) 3 . G a s i f i c a t i o n - p r o d u c t i o n o f s y n t h e s i s gasa r t i f i c i a l p e t r o l and methanol 4 . Liquefaction (methanol)

-

5. F a s t - p y r o l y s i s

p r o d u c t i o n of l i q u i d f u e l

-

production of l i q u i d fuel

-

6 . Synthesis - gasification i n t h e presence o f catalysts production o f liquid fuel (methanol, a r t i f i c i a l p e t r o l )

Fig.

I

1. P r o d u c t i o n o f F u e l a n d E n e r g y f r o m B i o m a s s by B i o c o n v e r s i o n a n d T h e r m o c h e m i c a l C o n v e r s i o n .

w

0 I

d u c t i o n o f non-vood

331

-

biomass, growth of aquatic biomass,

and t h e

u t i l i z a t i o n of municipal wastes;

-

biotechnological conversion, i.e.

p r o d u c t i o n o f e t h a n o l or o t h e r

a l c o h o l s , o r g a n i c a c i d s , s o l v e n t s , b i o g a s and hydrogen from d i f f e rent kinds of biomass;

-

t h e r m o c h e m i c a l c o n v e r s i o n ( d i r e c t c o m b u s t i o n , g a s i f i c a t i o n , PY r o lysis, liquefaction, fast-pyrolysis, synthesis) t o produce iquid s o l i d and gaseous fuels.

(Figure)

T h e b i o m a s s c o n t e n t i n t h e b i o s p e r e i s e s t i m a t e d a t 8.10

tons

9 0 7; o f t h i s i s a c c o u n t e d f o r b y w o o d . A n n u a l l y 2.1011 t o n s a r e r e n e w e d , o f w h i c h 7.1010 t o n s a c c u m u l a t e i n c o n t i n e n t a l f o r e s t s w i t h a t o t a l e n e r g y c o n t e n t e x c e e d i n g t h r e e times o f t h e u p - t o - d a t e (21,

world energy consumption.

I t is noteworthy t h a t prospected r e s e r v e s of coal a r e estima11 o f o i l - 2.10l1 t o n s , o f n a t u r a l g a s - 1 . 1 0

t e d a t 5.1011 t o n s ,

tons (coal equivalent)

(2).

I n a number o f d e v e l o p e d a n d d e v e l o p i n g c o u n t r i e s , b i o m a s s i s a l r e a d y used t o produce f u e l and energy. About 1 b i l l i o n people i n t h e w o r l d u s e wood a s f u e l ; t h e o n e - s e v e n t h p a r t o f t h e e n e r g y u s e d o r i g i n a t e s from biomass ( 3 ) .

I n such developing c o u n t r i e s a s Ethio-

p i a , Nepal and T a n z a n i a , power e n g i n e e r i n g d e p e n d s on b i o m a s s u t i l i z a t i o n b y 9 5 %,:i n N i g e r , S o m a l i a a n d S u d a n b y 8 5 7;;

i n Bangladesh

a n d K e n y a b y 7 5 X , i n T h a i l a n d b y 6 5 X ; i n I n d i a b y 5 5 Z; i n B o l i v i a b y 45 %;

i n C h i n a b y 35 X ; i n B r a z i l by 2 5 L ( 3 ) .

I n 1 9 8 3 B r a z i l p r o d u c e d 6.3 m i l l i o n t o n s o f e t h a n o l f r o m s u g a r cane wastes,

a n d i n t h e s a m e y e a r i n B r a z i l , 9.3 m i l l i o n c a r s r a n o n

g a s o h o l , a mixture of p e t r o l (80 L) and e t h a n o l (20

XI, o f which

1 m i l l i o n c a r s used gasohol c o n t a i n i n g e t h a n o l with 5 Z o f water. B i o m a s s a s a n e n e r g y s o u r c e i s a l s o u s e d i n some d e v e l o p e d coun-

3.5 X , i n C a n a d a - 5 X , i n Swe17 X , a n d i t i s c o n s i d e r e d t o b e a v e r y den - 10 X, and i n F i n l a n d p r o i n i s i n g e n e r g y s o u r c e . By t h e y e a r 2000, i t s c o n t r i b u t i o n t o t h e US n a t i o n a l e n g i n e e r i n g p o w e r i s e x p e c t e d t o i n c r e a s e u p t o 4-5 Z, w h i c h w i l l make u p 156 m i l l i o n t o n s o f i d e a l f u e l . tries, e.g.

i n t h e United S t a t e s

-

-

The US D e p a r t m e n t o f P o w e r , E n g i n e e r i n g a n d A g r i c u l t u r e r e p o r t e d t h a t i n 1 9 8 2 1.6.1Ol5

B t u were p r o d u c e d f r o m b i o m a s s ,

p l a n s t o g e t u p t o 4.lOl5 B t u .

i n o r d e r t o r e p l a c e l i q u i d f u e l , e q u i v a l e n t l y t o &.lo3

f o r w h i c h 1 5 2 m i l l i o n b u s h e l s o f g r a i n were u s e d , used a s a fodder ( 3 ) .

a n d i n 1990 i t

The U n i t e d S t a t e s p r o d u c e d e t h a n o l , E t u i n 1983,

the protein being

-

332

I n t h e European Communities,

-

t h e c o n t r i b u t i o n o f biomass t o po-

wer e n g i n e e r i n g by 1990 s h o u l d make u p t h e same f i g u r e , e n a b l e o i l i m p o r t s t o be r e d u c e d by 100 m i l l i o n t o n s , o f 10 b i l l i o n U S 0 p e r annum.

P r i o r t o 1983,

which w i l l

w i t h savings

t h e EEC a l l o t t e d u p t o

5 m i l l i o n ECU a n n u a l l y t o s o l v e t h e problem o f f u e l p r o d u c t i o n from biomass,

while

f r o m 1 9 8 4 t o 1987 t h e y a s s i g n e d u p t o 1 3 0 m i l l i o n

ECU. The West German g o v e r n m e n t p l a n n e d t o p r o d u c e 5.105 e t h a n o l w h i c h was a d d e d ( 2 . 5

%)

t o motor f u e l .

tons o f

The b i o m a s s c o n t r i -

b u t i o n t o n a t i o n a l p o w e r e n g i n e e r i n g o f West Germany h a d t o b e 2.5

X

o f t h e t o t a l i n 1985. G r e a t B r i t a i n e x p e c t s t h e same l e v e l t o b e r e a c h e d by 1 9 9 0 , and b i o g a v w i l l have t o c o v e r a l l t h e energy needs o f a g r i c u l t u r e . The F r e n c h g o v e r n m e n t p l a n n e d t h a t b e g i n n i n g f r o m 1985 i t w o u l d u t i l i z e 14-15 m i l l i o n t o n s o f s t r a w a n n u a l l y t o produce m e t h a n o l f o r a g r i c u l t u r a l vehicles.

I n the 21st century,

France e x p e c t s a l l t h e energy t o be d e r i -

ved f r o m r e n e w a b l e s o u r c e s ,

w i t h b i o m a s s m a k i n g u p 25 X.

A c c o r d i n g t o A p p l i e d Power T e c h n o l o g y I n c .

(California),

the

d r o p o f o i l p r i c e s o b s e r v e d i n 1 9 8 5 - 1 9 8 6 on t h e w o r l d m a r k e t w i l l hardly i n f l u e n c e the production o f bioenergy. The i n t e r e s t i n b i o m a s s as a n e n e r g y s o u r c e i s e x p l a i n e d b y the following facts:

-

biomass i s renewable; energy

s t o r e d i n biomass can be p r e s e r v e d and used d u r i n g a l o n g

period;

-

i t can be c o n v e r t e d t o d i f f e r e n t f u e l s ;

-

many m o d e r n t e c h n o l o g i e s o f b i o e n e r g y p r o d u c t i o n a v a i l a b l e

for

i m m e d i a t e a p p l i c a t i o n have a l r e a d y been d e v e l o p e d and t e s t e d ,

-

biomass p r o d u c t i o n i s a v e r y p r o m i s s i n g b r a n c h o f power e n g i n e e ring;

-

v a r i o u s t y p e s o f o r g a n i c wastes can be used e f f i c i e n t l y ; f o r some r e g i o n s ,

b i o m a s s i s more a d v a n t a g e o u s when c o m p a r e d t o

c o n v e n t i o n a l f u e l s or i t i s even t h e b a s i c t y p e o f e n e r g y ;

-

b i o f u e l i s e c o l o g i c a l l y advantageous,

s i n c e n o h a r m f u l gaseous

s u l p h u r o x i d e s form and t h e C o g b a l a n c e i n t h e b i o s p h e r e i s n o t affected. However,this disadvantages:

new b r a n c h o f p o w e r e n g i n e e r i n g h a s a number o f

-

333

-

l a r g e l a n d areas a r e r e q u i r e d t o produce t h e biomass, water,

fertilizers,

as w e l l as

etc;

-

t h e b i o f u e l c o s t v a r i e s w i t h i n a wide range,

-

o n l y l o c a l u t i l i z a t i o n o f biomass i s p r o f i t a b l e ;

and sometimes i t i s

s i g n i f i c a n t l y higher than t h a t o f conventional fuels;

biomass,

as a r u l e ,

c o n t a i n s more t h a n 5 0

X

water,

which makes

t h e t e c h n o l o g y and i t s c o n v e r s i o n t o f u e l and energy more expen-

si ve ;

-

b i o m a s s p r o d u c t i v i t y s i g n i f i c a n t l y depends o n a g r i c u l t u r a l c l i m a t i c conditions;

-

some t y p e s o f b i o m a s s a r e s e a s o n a l ; e f f i c i e n c y o f p h o t o s y n t h e s i s i s v e r y low; some t e c h n o l o g i e s o f t h e b i o m a s s c o n v e r s i o n t o f u e l a r e s t i l l in e f f ic i e n t ;

-

b i o m a s s g r o w i n g r e q u i r e s some c h a n g e s i n f a r m i n g a n d f o r e s t r y m e t hods;

-

i t i s more d i f f i c u l t t o s t o r e b i o m a s s t h a n o i l o r n a t u r a l gas.

Amounts and t y p e s o f b i o m a s s - d e r i v e d the

f u e l s depend n o t o n l y on

b u l k o f r e n e w a b l e b i o m a s s b u t a l s o on i t s q u a l i t y ,

composition o f organics,

physical characteristics,

i.e.

Biomass i n v o l v e s a l l k i n d s o f p l a n t and a n i m a l t i s s u e s , d u c t s o f human a n d a n i m a l m e t a b o l i s m , I n t h e f o r t h c o m i n g 10-15

dampness,

etc. pro-

and o r g a n i c wastes.

y e a r s v a r i o u s o r g a n i c w a s t e s seem t o

b e m a j o r r a w m a t e r i a l t o p r o d u c e f u e l and e n e r g y ,

since i n developed

c o u n t r i e s a b o u t 5 t o n s o f o r g a n i c wastes ( d r y w e i g h t ) p e r head accum u l a t e i n one y e a r .

F u r t h e r i n t e n s i f i c a t i o n and u r b a n i z a t i o n w i l l

causes h i g h e r c o n c e n t r a t i o n o f w a s t e s , w h i c h w i l l r e q u i r e , o n t h e one hand,

u r g e n t ways t o u t i l i z e t h e m and d e v e l o p t h e m i n o r d e r t o

p r o t e c t the e x t e r n a l environment. t i o n o f promising high-efficient

I t w i l l also require the introduct e c h n o l o g i e s o f biomass p r o c e s s i n g

w i t h p o s s i b l e secondary a p p l i c a t i o n s i n p a r t i c u l a r , gy,

o r g a n i c and m i n e r a l f e r t i l i z e r s ,

t o produce ener-

and o t h e r v a l u a b l e p r o d u c t s .

B i o m a s s c a n b e t r a n s f o r m e d t o f u e l a n d e n e r g y by b i o l o g i c a l a n d thermochemical conversion. The f o l l o w i n g

two methods o f b i o l o g i c a l c o n v e r s i o n e x p e c t t o

be d e v e l o p e d i n t h e n e a r f u t u r e ,

namely,

and e t h a n o l ,

first,

which i s explained,

o f the fuels;

second,

technologies;

and f i n a l l y ,

the production o f biogaa by p h y s i c a l c h a r a c t e r i s t i c s

b y t h e r e l a t i v e s i m p l i c i t y and e f f i c i e n c y o f by s p e c i f y i n g t h e p r o c e s s e s w h i c h e f f i -

c i e n t l y r e n d e r wastes which a r e h a r m f u l f o r t h e f o l l o w i n g u t i l i z a t i o n .

I n most c o u n t r i e s , tion,

-

334

p r e f e r e n c e w i l l be g i v e n t o b i o g a s p r o d u c -

coming m a i n l y f r o m a g r i c u l t u r a l ,

i n d u s t r i a l and m u n i c i p a l was-

tes. C o n v e r s i o n o f wastes from p l a n t g r o w i n g and s t o c k - r a i s i n g

could

s u p p l y 10 76 o f t h e w o r l d a n n u a l e n e r g y n e e d s ( 4 ) . Biogas, de ( 3 0

composed o f m e t h a n e ( o n a v e r a g e 7 0 X ) a n d c a r b o n d i o x i -

X ) i s a product

n i c wastes.

o f anaerobic b a c t e r i a l decomposition o f orga-

Biogas competes w i t h t h e c a l o r i f i c v a l u e ( a b o u t 5.5-6.0 3 ) w i t h t h e b e s t t y p e s o f c o n v e n t i o n a l f u e l s , such

thousand kcal/m

as k e r o s e n e ( 9 0 0 0 k c a l / l ) ,

c o a l (7000 k c a l / k g ) ,

k g ) , b u t a n e 11 0 0 0 k c a l / k g ) ,

f i r e w o o d (5000 k c a l /

and manure b r i q u e t t e s ( 2 0 0 0 k c a l ( g )

(4). Methane f e r m e n t a t i o n o r b i o g a s p r o d u c t i o n p r o c e e d s i n temperat u r e s r a n g i n g f r o m 10-15' mesophilic used.

(30-40')

o r even a t

t o 55-60'

and t h e r m o p h i l i c

(55-60')

looo,

b u t as a r u l e

processes are widely

Methane f e r m e n t a t i o n i s c a r r i e d o u t i n m e t h a n e t a n k s w i t h a v o -

l u m e o f 1 t o some t h o u s a n d c u b i c m e t r e s . One t o n o f f e r m e n t e d o r g a n i c w a s t e s ( d r y w e i g h t ) g i v e s f r o m 2 0 0 t o 500-600

m3

o f biogas.

I n d u s t r i a l b i o g a s p r o d u c t i o n f r o m o r g a n i c w a s t e s h a s a number o f advantages:

-

b i o g a s p r o d u c t i o n i s a c c o m p a n i e d by e f f i c i e n t t r e a t m e n t o f sewage; o r g a n i c w a s t e s a r e c o n v e r t e d t o v a l u a b l e f u e l a n d t h e i r amount i n wastewaters decreases 1 0 times;

anaerobic decomposition i s a t the

same t i m e s a n i t a r y t r e a t m e n t o f w a s t e w a t e r s , se d e r i v e d f r o m a n i m a l husbandry

s t r o y s h e l m i n t h eggs,

-

i n p a r t i c u l a r o f tho-

and m u n i c i p a l sewage,

w h i c h de-

p a t h o g e n i c m i c r o f l o r a a n d weed s e e d s ;

anaerobic treatment o f sludge,

a n i m a l h u s b a n d r y and p l a n t g r o w i n g

w a s t e s r e s u l t s i n t h e m i n e r a l i z a t i o n o f n i t r o g e n and p h o s p h o r u s t h e main components o f f e r t i l i z e r s , be preserved,

-

and a l l o w s t h e s e compounds t o

i n c o n t r a s t t o t h e t r a d i t i o n a l way o f o r g a n i c f e r t i -

l i z e r p r o d u c t i o n by c o m p o s t i n g ,

when a b o u t 3 0 - 4 0

X o f the n i t r o -

gen i s l o s t ;

-

e f f i c i e n c y o f c o n v e r s i o n o f o r g a n i c w a s t e s t o b i o g a s is v e r y h i g h

-

b i o g a s c a n b e u s e d w i t h h i g h e f f i c i e n c y as a f u e l or c a n b e t r a n s -

i n t h e c a s e o f methane f e r m e n t a t i o n a n d r e a c h e s up t o 8 0 - 9 0

f o r m e d w i t h t h e h e l p o f g a s p r o d u c e r s w i t h an e f f i c i e n c y o f e l e c t r i c p o w e r ( 3 3 ?A)

5; 83 %

o r h e a t energy ( 5 0 X ) and can be used i n i n -

t e r n a l conbustion engines;

-

b i o g a s p l a n t s c a n b e i n s t a l l e d i n any r e g i o n s a n d do n o t r e q u i r e e x p e n s i v e gas p i p e - l i n e s .

- 335 -

N o w a d a y s b i o g a s is s u c c e s s f u l l y p r o d u c e d i n a n u m b e r o f c o u n -

tries. F o r i n s t a n c e , China had about 7 m i l l i o n b i o g a s p l a n t s w i t h a v o l u m e o f 8 - 1 0 m 3 i n 1 9 8 3 , i . e . w i t h a t o t a l v o l u m e o f a b o u t 600 m i l 3 lion m T h i s e n a b l e d a b o u t 90-100 b i l l i o n t o n s o f b i o g a s t o b e p r o -

.

d u c e d a n n u a l l y or up t o 80 m i l l i o n t o n s o f i d e a l f u e l t o b e r e p l a c e d , and a b o u t 200-250 m i l l i o n t o n s ( d r y w e i g h t ) o f o r g a n i c w a s t e s t o be processed.

This p r o v i d e s 30 m i l l i o n p e a s a n t s w i t h f u e l . By t h e

year 2000, C h i n a p l a n s t o c o n s t r u c t 30 m i l l i o n b i o g a s p l a n t s t o p r o d u c e up t o 4 0 0 b i l l i o n m3 o f b i o g a s .

Besides s m a l l b i o g a s p l a n t s Chi-

na h a s 36000 l a r g e p l a n t s . I n d i a had 58000 f a m i l y p l a n t s i n 1985 and planned t o c o n s t r u c t an

e x t r a 400000 p l a n t s . I n t h e European Communities, 41 p l a n t s operated i n 1978; i n 1983,

5 7 1 p l a n t s p r o c e s s e d l i q u i d w a s t e s a n d 1 7 p l a n t s c o n v e r t e d rnunicip a l g a r b a g e t o b i o g a s ( u p t o 100 m i l l i o n m3

p e r annum).

Large biogas p l a n t s a r e widely used i n the United S t a t e s . p r o c e s s m u n i c i p a l g a r b a g e t o p r o d u c e up t o 100 m i l l i o n m3

They

of biogas

annually. T h e S o v i e t U n i o n h a s a g o o d p o t e n t i a l i n terms o f r a w m a t e r i a l and t e c h n i c a l p r o v i s i o n t o produce s i g n i f i c a n t amounts of b i o g a s , a n d t h i s w i l l e n a b l e f o s s i l f u e l s t o b e r e p l a c e d i n some i n d u s t r i e s . I n t h e S o v i e t Union,

t h e m a i n wastes t o b e u t i l i z e d a r e m u n i c i -

p a l sewage and garbage, w a s t e s of a g r i c u l t u r e and wood-workings.

Va-

r i o u s i n d u s t r i e s a n n u a l l y g i v e m o r e t h a n 500 m i l l i o n t o n s o f o r g a n i c wastes (dry weight), which s t o c k - r a i s i n g

i n c l u d i n g a g r i c u l t u r e - 360 m i l l i o n t o n s , o f g i v e s 230 m i l l i o n t o n s and p l a n t - g r o w i n g

g i v e s 130 m i l l i o n t o n s ; w o o d - w o r k i n g bage

-

weight)

-

70 million tons;

60 m i l l i o n t o n s ; m u n i c i p a l sewage

-

which

municipal gar-

10 m i l l i o n t o n s ( d r y

(1).

T h e p r o c e s s i n g o f a s i g n i f i c a n t p a r t o f t h e o r g a n i c w a s t e s by m o d e r n t e c h n o l o g i e s c o u l d g i v e u p t o 1 2 0 b i l l i o n m3 o f b i o g a s p e r annum o r r e p l a c e up t o 1 0 0 m i l l i o n t o n s o f i d e a l f u e l . t h e coming 20-25

However,

in

years it w i l l be possible i n f a c t t o produce only

1 5 - 1 6 b i l l i o n rn3 o f b i o g a s p e r a n n u m ,

a t l a r g e s t o c k - r a i s i n g and po-

u l t r y farms and i n c i t i e s , where i t i s n e c e s s a r y t o t r e a t sewage. As a r u l e , b i o g a s p l a n t s p a y

f o r t h e m s e l v e s f o r 3-5

years.

The l a r g e r

t h e p l a n t , t h e higher its economic e f f i c i e n c y . A n i m a l m a n u r e i s t h e most w i d e l y s p r e a d t y p e o f o r g a n i c w a s t e t h a t r e q u i r e s e f f i c i e n t methods t o u t i l i z e it under t h e c o n d i t i o n that it preserves the fertilizing capacity.

I n t h e S o v i e t Union,

336 -

i t i s p o s s i b l e t o p r o d u c e up t o 100 b i l l i o n

m3 o f b i o g a s p e r annum, w h i c h makes up 1 0 % o f t h e a n n u a l m e t h a n e production. Besides, organo-mineral from o r g a n i c wastes.

f e r t i l i z e r s can a l s o be obtained

O f t h e m e n t i o n e d amount of s t o c k - r a i s i n g

wastes,

i t i s p o s s i b l e t o r e t u r n t o a g r i c u l t u r e o f which up t o 5 m i l l i o n t o n s of n i t r o g e n ,

2.5 m i l l i o n t o n s o f phosphorus o x i d e , and 1 0 m i l l i -

on t o n s o f p o t a s s i u m o x i d e , e n a b l e s t h e p r o d u c t i o n o f m i n e r a l fert i l i z e r s to be reduced. Municipal garbage and sewage a r e i m p o r t a n t s o u r c e s f o r b i o g a s production.

I n l a r g e c i t i e s i n t h e S o v i e t Union, b i o g a s p l a n t s a r e

c o m p o n e n t s o f systems f o r w a s t e w a t e r t r e a t m e n t .

For i n s t a n c e , bio-

g a s p l a n t s i n Moscow a n n u a l l y p r o d u c e u p t o 1 0 0 m i l l i o n m 3 o f b i o g a s which c o v e r s t h e h e a t and energy needs o f t h e p l a n t s themselves. P o t e n t i a l i t i e s of p r o c e s s i n g s e d i m e n t s and s l u d g e from t h e municip a l s e w a g e o f l a r g e S o v i e t c i t i e s o n l y a r e 700-1000 m i l l i o n m3

of

b i o g a s p e r annum. As w a s m e n t i o n e d a b o v e ,

some c o u n t r i e s s u c c e s s f u l l y p r o d u c e

b i o g a s from m u n i c i p a l g a r b a g e .

P o t e n t i a l i t i e s o f t h e S o v i e t Union

b i o g a s p r o d u c t i o n f r o m t h i s t y p e o f waste a m o u n t s u p t o 20 b i l l i o n

m 3 p e r annum. I m p o r t a n t raw m a t e r i a l f o r b i o g a s p r o d u c t i o n i s f r o m t h e wast e s o f wood-workinq,

wood-pulp

and the paper industry, microbiolo-

g i c a l and food i n d u s t r i e s . P o t e n t i a l i t i e s o f t h e S o v i e t Union f o r p r o c e s s i n g a l l t h e org a n i c w a s t e s formed a n n u a l l y u s i n g modern m e t h o d s o f b i o l o g i c a l a n d t h e r m o c h e m i c a l c o n v e r s i o n a r e e s t i m a t e d a t 150 m i l l i o n t o n s o f i d e a l fuel. I n a number o f c o u n t r i e s ,

i n c l u d i n g t h e S o v i e t Union, b i o g a s is

w i d e l y u s e d e v e n now t o g e t h e a t e n e r g y t h a t c a n b e s u p p l i e d t o t h e a g r i c u l t u r a l i n d u s t r y t o h e a t appartments and s e r v i c e rooms, t o g e t hot water and steam, e l e c t r i c power.

t o d r y g r a i n and hay, and f i n a l l y ,

I n some c o u n t r i e s ,

for

t o produce

i n s t a n c e i n C h i n a , Romania

a n d West G e r m a n y , b i o g a s i s u s e d a s e x t r a m o t o r f u e l (4). Liquid sludge produced during fermentation is successfully used a s an organo-mineral

f e r t i l i z e r . Moreover,

p h o t o b a c t e r i a and a l g a e

g r o w n on t h e s l u d g e e f f i c i e n t l y p u r i f y i t ; c l e a n w a t e r t h e n i s e i t h e r r e l e a s e d i n t o r e s e r v o i r s or used f o r i n d u s t r i a l p u r p o s e s , w h i l e t h e grown b i o m a s s i s u s e d a s a f o d d e r . Thus b i o g a s p r o d u c t i o n from o r g a n i c w a s t e s c a n o n l y h e l p t o a

-

337

-

c e r t a i n extent i n solving t h e t h r e e problems simultaneously: prot e c t i o n o f e x t e r n a l environment and supply o f energy and food (production of organo-mineral

fertilizers).

Ethanol is the o t h e r type of b i o f u e l t h a t i n t h e near f u t u r e

w i l l c o n t r i b u t e t o t h e power e n g i n e e r i n g o f some c o u n t r i e s a n d w i l l b e used a s a modern m o t o r f u e l . starch-

Nowadays i t i s m a i n l y d e r i v e d from

and s u g a r c r o p s or from v a r i o u s w a s t e s i n c o u n t r i e s w i t h

t r o p i c a l and s u b t r o p i c a l c l i m a t e s ,

o r f r o m wood ( l i g n o c e l l u l o s e ) i n

c o u n t r i e s w i t h a moderate c l i m a t e and i n zones or r i s k farming, including Russia. I n a d d i t i o n , b i o f u e l c a n b e p r o d u c e d by t h e r m a l c o n v e r s i o n o f wood t o l i q u i d , s o l i d a n d g a s e o u s f u e l . One o f t h e m e t h o d s w i d e l y used t o date is pyrolysis, a t 450-550

OC

t h a t is a n a e r o b i c c o n v e r s i o n o f biomass

t o charcoal, methanol, a c e t i c acid, combustible gases

such as methane, hydrogen, carbon monoxide. The o t h e r p r o m i s i n g m e t h o d i s g a s i f i c a t i o n , t h a t i s t h e b u r n i n g o f s o l i d biomass a t 900-1500

OC

t o produce synthetic gas ( a mixture

o f h y d r o g e n a n d c a r b o n m o n o x i d e ) , w h i c h c a n t h e n b e u s e d t o g e t methanol or a r t i f i c i a l petroleum.

L i q u e f a c t i o n is a l s o a v e r y promi-

s i n g method u s e d f o r t h e p r o d u c t i o n o f l i q u i d f u e l s from s o l i d b i o mass.

I n c o n t r a s t t o g a s i f i c a t i o n , t h e p r o c e s s r e q u i r e s s p e c i a l ca-

t a l y sts. Thus t h e n a t i o n a l p r o g r a m s i n c l u d i n g v a r i o u s t y p e s o f biomass c o n v e r s i o n t o f u e l a r e v e r y e x t e n s i v e a n d p r o m i s i n g ; many o f t h e m

can b e r e a l i z e d i n t h e n e a r f u t u r e , w h i l e t h e o t h e r s a r e a l r e a d y i m plemented, But biomass w i l l never b e a b l e t o completely r e p l a c e fo-

ssil f u e l s . Biomass is not only a s o u r c e of f u t u r e energy, b u t ,

i s l i f e on

the Earth. Biomass c o n t r i b u t i o n t o power e n g i n e e r i n g w i l l v a r y d e p e n d i n g on t h e biomass y i e l d and p r o d u c t i v i t y i n each i n d i v i d u a l country, and w i l l h a r d l y exceed 5-15 X o f t h e t o t a l . I t is q u i t e o b v i o u s t h a t t h e r m o n u c l e a r f u s i o n w i l l b e t h e main energy s o u r c e i n t h e n e a r f u t u r e , b u t t h i s d o e s not exclude t h e development of o t h e r p r o m i s i n g energy s o u r c e s , i n p a r t i c u l a r d i r e c t catalytic bioconversion developed.

Of

solar energy t o f u e l t h a t is already being

A good example is w a t e r p h o t o l y s i s a n d t h e t r a n s f o r m a t i o n

of t h e r e l e a s e d e n e r g y u s i n g v a r i o u s systems t o p r o d u c e f u e l s .

-

338

-

REFERENCES

1

2 3 4

E.S. Pantskhava and I . V . B e r e z i n , T e c h n i c a l b i o e n e r g e t i c s , 1986, B i o t e c h n o l o g y , N 2, p. 1 - 1 2 , and N 3 , p. 8 - 1 5 . D.O. H a l l , B i o m a s s d o e s e n e r g y f u e l s now a n d i n t h e f u t u r e . I n s t . Chem. Eng. Symp., Sep., 1 9 8 2 , N 7 8 , T 6 / 1 - T / 1 3 . I n t e r n a t i o n a l B i o e n e r g y D i r e c t o r y Handbook, W a s h i n g t o n , Amer, C o u n c i l B i o e n e r g y , 1984, pp. 1000. U n i t e d Nations,New-York, 1 9 8 3 , A d a p t e d G u i d e b o o k on B i o g a s D e v e l o p m e n t , ESCAP, B a n g k o k , T a y l a n d .

I11. Possible Positive and Negative Impacts of Biotechnology on Environment

This Page Intentionally Left TBlank

P O S I T I V E AND N E G A T I V E I M P A C T S OF BIOTECHNOLOGY O N THE E N V I R O N M E N T

D r . L . HUBER Bayerische Landesanstalt D-8000 Munchen 2 2 , F.R.G.

fgr

Wasserforschung,

K a u l b a c h s t r a s s e 37,

INTRODUCTION From t h e h i s t o r i c a l p o i n t o f v i e w ,

f e r m e n t a t i o n i s one o f man-

k i n d ’ s e a r l i e s t achievements i n t h e f i e l d o f a p p l i e d m i c r o b i o l o g y

It involved the deliberate application o f the

and b i o t e c h n o l o g y .

f o r t h e c o n v e r s i o n o f one p r o d u c t o r

a c t i v i t i e s o f micro-organisms substance i n t o another. such as cheese,

The e a r l i e s t p r o d u c t s w e r e f o o d s a n d d r i n k s

b e e r and wine.

t h e r o l e o f micro-organisms

These were b e i n g made l o n g b e f o r e

had been r e c o g n i z e d .

Modern b i o t e c h n o l o g y i n t h e c o n t e x t o f t h i s p a p e r i s u n d e r s t o o d as t h e b r a n c h o f a p p l i e d m i c r o b i o l o g y a n d b i o c h e m i s t r y i n w h i c h b i o l o g i c a l methods a r e a p p l i e d i n i n d u s t r i a l - s c a l e cesses.

production pro-

A l l b i o t e c h n i c a l processes a r e i n t e n d e d t o o p t i m i z e biomass

p r o d u c t i o n and t h e s p e c i f i c b i o c h e m i c a l c a p a c i t y o f b i o l o g i c a l systems,

a n d t o make maximum e c o n o m i c u s e o f them.

Figure 1 outlines

t h e b a s i c s t r u c t u r a l i n t e r r e l a t i o n s and f u n c t i o n s o f b i o t e c h n o l o g y . L i k e any o t h e r man-made

technology,

b i o t e c h n o l o g y has b o t h

p o s i t i v e and n e g a t i v e e f f e c t s on t h e e n v i r o n m e n t . t e c h n i c a l methods sms

-

-

The u s e o f b i o -

including genetically-engineered micro-organi-

is i n d i s p e n s a b l e f o r t h e m a n u f a c t u r e o f many p r o d u c t s e s s e n -

t i a l t o mankind.

I t i s t h u s i m p o r t a n t t h a t t h e u n q u e s t i o n a b l e bene-

f i t s o f t h i s technology be exploited.

A t t h e same t i m e ,

it

however,

i s n e c e s s a r y t o m i n i m i z e t h e n e g a t i v e e n v i r o n m e n t a l e f f e c t and hezards which r e s u l t from i t s a p p l i c a t i o n .

It i s significant that

b i o t e c h n o l o g i c a l p r o c e s s e s t h e m s e l v e s a r e w i d e l y used t o combat t h e d e t r i m e n t a l e f f e c t s o f t h e i n d u s t r i a l a p p l i c a t i o n o f such t e c h n o l o 9Y. APPLICATION OF BIOTECHNOLOGICAL P R O C E S S E S The e x t e n t t o w h i c h b i o t e c h n o l o g i c a l p r o c e s s e s a r e s p p l i e d

-

-

342

-

DISCIPLINES

[ BIOTEF~NOLDGYI

AREA Fig.

1. F u n c t i o n o f B i o t e c h n o l o g y .

- can be described i n very crude terms o n l y . C o m m e r c i a l o r i n d u s t r i a l a p p l i c a t i o n s a r e f o u n d p a r t i cularly i n the following fields:

which is nowadays c o n s i d e r a b l e

-

production of biomass and s p e c i f i c

compounds

fermented foods, beverages and food processing m i c r o b i a l c e l l s as raw m a t e r i a l s for t h e e x t r a c t i o n of b i o c h e micals, especially pharmaceutical products.

-

environmental protection

I t i s t y p i c a l o f t h i s b r a n c h o f i n d u s t r y t h a t t h e number of p r o d u c t s , which a r e produced i n l a r g e q u a n t i t i e s , i s r e l a t i v e l y s m a l l compared t o t h e i r v a r i e t y . Biomass p r o d u c t i o n c o m p r i s e s p r e d o m i n a n t l y t h e p r o d u c t i o n of y e a s t s grown o n c a r b o h y d r a t e s which s e r v e a s a n i m a l f e e d o r which a r e i n t e n d e d f o r human c o n s u m p t i o n . T h e p r o d u c t i o n o f s i n g l e - c e l l p r o t e i n s from d i f f e r e n t s o u r c e s can l i k e w i s e be mentioned i n t h i s c o n t e x t .

I n t h e f i e l d of produc-

-

343

-

t i o n o f s p e c i f i c compounds o r p r o d u c t s t h e m o s t i m p o r t a n t w i t h r e g a r d t o q u a l i t y and q u a n t i t y a r e : a l c o h o l s ( s u c h as e t h a n o l ,

butanol),

and o r g a n i c a c i d s ( a c e t i c a c i d ,

k e t o n e s ( s u c h as acetone)

l a c t i c acid,

propionic acid),

w h i c h a r e s y n t h e s i z e d by way o f i n c o m p l e t e o x i d a t i o n s o r t e c h n i c a l fermentations c i t r i c a c i d as a b i o s y n t h e s i s p r o d u c t o b t a i n e d f r o m c e r t a i n f u n g i ( A s p e r g i l l u s ) and o t h e r a c i d s o f t h e c i t r i c a c i d c y c l e fermented foods such as bread,

milk,

cheese,

sour p r o d u c t s and

beverages e x t r a c e l l u l a r polysaccharides o f the dextran type a n t i b i o t i c s s u c h as p e n i c i l l i n s ,

t e t r a c y c l i n e s and cephalospo-

r r n e s as secondary b i o s y n t h e s i s p r o d u c t s ,

m a i n l y from f u n g i

s t e r o i d s b y way o f s e l e c t i v e b i o t r a n s f o r m a t i o n a m i n o a c i d s a n d n u c l e o t i d e s by way o f s p e c i f i c f e r m e n t a t i o n methods t e c h n i c a l enzymes a n d b i o c a t a l y s t s ( p r o t e a s e s , ses),

e.g.

f o r detergents;

amylases,

lipa-

some o f t h e s e a r e a l r e a d y b e i n g p r o -

d u c e d u s i n g s t r a i n s t h a t h a v e b e e n m o d i f i e d b y way o f g e n e t i c engineering. Table 1 p r o v i d e s a qeneral i n d i c a t i o n i n terms o f annual p r o d u c t i o n volume o f t h e i m p o r t a n c e w o r l d w i d e o f f e r m e n t a t i o n p r o d u c t s .

TABLC 1 Economic i m p o r t a n c e o f b i o t e c h n o l o g i c a l p r o d u c t s (1979-82) ( a c c o r d i n g t o DELLWEG, 1 9 8 7 ) -.

mill.

Beer

97,5

Wine a n d s p a r k l i n g w i n e

37,l m i l l .

Antibiotics

18.000

l l c o h o l s (ethanol) Arnino a c i d s ( g l u t a m i c a c i d a n d l y a i n e ) Fodder y e a s t Baker's yeast C i t r i c acid Enzymes Vinegar

5,9

m3

t

mill.

342.000

m3 p.a. p.a.

t

t

m i l l .

t

1,4

mill.

t

26.000 1,6

t

mill.

p.a. p.a.

1,4

350.000 t

p.a.

p.a. p.a. p.a. p.a.

m3 p.e.

- 344

Last but not least,

-

b i o t e c h n o l o g y a l s o i n c l u d e s a e r o b i c and

a n a e r o b i c b i o l o g i c a l sewage p u r i f i c a t i o n p r o c e s s e s ,

c o m p o s t i n g and

anaerobic sludge digestion. NEGATIVE EFFECTS OF BIOTECHNOLOGY ON THE E N V I R O N M E N T S i n c e b i o t e c h n i c a l p r o d u c t i o n p r o c e s s e s t a k e p l a c e i n aqueous medium,

and t h e c o r r e s p o n d i n g t r a n s f o r m a t i o n s a r e f a i r l y s i m i l a r

t o chemical conversions,

t h e y a l s o g i v e r i s e t o t h e same p r o b l e m s

with:

-

i n c o m p l e t e u t i l i z a t i o n o f s u b s t r a t e s or s t a r t i n g p r o d u c t s (nutrient

solutions)

-

formation

o f i n t e r m e d i a t e s or end p r o d u c t s which cannot be

-

technological

exploited otherwise and b i o l o g i c a l l i m i t a t i o n s w i t h r e g a r d t o r e a l i -

z i n g maximum y i e l d s o r c o n v e r s i o n s . These p r o c e s s e s a c c o r d i n g l y r e s u l t i n e f f l u e n t s w h i c h a r e p o l l u t e d t o a g r e a t e r or l e s s e r e x t e n t .

The a c t u a l p o l l u t a n t s h e r e

are n u t r i e n t s o l u t i o n admixtures which cannot p l a y a r o l e i n the metabolic a c t i v i t i e s of t a i n non-fermentable

t h e microorganisms b e i n g used,

sugars i n molasses,

such as c e r -

or e l s e i n o r g a n i c substan-

c e s w h i c h h a v e t o b e p r o v i d e d i n e x c e s s b u t a r e o n l y p a r t i a l l y us e d up d u r i n g t h e c o n v e r s i o n s . As i s t h e c a s e w i t h c h e m i c a l s y n t h e s e s , processes a l s o l e a d t o by-products

biochemical production

w h i c h c a n n o t be u t i l i z e d and

w h i c h as a r u l e have t o be d i s p o s e d o f v i a t h e waste w a t e r . b i o l o g i c a l waste-water mixed c u l t u r e s ,

p u r i f i c a t i o n processes,

Even

w h i c h make u s e o f

p r o d u c e o r g a n i c e n d m e t a b o l i t e s w h i c h c a n n o t b e de-

graded f u r t h e r m i c r o b i a l l y

Important here i s the f a c t t h a t

the in-

t e r m e d i a t e and end p r o d u c t s o f b i o c h e m i c a l s y n t h e s e s a r e n o t a l l o wed t o have p r o p e r t i e s w h i c h w o u l d r e s u l t i n t h e i r b e i n g c l a s s i f i e d as dangerous substances. e.g.

There a r e c e r t a i n l y examples o f t h i s s o r t

i n t h e f i e l d o f b i o l o g i c a l sewage p u r i f i c a t i o n ;

as t h e n o n y l p h e n o l e t h o x y l a t e s ,

substances such

w h i c h a r e b r o k e n down t o t h e s i g n i -

f i c a n t l y more e c o t o x i c n o n y l p h e n o l . As b e i n g t y p i c a l o f t h e w a s t e - w a t e r

p o l l u t i o n r e s u l t i n g from

b i o t e c h n o l o g i c a l p r o c e s s e s m e n t i o n c a n be made h e r e o f t h e p o l l u t a n t c o n t e n t s d e r i v i n g f r o m t h e p r o d u c t i o n o f beer or yeast. B a s i c a l l y t h e y a r e a p p l i c a b l e t o many o t h e r b i o t e c h n o l o g i c a l p r o c e s s e s t o o , where t h e p o l l u t a n t c o n t e n t o f t h e e f f l u e n t w a s t e i s p r e d o m i n a n t l y organic.

-

345

-

D u r i n g t h e brewing o f beer s p e c i f i c waste-water q u a n t i t i e s o f 3 p r o d u c t a r e p r o d u c e d , w i t h an o r g a n i c p o l l u t a n t c o n 3 t e n t o f 3 - 6 k g BOD5/m T h i s c o r r e s p o n d s t o r e l a t i v e l y h i g h BOD5

-

4

1 2 m /m3

.

c o n c e n t r a t i o n s o f 500 de,

-

1100 mg/l.

amongst o t h e r t h i n g s ,

l e sugars,

The d i s s o l v e d p o l l u t a n t s i n c l u -

p r o t e i n compounds,

v a r i o u s non-fermentab-

e x t r a c t e d o r g a n i c s u b s t a n c e s and o r g a n i c a c i d s ,

which

f o r t h e most p a r t however a r e e a s i l y b i o l o g i c a l l y d e g r a d a b l e .

Solid

r e s i d u e s s u c h as y e a s t can be used as a n i m a l f e e d ( H U B E R ) . The p r o d u c t i o n o f y e a s t l i k e w i s e r e s u l t s i n w a s t e w a t e r w h i c h i s h i g h l y p o l l u t e d w i t h o r g a n i c compounds; o t h e r hand, r u l a yeast

a t t h e same t i m e ,

t h e y e a s t i n g o f c e r t a i n e f f l u e n t wastes

-

e.g.

on t h e

w i t h To-

( C a n d i d a u t i l i s ) - f r o m t h e p u l p i n d u s t r y i s an i m p o r -

t a n t way o f

s u b j e c t i n g such waste water a t l e a s t t o a p a r t i a l b i o l o -

gical purification. The h i g h c o n c e n t r a t i o n o f o r g a n i c p o l l u t a n t s i n t h e w a s t e wa-

t e r f r o m y e a s t p r o d u c t i o n i s due p r i m a r i l y t o t h e f a c t t h a t a b o u t one t h i r d o f t h e d r y m o l a s s e s s u b s t r a t e c o n s i s t s o f o r g a n i c o r i n o r g a n i c s u b s t a n c e s w h i c h c a n b e a s s i m i l a t e d by t h e y e a s t c e l l s .

These

compounds t h u s t a k e n o p a r t i n t h e f e r m e n t a t i o n o r y e a s t i n g p r o c e s s , and pass unchanged i n t o t h e waste w a t e r ,

3

m /t product.

245 k g C O D / t

w h i c h amounts t o 10

-

80

The s p e c i f i c p o l l u t a n t c o n t e n t r a n g e s f r o m 1 5 6 t o molasses.

The COD:80D5 r a t i o i n u n t r e a t e d w a s t e w a t e r

r a n g e s o n a v e r a g e f r o m 1:3 h i g h o x y g e n demand,

t o 1:5.

I n p a r t i c u l a r because o f t h e

comprehensive t r e a t m e n t o f e f f l u e n t d e r i v i n g

f r o m y e a s t p r o d u c t i o n i s n e c e s s a r y when i t i s d i s c h a r g e d i n t o s u r face water.

The u s e o f r e c o m b i n a n t y e a s t r e s u l t i n g i n h i g h e r s u b -

s t r a t e u t i l i z a t i o n p r o v i d e s an i m p o r t a n t o p e n i n g f o r gene t e c h n o l o gy i n r e s p e c t o f r e d u c i n g p o l l u t a n t s i n e f f l u e n t s .

The s i t u a t i o n i s

s i m i l a r i n t h e case o f c i t r i c a c i d p r o d u c t i o n from molasses u s i n g Aspe r q i11us. A new s i t u a t i o n i s c r e a t e d when u s e i s made o f m i c r o o r g a n i s m s

w i t h r e c o m b i n a n t DNA,

e s p e c i a l l y w i t h pathogenic properties,

*here

b e s i d e s t h e u s u a l o r g a n i c p o l l u t a n t c o n t e n t i n w a s t e w a t e r an a d d i t i o n a l r i s k p o t e n t i a l a r i s e s due t o c e r t a i n q u a l i t i e s o f t h e o r g a nisms used f o r

b i o t e c h n o l o g i c a l production processes,

The v e r y e x -

t e n s i v e a n d h i g h l y c o n t r o v e r s i a l d i s c u s s i o n s a b o u t t h e p o s s i b l e dang e r s i n t h i s case such as

-

t h e r e l e a s e o f r-DNA

o r r-DNA organisms i n t o t h e e n v i r o n m e n t

and t h e p o s s i b i l i t y o f t h e i r s u p p l a n t i n g w i l d s t r a i n s

-

346

-

u n w a n t e d h o r i z o n t a l gene t r a n s f e r

t o other microorganisms

the spreading o f pathogenic or otherwise g e n e t i c a l l y a l t e r e d and dangerous m i c r o o r g a n i s m s ,

w i t h r i s k s f o r man,

a n i m a l s and

plants h a v e r e s u l t e d i n b o t h t h e WHO a n d t h e EFB P a r t y on S a f e t y i n R i o technology i n t r o d u c i n g a & - c l a s s system i n b i o t e c h n o l o g y

which

a s s i g n s t h e i n d i v i d u a l microorganisms used t o c e r t a i n r i s k categories.

T a b l e 2 shows t h e EFB c l a s s e s (FROMMER,

KRAMER):

TABLE 2

EFB Classes

I I1 I11 I V

harmless low r i s k medium r i s k high r i s k

There i s v i r t u a l l y worldwide agreement t h a t t h i s system

S

s u f f i c i e n t f o r p r a c t i c a l purposes. Many o f t h e h a r m l e s s m i c r o o r g a n i s m s o f g r o u p h i s t o r y o f s a f e use i n l a r g e - s c a l e (beer,

antibiotics,

enzymes,

sumption ( d a i r y products)

I have

a long

biotechnological production

a m i n o a c i d s ) a s w e l l as i n human c o n -

or i n agriculture (Bacillus thuringien-

sis). Class I 1 organisms (low r i s k )

a r e weak p a t h o g e n s w h i c h a r e u -

s u a l l y employed i n t h e p r o d u c t i o n o f v a c c i n e s or i n d i a g n o s t i c s . The e x t e n t o f t h e i r u s e ( c o m p a r e d w i t h c l a s s

I) i s f a i r l y limited.

H a r d l y any u s e i s made i n i n d u s t r i a l p r o d u c t i o n o f medium a n d high-risk

micro-organisms.

As f a r a s r-DNA

organisms a r e concerned,

t h e r e i s now a more o r l e s s g e n e r a l c o n s e n s u s o f o p i n i o n t h a t n o f u n d a m e n t a l l y o r q u a l i t a t i v e l y new r i s k s a r e t o b e e x p e c t e d i n research or production.

A r e l i a b l e r i s k assessment i s p o s s i b -

l e i f t h e p r o p e r t i e s o f t h e d o n o r s , r e c i p i e n t and v e c t o r a r e known

r-DNA

and r-DNA

o r g a n i s m s c a n n o t be c l a s s i f i e d p e r se as p a t h o -

g e n i c or hazardous The t r a n s f e r o f p l a s m i d s a n d g e n e s i s a n a t u r a l p r o c e s s w h i c h occurs i n every h a b i t a t .

-

347

-

I n v i e w o f t h i s t h e WHO p u b l i s h e d a s t a t e m e n t a s l o n g ago as 1983 w h i c h r e a d as f o l l o w s : "There a r e no u n i q u e o r s p e c i f i c s a f e t y r i s k s a s s o c i a t e d w i t h r e c o m b i n a n t DNA work

(genetic engineering);

t h e r i s k s a r e no g r e -

a t e r t h a n t h o s e a s s o c i a t e d w i t h w o r k w h e r e u s e i s made o f known pathogens,

a n d t h e y do n o t n e c e s s i t a t e s p e c i a l l a b o r a t o r y d e s i g n

or practice. P O S I T I V E I M P A C T S OF BIOTECHNOLOGY ON T H E E N V I R O N M E N l The p o s i t i v e i m p a c t w h i c h b i o t e c h n o l o g y h a s on t h e e n v i r o n m e n t derives

p r i m a r i l y f r o m t h e u s e o f numerous t r e a t m e n t m e t h o d s f o r was-

t e w a t e r and r e f u s e and,

t o an i n c r e a s i n g e x t e n t o f l a t e ,

t h e s o i l and f o r g a s e o u s w a s t e s .

also for

The q r e a t b e n e f i t t o b e r e a p e d f o r

t h e environment t h r o u g h t h e use o f b i o t e c h n o l o g i c a l p r o c e s s e s i n t h e n i e d i c a l and a g r i c u l t u r a l t i o n e d here.

f i e l d s w i l l be o n l y v e r y b r i e f l y men-

T h e r e i s nowadays a g r e a t v a r i e t y

of biotechnological

p u r i f i c a t i o n p r o c e s s e s a v a i l a b l e w h i c h make u s e o f m i c r o b i a l d e g r a dations i n order t o e l i m i n a t e p o l l u t a n t s r e s u l t i n q predominantly f r o m human a c t i v i t i e s .

These t e c h n o l o g i e s a r e d e s i g n e d t o r e n d e r

harmless a l l those substances which a r e g e n e r a l l y mixed t o g e t h e r i n l a r g e numbers t o c o n s t i t u t e w a s t e a n d w h i c h c a n n o t be u t i l i z e d o t herwise.

A b r i e f s u r v e y o f t h o s e p r o c e s s e s u s e d i n t h e f i e l d s o f domest i c and i n d u s t r i a l w a s t e w a t e r s i s g i v e n i n T a b l e 3 .

TABLE 3 Waste-water

t r e a t m e n t methods

A c t i v a t e d sludge processes Trickling f i l t e r s Rotating b i o l o g i c a l contactors Anaerobic contactors Waste s t a b i l i z a t i o n p o n d s

The a c t i v a t e d s l u d g e p r o c e s s i n p a r t i c u l a r ,

with i t s various

m o d i f i c a t i o n s , has a v e r y wide r a n g e o f a p p l i c a t i o n f o r a l a r g e number o f p o l l u t a n t s ;

one m i g h t a l m o s t s p e a k o f a p h y s i o l o g i c a l om-

nipotence i n respect o f t h e metabolism o f those micro-organisms

in-

v o l v e d i n t h e e l i m i n a t i o n o f o r g a n i c and p a r t i a l l y a l s o i n o r g a n i c

substances.

348

-

T h e r e i s h a r d l y any t y p e o f w a s t e w a t e r w i t h o r g a n i c

compounds w h i c h c a n n o t b e t r e a t e d b i o l o g i c a l l y . D u r i n g t h e l a s t few y e a r s i t h a s a l s o b e e n p o s s i b l e t o i s o l a t e a number o f b a c t e r i a whose enzymes a r e a b l e t o r e l e a s e t h e h a l o g e n from c h l o r i n a t e d hydrocarbons,

a t t h e moment c o n s i d e r e d t h e m o s t

c r i t i c a l compounds i n t h e e n v i r o n m e n t . t h e dehalogenases, enzymes,

Some o f

t h e s e enzymes,

namely

r e p l a c e t h e halogen w i t h a h y d r o x i d e group.

the so-called

mono-

and di-oxygenases,

Other

make u s e o f m o l e c u -

l a r o x y g e n t o i n t r o d u c e one o f t w o h y d r o x y l g r o u p s r e s p e c t i v e l y . O f considerable importance i n t h i s f i e l d i s the a v a i l a b i l i t y ,

gst other things, cation,

amon-

o f t e c h n i c a l means o f n i t r i f i c a t i o n a n d d e n i t r i f i -

w h i c h make i t p o s s i b l e t o r e m o v e

-

i n t h e form o f N

t e d n i t r o g e n compounds f r o m w a s t e w a t e r a n d d r i n k i n g w a t e r . m i c a l methods o f r e m o v i n g phosphorous, i s l i k e w i s e becoming i n c r e a s i n g l y

-

unwan-

Bioche-

using Acinetobacter strains,

important.

G e n e t i c e n g i n e e r i n g c a n i n f u t u r e p l a y an i m p o r t a n t r o l e , mely by p r o v i d i n g t a i l o r e d micro-organisms,

na-

i n p a r t i c u l a r where t o -

x i c a n d p e r s i s t e n t compounds ( c h l o r i n a t e d h y d r o c a r b o n s ,

PAC) n e e d

a t t e n t i o n w i l l n a t u r a l l y ha-

t o be degraded t o h a r m l e s s components;

v e t o be p a i d t o t h e p r o b l e m o f t h e r e l e a s e o f such m i c r o - o r g a n i s m s i n t o the environment.

S u i t a b l e g e n e r a a p p e a r t o b e Pseudomonas,

v o b a c t e r i u m and A r t h r o b a c t e r

PAC)

(CHAKRABARTY);

have l i k e w i s e a l r e a d y been d e s c r i b e d .

Fla-

s u i t a b l e p l a s m i d s (B 13, However,

such p r o d u c t s

and p r o c e s s e s a r e s t i l l i n a f a i r l y e a r l y s t a g e o f development. Whereas a e r o b i c p r o c e s s e s p l a y a d o m i n a n t r o l e i n sewage p u r i fication,

a n a e r o b i c p r o c e s s e s p r e d o m i n a t e i n t h e f i e l d o f s o l i d was-

t e disposal;

i t must o f c o u r s e b e remembered,

though,

t h a t use has

b e e n made f o r o v e r a c e n t u r y o f b i o m e t h a n a t i o n i n t h e t r e a t m e n t o f sewage s l u d g e . I n these f i e l d s too,

due t o b e t t e r k n o w l e d g e o f t h e b i o c h e m i c a l

and m i c r o b i o l o g i c a l i n t e r r e l a t i o n s h i p s ,

t h e r e has been c o n s i d e r a b l e

i m p r o v e m e n t d u r i n g t h e l a s t few y e a r s i n t h e t e c h n i q u e s u s e d ; has r e s u l t e d i n t h e processes becoming more s t a b i l i z e d ,

this

thus a l l o -

wing t h e u n p r o b l e m a t i c a l t r a n s f e r o f such technology t o t h e f i e l d s o f s o i l conditioning,

composting o f garbage and t r e a t m e n t

o f other

s o l i d and l i q u i d waste p r o d u c t s (REHM). N o t a s m a l l number o f h a l o g e n a t e d compounds,

which are b u i l t

up i n k e e p i n g w i t h t h e p a r a m e t e r s A O X o r T O C I and w h i c h were h i t h e r -

t o r e g a r d e d a8 m o s t l y non-degradable, s i s t e n t t o such a h i g h degree.

h a v e now p r o v e d n o t t o b e p e r -

I t seems t o b e e a s i e r t o r e m o v e t h e s e

-

349

-

compounds f r o m t h e e n v i r o n m e n t u n d e r a n a e r o b i c c o n d i t i o n s ,

although

t h e speed o f d e g r a d a t i o n i n t h e case o f i n t r i n s i c c o m e t a b o l i s m i s r a t h e r s l o w (REHM).

LEGAL A S P E C T S AND R E Q U I R E M E N T S The momentous p r o g r e s s i n b i o t e c h n o l o g y a n d e s p e c i a l l y i n g e n e t i c e n g i n e e r i n g r e q u i r e s t h e development o f a p p r o p r i a t e r e g u l a tory

frameworks t o p r e c l u d e r i s k s f o r t h e environment as a whole.

Work i s now b e i n g c a r r i e d o u t t o w a r d s t h i s e n d i n m o s t d e v e l o p e d countries.

The F . R . G .

nik-Cesetz"

har3 j u s t i m p l e m e n t e d t h e s o - c a l l e d

( G e n e t i c s Law),

"Gentech-

w h i c h f o r t h e moment c o n t a i n s 7 s p e c i -

f i c regulations covering a l l important negative aspects which might a r i s e from t h e i n d u s t r i a l use o f g e n e t i c a l l y m o d i f i e d organisms (ANONYMOUS).

W o r k i n g on t h e b a s i s o f 4 r i s k l e v e l s ,

m e a s u r e s a r e d e s c r i b e d e.g.

the necessary

f o r e n s u r i n g t h a t such o r g a n i s m s con-

t a i n e d i n e f f l u e n t waste a r e rendered i n a c t i v e .

Discharges from

p1ant.s w o r k i n g w i t h o r g a n i s m s o f r i s k l e v e l s I 1 1 o r I V m u s t b e s t e -

o r e l s e i n a c t i v a t e d u-

r i l i i e d a t 1 2 1 C f o r a p e r i o d o f 20 m i n u t e s ,

s i n g o t h e r e q u a l l y e f f e c t i v e methods (SANDERS e t a l l . E f f l u e n t and r e f u s e c l a s s i f i e d a s r i s k l e v e l I 1 m u s t l i k e w i s e be p r e t r e a t e d such t h a t t h e q e n e t i c a l l y m o d i f i e d organisms c o n t a i ned t h e r e i n a r e i n a c t i v a t e d t o t h e e x t e n t where no danger expected.

-

i s t o be

The most l i k e l y m e t h o d s h e r e a r e :

i n a c t i v a t i o n through the e f f e c t

o f s u i t a b l e chemicals under g i -

ven c o n d i t i o n s

-

i n a c t i v a t i o n by means o f p h y s i c a l m e t h o d s .

SUMMARY B i o t e c h n o l o g y as a whole, genetic engineering, human,

i n c l u d i n q t h e l a t e s t developments i n

i s h a v i n g a tremendous e f f e c t on a l l s t a g e s o f

a n i m a l and p l a n t l i f e .

For b e t t e r or

f o r worse,

i t i s our

t a s k t o t a c k l e t h e problems which a r e a s s o c i a t e d w i t h t h e use o f t h i s t e c h n o l o g y and w h i c h t o a h i g h degree a r e l o c a t e d i n t h e f i e l d o f unwanted e n v i r o n m e n t a l impacts.

A t t h e same t i m e ,

biotechnologi-

c a l p r o c e s s e s t h e m s e l v e s p r o v i d e u s w i t h t h e means a n d p o w e r f u l t o o l s t o c o m b a t t h e n e g a t i v e e f f e c t s w h i c h m i g h t r e s u l t f r o m t h e app l i c a t i o n o f these processes.

The b e s t t i m e i s now,

before things

become e v e n m o r e c o m p l i c a t e d a n d p o s s i b l y g e t o u t o f c o n t r o l ( W R A G G , ZECHENDORF).

I n my o p i n i o n ,

the general b e n e f i t s o f bio-technologi-

- 350

-

c a l processes f u l l y outweigh t h e i r r i s k f o r t h e environment. ver,

Moreo-

o r g a n i s m s p r o d u c e d by way o f m o l e c u l a r b i o t e c h n o l o g y a r e e x -

p e c t e d t o h a v e a b e n e f i c i a l i m p a c t on many o f o u r e n v i r o n m e n t a l p r o blems,

s u c h as w a t e r p o l l u t i o n ,

dous s u b s t a n c e s i n e f f l u e n t s ,

t h e d e g r a d a t i o n o f t o x i c and h a z a r -

r e f u s e and s o i l s ,

and maybe e v e n t h e

e l i m i n a t i o n o f atmospheric p o l l u t a n t s .

I t can g e n e r a l l y b e s t a t e d t h a t b y a p p l y i n g t h e b e s t p h y s i c a l , c h e m i c a l and b i o l o g i c a l t r e a t m e n t methods a v a i l a b l e ,

practically

a l l r i s k s c o n n e c t e d w i t h t h e u s e o f b i o t e c h n o l o g y c a n be m i n i m i z e d

o r e l i m i n a t e d t o t h e e x t e n t t h a t t h e r e need be n o s e v e r e l i m i t a t i o n s t o i t s common u s e .

REFERENCES

Anonymous B u n d e s r a t p u b l i c a t i o n 226/90 - V e r o r d n u n g f i b e r d i e S i c h e r h e i t s s t u f e n u n d S i c h e r h e i t s m a nahmen b e i g e n t e c h n i s c h e n A r b e i t e n i n g e n t e c h n i s c h e n A n l e g e n - (GenTSV) 29.3.90. A.M. C h a k r a b a r t y , " G e n e t i c E n g i n e e r i n g and P r o b l e m s o f E n v i r o n m e n t a l P o l l u t i o n " - B i o t e c h n o l o g y , V o l . 8 , p u b l . b y V C H , Weinheim 1986. H. D e l l w e g , " B i o t e c h n o l o g i e " - p u b l . b y VCH, Weinheim 1987. W . Frommer a n d P . K r a m e r , " S a f e t y Aspects i n Biotechnology: C l a s s i f i c a t i o n s and S a f e t y P r e c a u t i o n s f o r H a n d l i n g o f B i o l o g i c a l A g e n t s " - C o m e t t Course Management o f B i o t e c h n o l o g i c a l R i s k s , 3-5 O c t o b e r 1 9 8 9 , P a r i s . L . Huber, "Untersuchungen uber A b w a s s e r v e r h a l t n i s s e i n Brauer e i e n " - Munchen. B e i t r . z. Abw., F i s c h . - Und F l u O b i o l o g i e , Vol. (1969). E . Sanders, R . A . K . E g y p t i e n and W.D. Deckwer, "Thermische I n a k t i v i e r u n g r e k o m b i n a n t e r D N A " - B i o e n g i n e e r i n g 2 / 9 0 , V o l . 6 29-33. P . Wragg a n d B. Z e c h e n d o r f , " E t h i c s a n d t h e New B i o l o g y " - BFE, V o l . 7 , 1 March 1990, 57-59.

la

R E L E A S E OF GENETICALLYENVIRONMENT:

W.P.M.

ENGINEERED M I C R O O R G A N I S M S I N THE

R I S K OF HORIZONTAL GENETIC-TRANSFER

HOEKSTRA

Department f o r M o l e c u l a r C e l l B i o l o g y , U n i v e r s i t y o f U t r e c h t , 3 5 8 4 C H U t r e c h t , The N e t h e r l a n d s Padualaan 8 INTRODUCTION G e n e t i c a l l y - e n g i n e e r e d m i c r o o r g a n i s m s (GEMs) f o r more t h a n one decade

i n research laboratories.

t h e y a r e a l s o employed f o r i n d u s t r i a l purposes. large scale,

have been a p p l i e d S i n c e a few y e a r s

I n these,

s m a l l and

a p p l i c a t i o n s t h e GEMs a r e r e a d i l y c o n t r o l l e d b y e f f e c -

t i v e b i o l o g i c a l and p h y s i c a l c o n t a i n m e n t .

E f f e c t i v e containment w i l l

m i n i m i z e t h e p o s s i b i l i t y o f random s p r e a d o f GEMs. Q u i t e r e c e n t l y GEMs a r e u s e d o r p l a n n e d t o b e u s e d f o r d e l i b e r a t e release i n the environment,

u n d e r c o n d i t i o n s where s t r i c t b i o -

l o g i c a l c o n t a i n m e n t i s n o t d e s i r a b l e and p h y s i c a l c o n t a i n m e n t i s n o t feasible. D e l i b e r a t e r e l e a s e o f GEMs ( f o r b i o l o g i c a l c o n t r o l i n a g r i c u l -

o r waste water d e t o x i f i c a t i o n e t c . )

ture,

for soil

tion:

how t o p r e v e n t u n c o n t r o l l e d s p r e a d o f t h e GEMs o r t h e i r DNA.

r a i s e s t h e ques-

I n t h i s p a p e r I w i l l d i s c u s s t h i s q u e s t i o n and f o c u s m a i n l y on t h e possible horizontal transfer

o f DNA b y t h e GEMs.

WHY DELIBERATE RELEASE OF GEMs? T h e r e a r e many b i o l o g i c a l i n t e r a c t i o n s i n t h e e n v i r o n m e n t w h e r e microorganisms

play a v i t a l role.

I f one i s f a m i l i a r w i t h t h e p r e -

cise nature o f

such i n t e r a c t i o n s and i f t h e i n v o l v e d m i c r o o r g a n i s m s

a r e w e l l known,

e n g i n e e r i n g o f t h e m i c r o o r g a n i s m s c o u l d h e l p t o ma-

nipulate the interactions.

A w e l l known e x a m p l e t o i l l u s t r a t e t h i s i s t h e a p p l i c a t i o n o f a n I c e - m i n u s m u t a n t o f Pseudomonas s y r i n q a e t o p r e v e n t f r o s t damage

i n p l a n t s ( H i r a n o and Upper,

1985;

L i n d o w and P a n o p o u l o s ,

1988). I n

f a c t t h i s i s t h e f i r s t example o f an approved a p p l i c a t i o n o f i n a f i e l d experiment.

an GEM

-

352 -

F r o s t damage t o p l a n t s i s s t i m u l a t e d by t h e p r e s e n c e o f i c e nucleating sites, p l a n t leaves.

p r o v i d e d by many p a t h o v a r s o f Ps.

s y r i n q a e on

B a s e d on t h e k n o w l e d g e o f t h e b a c t e r i a l i c e - n u c l e a t i n g

p r o t e i n and t h e gene c o d i n g f o r i t , a v a r i a n t l a c k i n g t h i s p r o t e i n h a s b e e n c o n s t r u c t e d by DNA t e c h n o l o g y .

The I c e - m i n u s m u t a n t when

a p p l i e d i n t h e f i e l d under a p p r o p r i a t e c o n d i t i o n s c o u l d e x l u d e t h e w i l d type Ice-plus

bacteria.

I n t h a t way

f r o s t damage t o p l a n t s

c o u l d be p r e v e n t e d . T h e r e a r e many m o r e e x a m p l e s f o r a p p l i c a t i o n o f GEMs i n t h e environment.

A v e r y i n t e r e s t i n g and e x t e n s i v e l y

studied issue i s

the microbial degradation o f xenobiotic pollutants.

By g e n e t i c e n -

g i n e e r i n g o f v a r i o u s Pseudomonads i t i s p o s s i b l e t o c r e a t e new c a t a b o l i c p a t h w a y s by e x p a n d i n g e x i s t i n g p a t h w a y s ,

s o t h a t some r e c a l -

c i t r a n t p o l l u t a n t s c a n be u s e d a s s u b s t r a t e s (Ramos a n d T i m m i s ,

1987).

B e s i d e s g e n e t i c e n g i n e e r i n g c o u l d c r e a t e new c a t a b o l i c p a t h w a y s i n Pseudomonads by a s s e m b l i n g enzymes f r o m d i f f e r e n t s t r a i n s al.,

(Rojo e t

1987).

C O N C E R N ABOUT T H E DELIBERATE RELEASE E c o l o g i s t s and e n v i r o n m e n t a l i s t s

O F GEMS have shown c o n c e r n a b o u t t h e

d e l i b e r a t e r e l e a s e o f GEMS i n t h e e n v i r o n m e n t .

Their main concern

i s t h e u n p r e d i c t a b l e e c o l o g i c a l e f f e c t o f t h e GEMs.

The o r g a n i s m s

w h i c h a r e r e l e a s e d i n t h e e n v i r o n m e n t a r e m e a n t t o be a c t i v e f o r a w h i l e (some d a y s o r e v e n a w h o l e s e a s o n ) m i s s i o n and a r e ,

moreover,

i n order t o f u l f i l t h e i r

m o s t l y a p p l i e d i n h i g h numbers.

therefore a l t e r the pre-existing ecological niches. e x e r t c o n t r o l on t h e i n t r o d u c e d GEMs,

I n order t o

p r o p o s a l s t o endow a n GEM w i t h

a c o n d i t i o n a l l e t h a l g e n e t i c d e t e r m i n a n t h a v e b e e n made. t y p e o f c o n t r o l m i g h t be t h e s e l f - c o n t a i n e d mid-borne

k i l l i n g gene.

may s e r v e a s an e x a m p l e . plasmid R1.

T h i s gene

(e). I t i s however

They may

An u s e f u l

system based on a p l a s -

The s y s t e m d e s c r i b e d b y M o l i n e t al.

(1988)

They a p p l y a k i l l i n g gene d e r i v e d f r o m t h e

(m)i s

n o r m a l l y c o n t r o l l e d by a n o t h e r gene

p o s s i b l e t o s e p a r a t e t h e k i l l e r gene f r o m i t s

c o n t r o l l i n g gene and t o i n t r o d u c e i t i n an GEM u n d e r c o n t r o l o f a s p e c i f i c p r o m o t e r t h a t can be s w i t c h e d "on"

or " o f f " a t w i l l .

The GEM w i l l n o r m a l l y g r o w a n d f u n c t i o n when t h e s w i t c h i s " o f f " , b u t t h e G E M i s k i l l e d when t h e s w i t c h i s " o n " , T h e r e a r e v a r i o u s t y p e s o f s w i t c h e s t h a t c a n b e a p p l i e d i n o r d e r t o c o n t r o l t h e GEM a f t e r i t s release.

Whether t h i s t y p e o f "eco-

safeguard",

and a l l

-

353

-

o t h e r systems t h a t h a v e b e e n p r o p o s e d ,

are effective outside the

l a b o r a t o r y s i t u a t i o n i s n o t known. A n o t h e r c o n c e r n d e a l s w i t h t h e f a t e o f t h e m i c r o b i a l DNA i n t r o d u c e d by t h e G E M s .

Even i f , a f t e r a w h i l e ,

t h e GEMs a r e e f f e c t i v e l y k i l -

l e d , t h e r e w i l l s t i l l be t h e DNA o f t h e s e m i c r o o r g a n i s m s .

T h a t DNA

o r p a r t s o f t h a t D N A m a y f i n d a way t o n a t u r a l o c c u r i n g m i c r o o r g a n i s m s by v a r i o u s t r a n s m i s s i o n m e c h a n i s m s ( h o r i z o n t a l g e n e t i c - t r a n s fer).

TRANSI-ER OF D N A T h i s s e c t i o n w i l l be r e s t r i c t e d t o t r a n s f e r o f DNA i n b a c t e r i a , s i n c e f o r t h e s e o r g a n i s m s most k n o w l e d g e i s a v a i b l e . B e f o r e d i s c u s s i n q t h e v a r i o u s p o s s i b i l i t i e s f o r g e n e t i c t r a n s f e r from g e n e t i cally-engineered bacteria, it is important t o consider the nature of the genetic modification in the bacteria.

I t may b e t h a t t h e mo-

d i f i c a t i o n is l o c a t e d i n t h e b a c t e r i a l chromosome, l i k e i n t h e c a s e o f t h e Ice-minus m u t a n t o f Ps. s y r i n q a e r h e r e a chromosomal d e l e t i o n was i n t r o d u c e d .

The m o d i f i c a t i o n c o u l d a l s o b e i m p o s e d o n a b a c t e -

r i u m by i n t r o d u c t i o n o f a n a u t o n o m o u s r e p l i c a t i n g D N A v e h i c l e ( a plasmid or a phage).

As we w i l l s e e

later, a modification i n the

chromosome w i l l i n g e n e r a l c a u s e l e s s c o n c e r n f o r r i s k o f h o r i z o n t a l g e n e t i c t r a n s f e r t h a n m o d i f i c a t i o n s i n t r o d u c e d by p l a s m i d s o r p h a ges. There a r e s e v e r a l ways f o r h o r i z o n t a l g e n e t i c teria.

t r a n s f e r i n bac-

Table 1 presents a survey of t h e c h a r a c t e r i s t i c s of i ) trans-

f e c t i o n a l s o termed transformation i i ) conjugation and i i i ) transduction.

The d e t a i l s a b o u t t h e s e p r o c e s s e s h a v e b e e n r e v e a l e d f r o m

s t u d i e s under laboratory conditions.

I t is o n l y s i n c e a few years

t h a t i n v e s t i g a t i o n s have been performed t o test whether t h e s e t r a n s m i s s i o n r o u t e s a r e e f f e c t i v e " i n e c o " ( a term t o r e f e r t h a t n o r m a l ecological situations a r e involved). The r a n g e o f b a c t e r i a l g e n e r a f o r which g e n e t i c -

t r a n s f e r sys-

tems a r e d e m o n s t r a t e d ,

at l e a s t under laboratory conditions, is q u i t e e x t e n s i v e a n d i n c l u d e s G r a m - p o s i t i v e as w e l l a s G r a m - n e g a t i v e species.

I n g e n e r a l DNA t r a n s f e r o c c u r s e x c l u s i v e l y b e t w e e n members

b e l o n g i n g t o t h e same g e n u s .

S u c h s p e c i f i c i t y i s d e t e r m i n e d by s p e -

c i f i c r e c o g n i t i o n p r o c e s s e s a t t h e c e l l e n v e l o p e l e v e l a s well a s b y s p e c i f i c D N A r e c o g n i t i o n p r o c e s s e s by r e s t r i c t i o n e n d o n u c l e a s e s . However, t h e r e a r e m a n y r e p o r t s w h i c h s h o w t h a t some c o n j u g a t i o n , t r a n n d u c t i o n a n d t r a n s f e c t i o n systems h a v e l e s s r e s t r i c t e d o r e v e n

- 354

-

TABLE 1 Trans fer-System transfect ion

1)

(transformation)

mechanism

remarks

transmission

natural

o f f r e e DNA

artificial

2)

systems

conjugation

transduction

transmission

s e l f transmission

o f DNA by

mobilization

c e l l - c e l l contact

conduction

transmission o f

3)

g e n e r a l and 4 )

DNA by phage

restricted

particles

systems

1)

t o a v o i d c o n f u s i o n w i t h t h e term t r a n s f o r m a t i o n , used i n c e l l b i o l o g y t o d e s c r i b e t h e t r a n s i t i o n o f a normal c e l l t o a tumor c e l l , the term t r a n s f e c t i o n i s preferable.

2) n a t u r a l s y s t e m s a r e t h o s e s y s t e m s where DNA u p t a k e i s p o s s i b l e d u r i n g t h e l i f e c y c l e o f t h e b a c t e r i u m . A r t i f i c i a l systems a r e t h o s e systems where e x p e r i m e n t a l c o n d i t i o n s s h o u l d b e c r e a t e d t o f o r c e c e l l s t o DNA u p t a k e .

3)

Some p l a s m i d s c a r r y a l l i n f o r m a t i o n f o r c o n j u g a l t r a n s f e r , t h e y a r e s e l f - t r a n s m i s s i b l e . O t h e r s l a c k some t r a n s f e r f u n c t i o n s b u t c a n b e m o b i l i z e d t h r o u g h c o m p l e m e n t a t i o n by o t h e r ( s e l f t r a n s m i s s i b l e ) + n o n - m o b i l i z a b l e p l a s m i d c a n be plasmids. A non-self-transmissible, conjugally transferred a f t e r recombination w i t h a self-transmissible plasmid (conduction).

4 )

Some p h a g e s a r e a b l e , a f t e r l y t i c i n f e c t i o n , t o p i c k up any gene from t h e h o s t ( g e n e r a l t r a n s d u c t i o n ) . Other phages a r e a b l e , a f t e r l y s o g e n i c i n d u c t i o n , t o t r a n s d u c e o n l y t h o s e genes t h a t a r e i n c l o s e v i c i n i t y t o t h e l y s o g e n i c p h a g e ( s p e c i a l i z e d or r e s t r i c t e d t r a n s d u c tion).

r a t h e r broad host ranges.

A r e c e n t o b s e r v a t i o n shows t h a t ,

d i t i o n s w h i c h a b o l i s h e f f e c t i v e DNA r e s t r i c t i o n , negative (E. i s possible

c o l i ) t o Gram-positive (Schafer e t e l . ,

1990).

transfer

under conf r o m Gram-

b a c t e r i a (coryneform bacteria)

A v e r y f a s c i n a t i n g example o f

b r o a d h o s t r a n g e i s t h e t r a n s f e r o f DNA b y c o n j u g a t i o n f r o m A q r o b a c t e r i u m tumefaciens t o p l a n t s ( s e e Zambryski,

1988 f o r a r e v i e w ) .

When D N A i s t r a n s f e r r e d ,

355

-

whatever t h e t r a n s f e r mechanism,

the

f a t e o f t h e t r a n s f e r r e d D N A i s d e t e r m i n e d f i r s t o f a l l by t h e f a c t whether i t i s a r e p l i c o n or n o t .

DNA n o t i n t h e form o f a r e p l i c o n

can only be maintained i n t h e c e l l a f t e r recombination w i t h a r e p l i c o n i n t h e c e l l . DNA i n t h e f o r m o f a r e p l i c o n , h o w e v e r , m a y b e maintained a s such i n a cell. pect t o horizontal genetic ments.

-

I n t e r e s t i n g DNA m o l e c u l e s w i t h rest r a n s f e r a r e f o r m e d by t r a n s p o s o n e l e -

T h e s e DNA e l e m e n t s , w h i c h a r e f r e q u e n t l y u s e d f o r e n g i n e e r i n g

b a c t e r i a , a r e a b l e t o move a u t o n o m o u s l y t o a l l k i n d s o f DNA m o l e c u l e s (see Berg, 1989 f o r a review).

I n t h a t way s u c h D N A e l e m e n t s

may b e s p r e a d v e r y e x t e n s i v e l y e v e n o v e r b r o a d h o s t r a n g e s .

A spe-

c i a l group o f transposons ( T n w , and r e l a t e d elements) codes f o r t h e i r . own c o n j u g a l t r a n s f e r , w h i c h e n a b l e s a n e f f e c t i v e s p r e a d ( C l e w e l l and Gawron-Burke,

1986).

D N A TRANSFER F R O M BACTERIA IN T H E E N V I R O N M E N l A s s t a t e d b e f o r e most DNA t r a n s f e r p r o c e s s e s have b e e n s t u d i e d However, e p i d e m i o l o g i c a l s t u -

i n d e t a i l under laboratory conditions.

d i e s i n t h e s i x t i e s o n t h e s p r e a d o f a n t i b i o t i c - r e s i s t a n c e s i n Ent e r o b a c t e r a c i a e , s u g g e s t e d t h a t c o n j u g a t i o n mechanisms c o u l d opera-

t e i n t h e c o l o n or i n t h e b l a d d e r . G r i f f i t h i n h i s p i o n e e r i n g exper i m e n t s on t r a n f o r m a t i o n i n S t r e p t o c o c c u s p n e u m o n i a e i n f a c t t a u g h t us,

in retrospect,

t h a t t r a n s f o r m a t i o n t o o k p l a c e i n t h e body o f

t h e mice u s e d i n h i s e x p e r i m e n t s .

Later transformation of Bacillus

s u b t i l i s i n t h e s o i l was d e m o n s t r a t e d (Graham a n d I s t o c k ,

19781,

w h i l e Wackernagel and h i s c o w o r k e r s c l e a r l y showed t h a t DNA i n t h e s o i l c a n b e very s t a b l e a n d c a n e f f i c i e n t l y b e t a k e n u p by r e c i p i e n t b a c t e r i a (Lorenz e t a l . ,

1 9 8 8 ) . T r a n s d u c t i o n a s a mean t o t r a n s f e r

g e n e t i c i n f o r m a t i o n i n t h e n a t u r a l environment h a s been demonstrated f o r e x a m p l e by M o r r i s o n e t e l . ,

(1978).

T h e r e a r e v e r y many e x a m p l e s

d e s c r i b e d f o r t r a n s f e r o f DNA i n t h e n a t u r a l e n v i r o n m e n t ,

the inte-

r e s t e d r e a d e r i s r e f e r r e d t o a r e c e n t book i n t h e s e r i e s E n v i r o n m e n t a l Biotechnology: and R.V.

Gene t r a n s f e r i n t h e e n v i r o n m e n t .

eds.

S.B.

Levy

Miller (1989).

For t h e d i s c u s s i o n a b o u t t h e p o s s i b i l i t i e s f o r g e n e t i c t r a n s f e r under environmental conditions, i t is of great importance t o understand t h e relevant microbial ecology.

I n f a c t o u r l i m i t a t i o n i n ma-

k i n g r i s k a n a l y s i s f o r t h e a p p l i c a t i o n o f GEMS i n f i e l d e x p e r i m e n t s ,

i s c a u s e d by t h e c o m p l e x i t y o f t h e e c o s y s t e m s . I n t h e r i s k a n a l y s i s

-

356

-

o f GEMs f o r l a b o r a t o r y o r i n d u s t r i a l u s e we a r e f a m i l i a r w i t h c o n s i d e r a t i o n s a b o u t t h e e n g i n e e r e d g e n e s as w e l l a s a b o u t t h e v e c t o r o f t h o s e genes and t h e n a t u r e o f t h e e n g i n e e r e d h o s t .

However,

as

s t a t e d c l e a r l y by M i l l e r a n d L e v y i n t h e r e f e r e n c e s t a t e d a b o v e ,

for

e n v i r o n m e n t a l r e l e a s e one h a s t o t a k e i n t o c o n s i d e r a t i o n a number o f e c o l o g i c a l f a c t o r s (e.9. anorganic surfaces,

water content,

temperature,

nature o f organic or

pH,

m i c r o b i a l competion etc.)

as w e l l

a s a number o f s p e c i f i c b i o l o g i c a l f a c t o r s w h i c h may i n f l u e n c e t h e transfer

p o t e n t i a l o f i n f o r m a t i o n f r o m a GEM t o i n d i g e n o u s b a c t e r i a

i n the s o i l , tinent

i n t i s s u e s or i n w a t e r .

considerations i s clear:

The c o n s e q u e n c e o f t h e s e p e r -

i t is i m p o s s i b l e t o p r e d i c t t h e f a -

t e o f an GEM a f t e r i t s r e l e a s e i n t h e e n v i r o n m e n t .

The f a t e w i l l

d i f f e r f r o m c a s e t o c a s e a n d e v e n when t h e same GEM i s u s e d u n d e r d i f f e r e n t conditions, analysis.

t h e s e c o n d i t i o n s may a f f e c t g r e a t l y t h e r i s k

A n o t h e r consequence o f t h e n o t i o n t h a t t h e e c o l o g i c a l con-

d i t i o n s a r e s o complex,

c o u l d b e t h a t one s h o u l d o n l y c o n s i d e r GEMs

f o r d e l i b e r a t e r e l e a s e t h a t do n o t d e v i a t e e x t e n s i v e l y from t h e wild,

n a t u r a l occuring,

organism.

Moreover s i n c e chromosomal modi-

f i c a t i o n s a r e l e s s l i k e l y t o be s p r e a d e f f e c t i v e l y ,

such m o d i f i c a -

t i o n s s h o u l d be p r e f e r r e d o v e r p l a s m i d or phage b o r n e m o d i f i c a t i o n s .

A C A S E S T U D Y ON THE FATE OF

I N THE

TnS

MODIFIED PSEUDOMONAS FLUORESCENS

FIELD

F o r t h e r o l e o f Ps.

fluorescens i n growth promotion o f potato-

e s i n f i e l d s t h a t a r e f r e q u e n t l y used t o c u l t i v a t e p o t a t o e s , r o p h o r e s p r o d u c e d b y pseudomonads may be v i t a l To t e s t t h i s i n a c r i t i c a l e x p e r i m e n t ,

(Geels e t a l . ,

side1986).

a TnS i n s e r t i o n was i n t r o d u -

c e d i n one o f t h e s i d e r o p h o r e g e n e s o f Ps.

fluorescens.

m i n u s m u t a n t s were l a t e r i n t r o d u c e d w i t h p o t a t o

Such S i d -

seed t u b e r s i n a

f i e l d a t t h e e x p e r i m e n t a l f a r m "de S c h r e e f " n e a r L e l y s t a d i n H o l l a n d . After introduction o f the genetically-engineered

s t r a i n JM3742,

the

f a t e o f t h i s b a c t e r i u m i n t h e f i e l d was s t u d i e d as w e l l a s p o s s i b l e t r a n s f e r o f the transposon t o other b a c t e r i a i n the rhizosphere. Although t h e a n a l y s i s i s n o t y e t complete, and H o e k s t r a ,

i n JM3742,

t h e r e s u l t s s o f a r (Bakker

u n p u b l i s h e d r e s u l t s ) show t h a t :

Tn5

i s stable i n stra-

t h e s u r v i v a l o f s t r a i n JM3742 a f t e r one s e a s o n i n t h e

f i e l d was p o o r and t r a n s f e r o f t h e t r a n s p o s o n t o o t h e r pseudomonads c o u l d n o t be detected.

Spread o f

s t r a i n 31.13742 t o f i e l d s n e i g h b o u r i n g

t h e e x p e r i m e n t a l f i e l d was r a r e b u t c o u l d b e d e t e c t e d .

From t h e s e

-

357

-

s t u d i e s we a l s o e x p e r i e n c e d t h a t i n o r d e r t o d r a w f i r m c o n c l u s i o n s , d e t e c t i o n o f Tn? r e q u i r e d m o l e c u l a r g e n e t i c a p p r o a c h e s dization)

(DNA h y b r i -

a n d c o u l d n o t b e b a s e d on t h e s p e c i f i c f e n o t y p i c c h a r a c t e -

r i s t i c s o f t h e transposon.

T h a t i m p l i e s t h a t r i s k a n a l y s i s a r e do-

omed t o b e e x p e n s i v e a n d t i m e c o n s u m i n g .

REFER tI NC E 5

1 2 3

4 5 6

7 8

9 10 11 12 13

14

D . E . B e r g , I n : Gene t r a n s f e r i n t h e e n v i r o n m e n t 5.8. L e v y a n d R . V . M i l l e r eds. Mc. G r a w - H i l l P u b l . Comp. New Y o r k 1 9 8 9 , p.99. D . B . C l e w e l l . a n d C . Grawon-Burke. Annu. Rev. M i c r o b i o l . 4 0 ( 1 9 8 6 ) 635. F . P . G e e l s . e t a l . N e t h . J. P 1 . P a t h o l . 92 ( 1 9 8 6 ) 257. J . B . Graham, a n d C . A . I s t o c k . M o l . Gen. G e n e t 1 6 6 ( 1 9 7 8 ) 287. S . S . H i r a n o , a n d C.D. U p p e r . B i o / t e c h n o l o g y 3 ( 1 9 8 5 ) 1 0 7 4 . S.E. Lindow, and N.J. P a n o p o u l o s . I n The r e l e a s e o f g e n e t i Collins, c a l l y - e n g i n e e r e d m i c r o - o r g a n i s m s . M. Sussman, C.H F . A . S k i n n e r a n d O . E . S t e w a r t - T u l l e d s . Acad. P r e s s L o n d o n , 1988. p. 1 2 1 . M.G. L o r e n z , e t a l . J. Gen. M i c r o b i o l . 1 3 4 ( 1 9 8 8 ) 107. R.V. M i l l e r a n d 5 . 8 . L e v y . I n : Gene t r a n s f e r i n t h e e n v i r o n m e n t . S . 8 . L e v y and R . V . M i l l e r e d s . Mc G r a w - H i l l P u b l . Comp. New Y o r k 1 9 8 9 , p. 405. S . M o l i n , e t a l . B i o / t e c h n o l o g y 5 (1987) 1315 M o r r i s o n e t a l . A p p l . E n v i r o n . M i c r o b i o l . 36 (1978) 724. J . L . Ramos, and K . N . Timmis. M i c r o b i o l o g i c a l S c i e n c e s 4 (1987) 228. R o j o e t a l . S c i e n c e 238 ( 1 9 8 7 ) 1 3 9 5 . A . S c h l f e r , , e t a l . J. Bacterial. 1 7 2 ( 1 9 9 0 ) 1653. P . Z a m b r y s k i , Annu. Rev. G e n e t . 22 ( 1 9 8 8 ) 1.

This Page Intentionally Left TBlank

THE ROLE OF CULTURE COLLECTIONS T O S A F E G U A R D N A T U R E ’ S MICROBIOLOGICAL RESOURCES

K.A.

MALIK

D S M - D e u t s c h e Sammlung v o n M i k r o o r g a n i s m e n u n d Z e l l k u l t u r e n GmbH M a s c h e r o d e r Weg 1 B , D - 3 3 0 0 B r a u n s c h w e i g , FRG

E a r l y man c o n s t a n t l y s t r u g g l e d them as d a n g e r o u s a n d u n d e s i r a b l e . agricultural

revolution,

t o destroy animals,

human b e i n g s f i r s t

u s e f u l n e s s a n d began t o d o m e s t i c a t e them. efforts,

considering

During the times o f the n e o l i t h i c s t a r t e d t o sense t h e i r

As a r e s u l t o f t h e i r h a r d

t o d a y we b e n e f i t f r o m t h e u s e f u l a n i m a l s t h e y d o m e s t i c a t e d

and need n o t f e a r t h e dangerous ones w h i c h have e i t h e r been d e s t r o y ed o r c o n t r o l l e d . U n f o r t u n a t e l y microorganisms had t o f a c e a s i m i l a r f a t e a f t e r t h e i r recognition i n the 17th century.

A t f i r s t c o n s i d e r i n g them as

d a n g e r o u s a n d p a t h o g e n s we s t a r t e d t o d e s t r o y t h e m i n t e n t i o n a l l y . And now, b y d i s t u r b i n g t h e e c o l o g i c a l b a l a n c e ,

by c r e a t i n g e n v i r o n -

m e n t a l p o l l u t i o n a n d b y t h e u s e o f e x t e n s i v e c h e m i c a l s we a r e c o n s t a n t l y d e s t r o y i n g them u n i n t e n t i o n a l l y .

However,

we know t h a t m i c r o -

organisms a r e i n d i s p e n s a b l e f o r t h e c o n t i n u e d e x i s t e n c e o f p l a n t and animal l i f e fore,

a n d we c a n n o t a f f o r d t o d e s t r o y t h e m any l o n g e r .

i t i s our responsibility t o collect,

There-

keep and p r e s e r v e t h e

m i c r o o r g a n i s m s f o r o u r own u s e and f o r t h e uae o f c o m i n g g e n e r a t i o n s . f’rof.

F r a n t i s e k K r a l ( 1 8 4 6 - 1 9 2 1 ) f r o m P r a g u e was t h e f i r s t who

r e a l i z e d t h e i m p o r t a n t n e e d o f c u l t u r e c o l l e c t i o n s more t h a n h u n d r e d y e a r s ago a n d he c o l l e c t e d c u l t u r e s w h i c h he made a v a i l a b l e f o r a f e e t o o t h e r w o r k e r s (1, 2 ) .

T h i s was t h e w o r l d ’ s

f i r s t known c u l t u -

r e c o l l e c t i o n w h i c h was l a t e r t r a n s f e r r e d t o t h e U n i v e r s i t y o f V i e n n a i n 1915 by P r o f e s s o r Ernst Pribram.

Later,

i n 1904 t h e f i r s t i n d e -

p e n c ‘ ? n t c o l l e c t i o n t o p r e s e r v e and s u p p l y a w i d e r a n g e o f f u n g a l c u l t u r e s wss e s t a b l i s h e d a n d i t s t i l l e x i s t s a s t h e C e n t r a l b u r e a u v o o r S c h i m m e l c u l t u r e s (CBS) many o f t h e m t h r o u g h o u t

a t Baarn,

The N e t h e r l a n d s ( 3 ) .

the world providing cultures,

Now t h e r e a r e information

-

360

-

and a wide r a n g e o f s e r v i c e s f o r t h e g e n e r a l s u p p o r t of m i c r o b i o l o gy a n d b i o t e c h n o l o g y

( 4 - 9).

The u s e o f m i c r o o r g a n i s m s a n d c e l l c u l t u r e s t o s o l v e a g r i c u l tural,

food,

h e a l t h , e n e r g y a n d e n v i r o n m e n t a l p r o b l e m s i s now r a -

p i d l y i n c r e a s i n g and w i t h t h i s t h e r e i s an e v e r growing need f o r t h e p r e s e r v a t i o n and s t o r a g e o f newly i s o l a t e d s t r a i n s ,

g e n e t i c a l l y en-

g i n e e r e d s t r a i n s , s t r a i n s w i t h p l a s m i d s and o t h e r u s e f u l microorganisms.

C u l t u r e c o l l e c t i o n s , which p l a y a v i t a l role t o s a f e g u a r d

natural microbiological resources, preserving representative,

are s p e c i a l i s e d i n keeping and

t y p e , r e f e r e n c e and b i o t e c h n o l o g i c a l l y

important s t r a i n s o f microorganisms and cell l i n e s i n v i a b l e and s t a b l e s t a t e and a r e a c o n s t a n t s o u r c e of s u p p l y f o r such a u t h e n t i c cultures. I n i t i a l l y , t h e m a i n r o l e s o f most f u n g a l c o l l e c t i o n s were i n a g r i c u l t u r e , brewing and medicine.

I t was d r a m a t i c a l l y e x t e n d e d i n

t h e 1930s t o t h e pharmaceutical industry following t h e discovery o f p e n i c i l l i n , and i n 1980’s, t o biotechnology

( 3 ) . U n t i l t h e 1920’s

t h e m a i n a r e a s o f a c t i v i t y o f b a c t e r i a l c u l t u r e c o l l e c t i o n s were f o r taxonomic and e p i d e m i o l o g i c a l s t u d i e s . I n 1930s t h e s t u d i e s o f microb i a l physiology and biochemistry extended t h e i r importance due t o t h e maintenance o f microorganisms w i t h s p e c i a l p r o p e r t i e s and p r o d u c t i v e q u a l i t i e s . Now c u l t u r e c o l l e c t i o n s a s s o c i a t e d w i t h b i o l o g i c a l a n d b i o t e c h n o l o g i c a l i n d u s t r y and i n s t i t u t i o n s have proved worthwhile f o r t h e p r e s e r v a t i o n and maintenance of important and d i v e r s e groups o f p r o k a r y o t i c and e u k a r y o t i c recombinant and non-recombinant

strains

(1, 2 ) . C u l t u r e c o l l e c t i o n s i n a d d i t i o n t o t h e c a r e f u l d e p o s i t o n , docum e n t a t i o n , m a i n t e n a n c e , p r e s e r v a t i o n and d i s t r i b u t i o n o f u s e f u l , rep r e s e n t a t i v e and t y p e s t r a i n s p l a y s e v e r a l o t h e r important roles. T h e m a i n i n t e r e s t o f some b a c t e r i a l a n d f u n g a l s e r v i c e c u l t u r e c o l l e c t i o n s and t h e i r t y p e o f h o l d i n g s and s e r v i c e s o f f e r e d t o microbiology a n d b i o t e c h n o l o g y h a s a l r e a d y b e e n summarized i n few a r t i l e s (1 - 3 , 10, 11).

A c c o r d i n g t o t h e W o r l d D a t a C e n t r e o f M i c r o o r g a n i s m s (WDC), a b o u t h a l f a m i l l i o n c u l t u r e s a r e o n r e c o r d s i n 360 c u l t u r e c o l l e c t i o n s from 60 c o u n t r i e s ( 1 2

-

1 4 ) . The number o f d i f f e r e n t i s o l a t e s o f f u n -

g i currently maintained i n t h e l i v i n g s t a t e throughout t h e world’s c o l l e c t i o n s i s i n e x c e s s o f 17000 w h i c h r e p r e s e n t s a b o u t 7000 s p e c i e s . A b o u t 6 0 % o f WDC r e g i s t e r e d c o l l e c t i o n s h a v e a p p l i e d m i c r o b i l o g y a s t h e i r s p e c i a l i n t e r e s t and mainly cover a r e a s o f a g r i c u l t u r e , i n d u s t r y ,

dairy,

food and

361

-

g e o m i c r o b i o l o g y . Such c o l l e c t i o n s a r e a w o r l d re-

s o u r c e , t h e s i g n i f i c a n c e o f w h i c h may o n l y b e r e c o g n i z e d i n t h e l i g h t of future s c i e n t i f i c discoveries.

The s t r a i n s c o l l e c t i v e l y

maintained between t h e fungal c u l t u r e c o l l e c t i o n s a l s o p r o v i d e a tremendous g e n e t i c r e s o u r c e o f b i o t e c h n o l o g i c a l importance f o r t h e p r e s e n t and c o u l d p r o v e t o be o f immense s i g n i f i c a n c e f o r t h e f u t u re. However,

a c c o r d i n g t o o n e e s t i m a t e , 2-4 m i l l i o n s p e c i e s o f o r -

g a n i s m s o c c u r i n l o w l a n d t r o p i c s a n d 50 X o f t h e s e may b e c o m e e x t i n c t by t h e y e a r 2 0 0 0 b e c a u s e o f t h e u s e o f i n a d e q u a t e m e t h o d s o f maintenance.

T h e r e f o r e , a s t a b l e n a t i o n a l and i n t e r n a t i o n a l germ

plasm base i s r e q u i r e d f o r maintaining our g e n e t i c r e s o u r c e s once we h a v e a c q u i r e d

we h a v e a c q u i r e d a n d a s s e m b l e d .

The m a i n t e n a n c e a n d p r e s e r v a t i o n o f a f u l l r a n g e o f i s o l a t e s i s an enormoua t a s k and e v e n t h e l a r g e s t c o l l e c t i o n s a r e g e n e r a l l y

n o t a b l e t o b u i l d up l a r g e h o l d i n g s o f s t r a i n s o f s i n g l e s p e c i e s , genera or groups.

T h e r e f o r e t h e c u l t u r e c o l l e c t i o n s b u i l d up g e n e

b a n k s t h a t t h e y j u d g e v a l u a b l e or t h a t would b e u s e f u l i n t h e f u t u -

re e.g.

strains including type strains, strains deposited for their

special properties,

assay s t r a i n s , genetically engineered strains,

i n d u s t r i a l s t r a i n s , plasmid bearing s t r a i n s and o t h e r s .

Once a c q u i -

r e d , such s t r a i n s a r e a u t h e n t i c a t e d , documented and s u b j e c t e d t o p r e s e r v a t i o n u s i n g most a p p r o p r i a t e methods i n o r d e r t o f u n c t i o n a s 1

a continuous source o f supply f o r v i a b l e and s t a b l e s t r a i n s (Fig. and 2 ) . 'The p r e s e r v a t i o n o f m i c r o o r g a n i s m s a n d a n i m a l o r p l a n t c e l l s i s a h i g h l y s p e c i a l i z e d a c t i v i t y c a l l i n g f o r e x p e r i e n c e d back up

and o f t e n very s p e c i a l i z e d equipment.

I t is not possible f o r the

c o i l e c t i o n s t o optimize t h e preservation of each c u l t u r e and put forward a u n i v e r s a l method of p r e s e r v a t i o n a s o f t e n a s i t h a s been observed becaus there is a g r e a t v a r i a t i o n i n s u s c e p t i b i l i t y of t h e v a r i o u s p r e s e r v a t i o n methods and u s u a l l y t h e method s u i t a b l e f o r one group, g e n e r a or s p e c i e s might n o t be s u i t a b l e for a n o t h e r (15

-

18).

D u r i n g i n a d e q u a t e p r e s e r v a t i o n , some m i c r o o r g a n i s m s c o u l d a l s o u n dergo changes i n t h e i r physiology, biochemistry,

p a t h o g e n i c i t y or

o t h e r d e s i r a b l e c h a r a c t e r s ( 1 9 , 20). T h u s t h e c o l l e c t i o n s t h a t a r e r e s p o n s i b l e t o m a i n t a i n a b r o a d s p e c t r u m o f i s o l a t e s o v e r l o n g per i o d s i n a v i a b l e and s t a b l e s t a t e must have a d e q u a t e equipment, pers o n n e l and f i n a n c i a l r e s o u r c e s t o o p e r a t e a v a r i e t y o f modern pres e r v a t i o n m e t h o d s . Major c u l t u r e c o l l e c t i o n s w i t h s p e c i a l i s t s b a c k

- 362

up t h e r e f o r e f r e q u e n t l y

(21

-

-

t e s t a n d a d o p t new m e t h o d s o f p r e s e r v a t i o n

28).

A l l p r e s e r v e d c u l t u r e s n o t o n l y s h o u l d r e t a i n maximum v i a b i l i t y o v e r p r o l o n g e d p e r i o d s o f s t o r a g e b u t t h e y must r e m a i n unchanged i n their properties.

I t has been o f t e n observed t h a t d u r i n g i n a d e q u a t e

maintenance and p r e s e r v a t i o n ,

s t r a i n d r i f t o c c u r s and i n d u s t r i a l

p r o d u c t i o n s t r a i n s f a i l t o show c o n s i s t a n c y o f p e r f o r m a n c e . vice culture collections

The s e r -

a c t as d e p o s i t o r i e s f o r a l l k i n d s o f m i c r o -

organisms t h a t a r e o f p a s t ,

p r e s e n t o r p o t e n t i a l i m p o r t a n c e and t h u s

f u n c t i o n as r e s o u r c e and i n f o r m a t i o n c e n t r e s f o r t h e g e n e r a l s u p p o r t o f m i c r o b i o l o g y and b i o t e c h n o l o g y . fundamental

For service collections i t i s o f

importance t h a t they adopt a s e l e c t i o n o f maintenance

m e t h o d s t h a t p r o d u c e maximum s u r v i v a l l e v e l s a n d g u a r a n t e e s t r a i n stability.

Fig.

1. S t o r a g e o f f r e e z e - d r i e d

ampoules a t 9

OC

a t t h e DSM.

The m a j o r a c t i v i t i e s common t o a l l s e r v i c e c u l t u r e c o l l e c t i o n s g e n e r a t e a q u a n t i t y o f e s s e n t i a l i n f o r m a t i o n w h i c h i s now s y s t e m a t i c a l l y recorded u s i n g computers ( 2 9 , 30). computer,

S t r a i n data,

stored i n a

a l l o w t h e s e a r c h and r e t r i e v a l o f s c i e n t i f i c i n f o r m a t i o n

- 363

-

a n d e s t a b l i s h a d a t a b a s e t h a t may b e u s e d s u b s e q u e n t l y f o r comp u t e r i d e n t i f i c a t i o n and f o r t h e p r e p a r a t i o n o f a catalogue o f s t r a ins.

An u p - t o

ffective

d a t e c a t a l o g u e i s a n e s s e n t i a l r e q u i r e m e n t f o r t h e e-

f u n c t i o n i n g o f a s e r v i c e c o l l e c t i o n and t h e t r a n s f e r o f

c a t a l o g u e d a t a t o a c o m p u t e r a l l o w s a r e a d y u p d a t i n g f o r new e d i t i ons.

The WDC w h i c h h a s b e e n r e l o c a t e d f r o m B r i s b a n e ,

RIKEN,

Australia t o

Saitama i n Japan,

i s a p a r t o f t h e World Federation f o r Cul-

t u r e C o l l e c t i o n s (WFCC).

I t p r o v i d e s i n f o r m a t i o n on c o l l e c t i o n s and

t h e i r h o l d i n g s world wide. provides a Directory and c u l t u r e d c e l l s .

The WDC makes s e v e r a l d a t a b a s e s a v a i l a b l e t h r o u g h

t h e MSDN n e t w o r k (MSDN, versity,

Cambridge,

The MSDN ( M i c r o b i a l S t r a i n D a t a N e t w o r k )

t o t h e sources o f i n f o r m a t i o n on microorganisms

I n s t i t u t e o f Biotechnology,

Cambridge Uni-

U.K.).

I n o r d e r t o have a b e t t e r access t o t h e w e a l t h o f i n f o r m a t i o n a v a i l a b l e i n European C u l t u r e C o l l e c t i o n s and t o p u b l i c i z e ,

various

i n f o r m a t i o n networks and databases such as M i c r o b i a l C u l t u r e I n f o r maticin S e r v i c e

(MiCIS)

and M i c r o b i a l I n f o r m a t i o n Network Europe

(MINE) a n d I C E C C ( I n f o r m a t i o n C e n t r e f o r E u r o p e a n C u l t u r e C o l l e c t i o n s ( b a s e d a t t h e DSM) h a v e b e e n s t a r t e d ( 3 0 ) .

Fig.

2.

L i q u i d . n i t r o g e n s t o r a g e o f s t o c k c u l t u r e s a t t h e DSM.

- 364 -

M i c r o b i a l c u l t u r e c o l l e c t i o n s were p r i m a r i l y a r e s o u r c e f o r m i c r o o r g a n i s m s b u t now many o f t h e s e h a v e a b r o a d e r r o l e a s w o r l d gene b a n k s ,

r e s u l t i n g f r o m new d e v e l o p i n e n t s i n m o l e c u l a r b i o l o g y .

C o l l e c t i o n s s e r v e as r e p o s i t o r i e s f o r o r g a n i s m s c i t e d o r u s e d i n s c i e n t i f i c p u b l i c a t i o n s so t h a t

t h e m i c r o o r g a n i s m s can be used

f o r r e f e r e n c e w o r k and t h a t t h e o r i g i n a l w o r k c a n b e r e p e a t e d o r confirmed.

Many c u l t u r e c o l l e c t i o n s f u n c t i o n a s I n t e r n a t i o n a l Depo-

sitory Authorities

(IDAs)

and a r e a b l e t o p r o v i d e necessary i n f o r -

m a t i o n t o p a t e n t p r o c e s s e s and t h e s t r a i n s used i n p a t e n t a p l l i c a t i o n s ( 3 1 ) . C u l t u r e c o l l e c t i o n s c a r r y o u t r e s e a r c h programmes t h a t c o m p l e m e n t t h e i r i n t e r e s t s and h a v e t h e a d v a n t a g e o f a c c e s s t o l a r ge number o f a u t h e n t i c a t e d m i c r o o r g a n i s m s a v a i l a b l e f o r s t u d y .

The

m a j o r i t y o f microorganisms i n a c u l t u r e c o l l e c t i o n are o f t e n n o t f u l l y c h a r a c t e r i z e d and provi.de t h e p o s s i b i l i t y o f b e i n g e x p l o r e d and screened f o r t h e i r

f u l l r e s e a r c h and i n d u s t r i a l p o t e n t i a l .

e x p e r t i s e developed a t the c o l l e c t i o n s , t o g i v e a d v i c e on t a x o n o m y , maintenance,

preservation,

enables the c o l l e c t i o n s t a f f

identification, supply,

The

isolation,

culturing,

d e p o s i t i o n and s e v e r a l o t h e r

c o l l e c t i o n related matters. The i n t e r a c t i o n and c o l l a b o r a t i o n b e t w e e n c u l t u r e c o l l e c t i o n s through national, creasing.

r e g i o n a l and i n t e r n a t i o n a l n e t w o r k i s r a p i d l y i n -

The W o r l d F e d e r a t i o n o f C u l t u r e C o l l e c t i o n s (WFCC) promo-

t e s and c o - o r d i n a t e s

such a c t i v i t i e s .

t i o n s O r g a n i z a t i o n (ECCO),

The E u r o p e a n C u l t u r e C o l l e c -

e s t a b l i s h e d i n 1 9 8 1 h a s now 4 1 members

and i t p r o v i d e s a chance f o r t h e C u r a t o r s t o meet a n n u a l y t o d i s c u s s c o l l e c t i o n s r e l a t e d problems i n Europe.

There a r e s e v e r a l r e g i o n a l

f e d e r a t i o n s o f c u l t u r e c o l l e c t i o n s (USFCC, collaborative actions.

UKFCC),

t o promote such

The u s e f u l n e s s o f c u l t u r e c o l l e c t i o n s i n t h e

w o r l d and t h e i r f u n d a m e n t a l r o l e i n m i c r o b i o l o g y h a s o f t e n b e e n p r o j e c t e d i n t h e I n t e r n a t i o n a l Cultures C o l l e c t i o n Conferences ( 3 2

-

34)

a n d b y t h e w o r l d n e t w o r k o f M i c r o b i a l R e s o u r c e s C e n t r e s (MIRCEN). Today,

t h e r e a r e 1 8 MIRCEN c e n t r e s w o r l d w i d e w h i c h p a r t i c i p a t e i n

a g l o b a l c o l l a b o r a t i v e network p a r t i c u l a r l y i n the harnessing o f t h e b e n e f i c i a l a p p l i c a t i o n s o f t h e m i c r o b i a l w o r l d f o r human p r o gress ( 3 5 ) . In short,culture

t h i n g s t o a l l men’,

c o l l e c t i o n s h a v e many r o l e s a n d t r y t o b e ’ a l l

b u t t h e conservation o f microorganisms remains

t h e p r i m a r y o b j e c t i v e o f f u n g a l and b a c t e r i a l c u l t u r e c o l l e c t i o n s , which c o n s t i t u t e s a fundamental sources.

resource f o r n a t u r e ’ s genetic re-

-

365 -

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K.A. Malik, Preservation o f biotechnologically important microiirganisms i n c u l t u r e c o l l e c t i o n s , In: Progress i n B i o t e c h n o l o g y v o l . 4 , ( I n t e r b i o t e c h 8 7 , Enzyme T e c h n o l o g i e s ) , A B l a z e j and J , Zemek ( E d i t o r s ) , E l s e v i e r S c i e n c e P u b l i s h e r s , Amsterdam, 145-186, (1988). K . A . M a l i k and D . C l a u s , B a c t e r i a l C u l t u r e C o l l e c t i o n s : T h e i r i m p o r t a n c e t o B i o t e c h n o l o q y and M i c r o b i o l o q y . I n t h e B i o t e c h n o R u s s e l Ed.), V o l . 5, l o g y and G e n e t i c E n g i n e e r i n g R e v i e w ( G . E . pp. 137-197, ( 1 9 8 7 ) . I n t e r c e p t L t d . Ferndown, D o r s e t , U K . D.L. Hawksworth, Fungus C u l t u r e C o l l e c t i o n s as a B i o t e c h n o l o g i c a l Resource. I n B i o t e c h n o l o g y and G e n e t i c E n g i n e e r i n g Reviews (G.E. R u s s e l , E d . ) , volume 3 , p p . 4 1 7 - 4 4 0 , ( 1 9 8 5 ) . I n t e r c e p t , P o n t e l a n d , N e w c a s t l e u p o n Tyne. S . M . M a r t i n and V . B . D . Skerman ( E d s ) World D i r e c t o r y o f C o l l e c t i o n s o f C u l t u r e o f M i c r o o r g a n i s m s , W i l e y - I n t e r s c i e n c e , New York, (1972). J.R. P o r t e r , The w o r l d v i e w o f c u l t u r e c o l l e c t i o n s . A m e r i c a n Type C u l t u r e C o l l e c t i o n 5 0 t h A n n i v e r s a r y Symposium. The R o l e Colo f c u l t u r e c o l l e c t i o n i n t h e E r a o f M o l e c u l a r B i o l o g y (R.R. w e l l , E d . ) , pp. 62-72, ( 1 9 7 6 ) , American S o c i e t y f o r M i c r o b i o l o gy, Washington, DC. M . Rogosa (Ed). N a t i o n a l Wnrk C o n f e r e n c e on M i c r o b i a l C o l l e c t i o n s o f Major Importance t o A g r i c u l t u r e . American P h y t o p a t h o l o g i c a l Society, S t . Paul. Minesota (1981). V . McGowan and V.B.D. Skerman ( E d s ) . W o r l d D i r , e c t o r y o f C o l l e c t i o n s o f C u l t u r e s o f M i c r o o r g a n i s m s , 2nd e d n . W o r l d D a t a C e n t r e f o r M i c r o o r g a n i s m s , B r i s b a n e . (15'82). W . A . C l a r k and W.O. L o e g e r i n g , F u n c t i o n s and maintenance o f a t y p e c u l t u r e c o l l e c t i o n . Annual Review o f P h y t o p a t h o l o g y 5, 319-342 ( 1 9 6 7 ) . E . J . Da S i l v a , A.C.J. B u r g e r s a n d R . J . Olembo, U N E S C O , UNEP a n d t h e i n t e r n a t i o n a l community o f c u l t u r e c o l l e c t i o n s . I n Proceedings o f t h e T h i r d I n t e r n a t i o n a l C o n f e r e n c e s on C u l t u r e C o l l e c t i o n s . P e r e i r a , Eds.) pp. 1071 4 - 1 9 M a r c h , 1977 ( F . F e r n a n d e s a n d R . C . 1 2 0 ( 1 9 7 7 ) . U n i v e r s i t y o f Bombay, Bombay. S.P. L a p a g e , J.E. S h e l t o n , T . G . M i t c h e l l and A.R. Mackenzie, C u l t u r e C o l l e c t i o n s and p r e s e r v a t i o n o f b a c t e r i a . I n Methods i n M j c r o b i o l o g y (J.R. N o r r i s a n d D.W. R i b b o n s , E d s ) , v o l u m e 3A, pp. 135-227 ( 1 9 7 0 ) . Academic P r e s s , London. L . I . S l y , The r o l e o f c u l t u r e c o l l e c t i o n s i n m i c r o b i o l o g y a n d b i o t e c h n o l o g y . I n : UNESCO/WFCC/ICY/TISR T r a i n i n g C o u r s e on Yeasts: T h e i r i d e n t i f i c a t i o n , p r e s e r v a t i o n and use i n B i o t e c h n o l o g y , pp. 67-74 ( 1 9 8 4 ) . Bangkok M i r c e n , T h a i l a n d . I n s t i t u t e o f S c i e n t i f i c and T e c h n o l o g i c a l R e s e a r c h , B a n g k o k . V.B.D. Skerman, W o r l d D a t a C e n t r e f o r M i c r o o r g a n i s m s . I n P r o c e e d i n g s o f t h e F o u r t h I n t e r n a t i o n a l C o n f e r e n c e on C u l t u r e C o l l e c t i o n , 20-24 J u l y 1 9 8 1 (M. K o c u r and E . J . da S i l v a , E d s ) , p p . 1 1 - 1 7 ( 1 9 8 4 ) . W o r l d F e d e r a t i o n o f C u l t u r e C o l l e c t i o n s , London. S.M. M a r t i n , R e g i o n a l c u l t u r e c o l l e c t i o n s i n t h e d e v e l o p i n g w o r l d . I n P r o c e e d i n g s o f t h e Second C o n f e r e n c e o n C u l t u r e C o l l e c Da S i l v a , V.B.D. Skerman t i o n s (A.F. Pestana de C a s t r o , E . J . L e v e r i t t E d s . ) , p p . 9 6 - 9 9 ( 1 9 7 6 ) . UNESCO/UNEP/WFCC/ and W . W . World Data Centre f o r Microorganisms, Brisbane, A u s t r a l i a . F.A. S k i n n e r , E . Hamatova a n d V.F. McGowan, I B P W o r l d C a t a l o g u e o f R h i z o b i u m C o l l e c t i o n s , 2nd e d n . ( 1 9 8 3 ) , (V.B.D. Skerman, E d ) . World Data Centre, Brisbane.

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15 16 17

18 19

20

21

22 23 24 25 26 27 28

29 30

31 32 33

K . A . Malik and P. Hoffmann, P r e s e r v a t i o n and s t o r a g e o f b i o t e c h n o l o g i c a l l y i m p o r t a n t m i c r o o r g a n i s m s . Chimica Oggi-June, 61-66 (1989). 8 . K i r s o p a n d J . J . S . S n e l l , M a i n t e n a n c e o f M i c r o o r g a n i s m s : A Man u a l o f L a b o r a t o r y M e t h o d s , ( 1 9 8 4 ) . Academic Press, London. H . H a t t , ( E d . ) , A m e r i c a n T y p e C u l t u r e C o l l e c t i o n M e t h o d s . 1. L a b o r a t o r y Manual on P r e s e r v a t i o n F r e e z i n g a n d F r e e z e - d r y i n g , ( 1 9 8 0 ) . American Type C u l t u r e C o l l e c t i o n , R o c k v i l l e , M a r y l a n d , USA. K . A . M a l i k , Modern M e t h o d s o f Gene C o n s e r v a t i o n , ( 1 9 8 5 ) . A Lab o r a t o r y M a n u a l . PASTIC P r e s s , P a k i s t a n S c i e n c e a n d T e c h n o l o g y Information Centre, Islamabad, Pakistan. A. D i e t z , C u l t u r e p r e s e r v a t i o n and i n s t a b i l i t y . I n B i o a c t i v e M i c r o b i a l P r o d u c t s : S e a r c h a n d D i s c o v e r y ( J . D . B u l o c k , L . J . Nisb e t and D.J. W i n s t a n e l y , E d s . ) , pp. 27-35 ( 1 9 8 2 ) . Academic P r e s s , N e w York. K . A . M a l i k , The r o l e o f c u l t u r e c o l l e c t i o n i n t h e s t a b i l i t y a n d p r e s e r v a t i o n o f m i c r o o r g a n i s m s , I n : 12eme C o l l o q u e o r g a n i s e p a r la S e c t i o n de Microbiologie I n d u s t r i e l l e e t de Biotechnologie d e l a SFM, S o c i e t e F r a n c a i s e d e M i c r o b i o l o g i e , S t a b i l i t e e t Cons e r v a t i o n d e s M i c r o o r g a n i s m e s , J . Amen, P . l e s s o n ( E d i t o r ) , 118-150 (1987). K . A . M a l i k , A new m e t h o d f o r l i q u i d n i t r o g e n s t o r a g e o f p h y t o t r o p h i c b a c t e r i a under a n a e r o b i c c o n d i t i o n s . J o u r n a l o f Microb i o l M e t h o d s 2, 4 1 - 4 7 ( 1 9 8 4 ) . K . A . M a l i k , A new f r e e z e - d r y i n g m e t h o d f o r t h e p r e s e r v a t i o n o f n i t r o g e n - f i x i n g a n d o t h e r f r a g i l e b a c t e r i a . J o u r n a l o f Microb i a l . M e t h o d s !, 2 5 9 - 2 7 1 ( 1 9 8 8 ) . P . H o f f m a n n , C r y o p r e s e r v a t i o n o f f u n g i . P u b l i c a t i o n No. 5 , ( 1 9 8 9 ) . UNESCO/WFCC T e c h n i c a l I n f o r m a t i o n S h e e t s . DSM, B r a u n s c h w e i g . J . Henry and 8 . K i r s o p , C r y o p r e s e r v a t i o n o f y e a s t s i n p o l y p r o p y l e n e s t r a w s . P u b l i c a t i o n No. 3 . UNESCO/WFCC T e c h n i c a l I n f o r m a t i o n S h e e t s . DSM, B r a u n s c h w e i g . K.A. Malik, Cryopreservation of bacteria with s p e c i a l reference t o a n a e r o b e s . P u b l i c a t i o n No. 4 , ( 1 9 8 9 ) . UNESCO/WFCC T e c h n i c a l I n f o r m a t i o n S h e e t s . DSM, B r a u n s c h w e i g . K . A . M a l i k , Use o f a c t i v a t e d c h a r c o a l f o r t h e p r e s e r v a t i o n o f a n a e r o b i c p h o t o t r o p h i c and o t h e r s e n s i t i v e b a c t e r i a by f r e e z e d r y i n g J o u r n a l o f M i c r o b i a l . Methods ( i n p r e s s ) , (1990). K . A . Malik, A s i m p l i f i e d l i q u i d - d r y i n g method f o r t h e p r e s e r v a t i o n o f microorganisms s e n s i t i v e t o f r e e z i n g and freeze-drying. J o u r n a l o f M i c r o b i a l Methods ( i n p r e s s ) , (1990). K . A . M a l i k , A new m e t h o d f o r l i q u i d - d r y i n g o f m i c r o o r g a n i s m s under a n a e r o b i c c o n d i t i o n s . J o u r n a l of M i c r o b i a l . Methods ( i n p r e s s ) , (1990). L . R . H i l l a n d M.I. K r i c h e v s k y , I n t e r n a t i o n a l S t r a i n D a t a Network. MIRCEN J o u r n a l 2 , 341-347 ( 1 9 8 6 ) . D. A l l s o p , D . L . Hawksworth a n d R . P l a t t , The CAB I n t e r n a t i o n a l Mycological I n s t i t u t e Culture Collection Database, Microbial C u l t u r e I n f o r m a t i o n S e r v i c e (MiCIS) and M i c r o b i a l I n f o r m a t i o n Network E u r o p e (MINE). I n t . B i o d e t e r . 2 5 , 1 6 9 - 1 7 4 , ( 1 9 8 9 ) . Guide t o t h e Deposit o f Microorganisms under t h e Budapest Treat y ; World I n t e l l e c t u a l P r o p e r t y O r g a n i z a t i o n Geneva, Second ed i t i o n , 1 9 8 9 , ISBN 9 2 - 8 0 5 - 0 1 9 5 - x . H . I i z u k a a n d T . H a s e g a w a ( E d s ) , P r o c e e d i n g s o f The F i r s t I n t e r n a t i o n a l C o n f e r e n c e on C u l t u r e C o l l e c t i o n s . U n i v e r s i t y Park Press. Baltimore (1970). M . Kocur a n d E . J . d a S i l v a ( E d s ) , P r o c e e d i n g o f The F o u r t h I n t e r n a t i o n a l C o n f e r e n c e on C u l t u r e C o l l e c t i o n s , 20-24 J u l y , 1 9 8 1 ,

-

34 35

367

-

Brno, C z e c h o s l o v a k a . World F e d e r a t i o n o f C u l t u r e Col e c t o n s , London ( 1 9 8 4 ) . Anonymous, P r o c e e d i n g s o f t h e Vth I n t e r n a t i o n a l C o n g r e s s o f C u l t u r e C o l l e c t i o n s , (1984). P o s t e r S e s s i o n A b s t r a c t s . Funny Press. Bangkok. MIRCEN News ( C o n t a c t Dr. E . J . d a S i l v a , D i v . S c i e n t i f i c R e s e a r c h a n d H i g h e r E d u c a t i o n , UNESCO, F - 7 5 0 0 7 P a r i s , F r a n c e .

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I S BIOTECHNOLOGY A BLESSING I N DISGUISE?

R.

KOKKE

R o y a l N e t h e r l a n d s Academy o f A r t s a n d S c i e n c e s , 1 0 0 0 G C , Amsterdam, The N e t h e r l a n d s We a r e c o n f r o n t e d w i t h t h e m a j o r p r o b l e m s : health,

t h e environment and p o l l u t i o n .

P.0.Box

19121,

population,

food,

I n many c a s e s b i o t e c h n o l o g y

can p r o v i d e l a s t i n g s o l u t i o n s and i s l i k e l y t o a f f e c t t h e d a i l y l i v e s o f a l l o f us. B e f o r e d i s c u s s i n g some n e g a t i v e a s p e c t s o f t h e a r e a s a l r e a d y mentioned, sently mation.

I want t o d r a w y o u r a t t e n t i o n t o a p r o b l e m t h a t h a s p r e -

received our greatest concern.

That i s t h e problem o f i n f o r -

B i o t e c h n o l o g y i s knowledge i n t e n s i v e .

We j u s t now know t h a t

we do n o t know e n o u g h a n d y e t we a r e s t i l l f o r c e d e.g. GEMS i n t o t h e e n v i r o n m e n t .

The q u e s t i o n i s :

t o release

When do we know enough?

I t i s n o t e a s y t o o b t a i n good k n o w l e d g e t h r o u g h l i t e r a t u r e s e a r c h e s

or by experiments. A l s o ,

knowledge once g a t h e r e d ,

i s p r o t e c t e d so

t h a t t h e p r o f i t s g a i n e d from i t w i l l b e n e f i t t h e r e s e a r c h e r s t o o . This i s i n turn,

a s t r o n g i n c e n t i v e f o r good r e s e a r c h .

There i s a l s o t h e q u a l i t y problem. posed t o t h e F l o o d o f f r e e i n f o r m a t i o n ,

Immediately a f t e r b e i n g exthe problem o f s e l e c t i n g

r e l e v a n t and r e l i a b l e i n f o r m a t i o n o c c u r s .

I t i s already d i f f i c u l t

t o c h o o s e f r o m t h e n u m b e r s o f b i o t e c h n o l o g i c a l s y m p o s i a . They h a v e increased logarithmically.

I n t h i s v e r y week t h e r e i s a l s o a n i n t e r -

n a t i o n a l b i o t e c h n o l o g y symposium j u s t n e x t d o o r t o me i n Amsterdam. N e x t week t h e r e i s one i n Copenhagen e t c . I n f o r m a t i o n h a s become a m a j o r c o n c e r n i n t h e w e s t e r n w o r l d a n d h a s s o m e t i m e s become " i n f o r m a t i o n

pollution".

I t i s often d i f f i -

c u l t t o f i n d o u t w h e t h e r one i s r e i n v e n t i n g t h e w h e e l o r , e v e n w o r -

s e , f o l l o w i n g a dead-end s t r e e t .

The p r o b l e m o f i n f o r m a t i o n o v e r l o -

ad i s o f c o u r s e n o t r e s t r i c t e d t o b i o t e c h n o l o g y .

B u t because o f i t s

m u l t i d i s c i p l i n a r y n a t u r e t h i s s c i e n c e i s p r o n e t o a l a r g e a n d sometimes errorneous i n f o r m a t i o n supply. The f a c t h a s b e e n r e c o g n i z e d t h a t t h e p r o l i f e r a t i o n o f i n f o r m a -

- 370 -

t i o n poses a g r e a t problem.

I t has e.g.

be come a c o n c e r n o f t h e Eu-

ropean Communities w i t h t h e r e s u l t s b e i n g t h a t i n f o r m a t i o n technol o g y and q u a l i t y c o n t r o l becoming a m a j o r p a r t o f t h e i r s c i e n c e programmes.

I n t h e p r o g r a m c a l l e d ESPRIT

11. ( E u r o p e a n S t r a t e g i c

Programme f o r R e s e a r c h a n d D e v e l o p m e n t i n I n f o r m a t i o n T e c h n o l o g i e s ) t h e e q u i v a l e n t o f about

I n another project

US d o l a r s i s s e t a s i d e f o r

2000 m i l l i o n

research s u p p o r t on t h i s m a t t e r

for

o f t h e EC,

a p e r i o d o f 5 years.

w i t h t h e acronym MINE

(standing

f o r M i c r o b i a l I n f o r m a t i o n Network Europe) c u l t u r e c o l l e c t i o n s o f m i c r o o r g a n i s m s a r e c o o p e r a t i n g t o c r e a t e a f u l l y i n t e g r a t e d and onl i n e a c c e s s i b l e common d a t a b a n k strains.

for a l l properties o f the stored

Q u a l i t y c o n t r o l i s an i n t e g r a l p a r t o f t h i s n e t w o r k .

European L a b o r a t o r y w i t h o u t w a l l s :

MINE].

A

[CEC

v a r i a t i o n f r o m o l d en-

glish dictionary states that information control i s l i k e diction a r i e s and watches,

y o u c a n n o t do w i t h o u t t h e m b u t e v e n t h e b e s t

cannot be expected t o always be acurate.

Q u a l i t y c o n t r o l and peer

r e v i e w a r e n e c e s s a r y c o n d i t i o n s f o r sound s c i e n t i f i c development s i n c e t h e y a v o i d i n f o r m a t i o n p o l l u t i o n a s much a s p o s s i b l e . hand,

On t h e o t h e r

they s h o u l d never v i o l a t e t h e p r i n c i p l e o f f r e e exchange o f

information.

You w i l l

agree t h a t t h i s i s a problem.

1990. Maintaining the growth o f science through infromation,

free

[C.W.

Gear;

exchange o f

A S M News 5 6 p p 6 4 a n d 6 5 1 .

SIDE-EFFECTS

I n t h i s context biotechnologists

should not solely

be concer-

ned about t h e r e l e a s e o f g e n e t i c a l l y manipulated organisms. mastered t h e n o n - l i v i n g

We h a v e

n a t u r e a n d a r e now w e l l i n t h e c o u r s e o f

d o m e s t i c a t i n g l i v i n g n a t u r e and t h e e n v i r o n m e n t .

Molecular biology

may r e f o r m l i v i n g n a t u r e t o c o n f o r m w i t h h uman w i l l s a n d n e e d s . have l e a r n e d from o u r s t r u g g l e t o m a s t e r p h y s i c a l n a t u r e .

We

We know

that i t i s impossible t o act without provoking counteraction.

And i n

b i o t e c h n o l o g y we m u s t l e a r n t o b a l a n c e o u r i n t e r e s t s a g a i n s t t h e negative side-effects.

F o r t h i s r e a s o n we n e e d t o p r o d u c e r e l i a b l e

r e s e a r c h d a t a and b e r e a l i s t i c .

Although the majority o f people to-

d a y c o n s i d e r t h e i n d u s t r i a l r e v o l u t i o n t o h a v e b e e n a p o s i t i v e movement,

the negative r e s u l t s should not be ignored.

h a s t a k e n many p e o p l e s l i v e s a n d h a p p i n e s s .

Industrialization

F o r example,

t h e number

o f p e o p l e b e i n g k i l l e d e v e r y y e a r b y c a r a c c i d e n t s i s many t i m e s h i g h e r t h a n t h e number o f p e o p l e d y i n g f r o m AIDS.

The i n d u s t r y a n d

i t s p r o d u c t s a l s o pose a g r e a t t h r e a t t o our environment.

Unfortu-

- 371

nately,

however,

-

a t the onset o f the i n d u s t r i a l revolution these

h a r m f u l e f f e c t s were n o t weighed a g a i n s t t h e b e n e f i t s .

Thus,

indus-

t r i a l d e v e l o p m e n t was d e t e r m i n e d b y t h e f r e e m a r k e t e c o n o m y . E v e n t o d a y many c o u n t r i e s s t i l l s t r i v e t o w a r d i n d u s t r i a l i z a t i o n , they consider

t o be t h e gateway t o p r o s p e r i t y .

which

I n t h i s context I

want t o remind you t h a t b i o t e c h n o l o g y i n t h e b r o a d e r sense i s n o t new.

A g r i c u l t u r a l development and t h e d o m e s t i f i c a t i o n o f a n i m a l s

h a v e gone o n f o r m i l l e n i a .

I r r e p a r a b l e n e g a t i v e e f f e c t s o f t h i s hu-

man i n t e r a c t i o n w i t h n a t u r e a r e v i s i b l e a l l o v e r t h e g l o b e . was f e r t i l e r e g i o n s , r i s delta for

What

as t h e M e d i t e r r a n e a n and t h e E u p h r a t e and T i g -

i n s t a n c e a r e now d e f o r e s t a t e d d r y l a n d .

A c e n t u r y ago

t h e l a s t g r e a t s l a v o n i a n oak f o r e s t s were c u t t o s u p p o r t t h e r a i l s o f w h a t was t h e n t h e new E u r o p e a n r a i l w a y s y s t e m .

W i t h t h e d e v e l o p m e n t o f b i o t e c h n o l o g y we h a v e t o t a k e i n t o account the negative side-effects. assesment.

This should be part o f our r i s k

It i s often j u s t a matter o f concentration,

though,

whet-

h e r w e c a l l i t a b e n i f i c i a l b i o l o g i c a l e f f e c t o r an unwanted s i d e effect.

[ F i g u r e 1. E f f e c t / d o s e

c u r v e f o r i n t a k e o f an e l e m e n t ] .

POPULATION AND FOOD The e n v i r o n m e n t a l r e l e a s e o f g e n e t i c a l l y m a n i p u l a t e d m i c r o o r g a n i s m s seems t o b e j u s t a m i n o r p r o b l e m when c o m p a r e d t o t h e n e e d f o r an i n c r e a s e i n c r o p p r o d u c t i o n . i s cultivated.

Nearly a l l agriculturable land

The c o m p a r a t i v e l y s m a l l a r e a o f t h e r e m a i n i n g n o t

y e t c u l t i v a t e d f e r t i l e l a n d s h o u l d be k e p t as r e s e r v a t i o n s o f b i o l o gical diversity.

A t t h e turn o f t h e ' c e n t u r y t h e r e w i l l be 6 b i l l i o n

p e o p l e o n t h i s g l o b e a n d t h e a g r i c u l t u r a b l e a r e a o f t h e e a r t h i s lim i t e d t o 1,5

b i l l i o n h e c t a r e s ( a b o u t t w i c e t h e s i z e o f t h e USA w i t -

hout Alaska).

Mankind i s r e a l i z i n g more and more t h a t r e s t r i c t i o n s

m u s t b e s e t o n i t s own g r o w t h . searchers,

A c c o r d i n g t o a g r o w i n g number o f r e -

t h e t h r e s h o l d o f what i s c o n s d e r e d t h e a c c e p t a b l e number

o f t h e human p o p u l a t i o n h a s b e e n c r o s s e d

The e x p l o i t a t i o n o f n a t u -

r a l r e s o u r c e s s u c h a s f o s s i l f u e l s was f r s t c o n s i d e r e d t h e m o s t important l i m i t a t i o n t o growth. impending on our c h i l d r e n ,

Although an energy shortage i s s t i l l

o u r a t t e n t i o n i s now f o c u s e d m o s t l y o n wa-

t e r and a i r p o l l u t i o n and t h e greenhouse e f f e c t . The e x t e n s i v e e x p l o i t a t i o n o f a g r i c u l t u r a l

land for

food produc-

t i o n l e a v e s l i t t l e room f o r t h e u s e o f b i o m a s s f o r o t h e r p u r p o s e s such as energy p r o d u c t i o n .

E x t e n s i v e a g r i c u l t u r a l g r o w t h i s needed

t o f e e d t h e human b i o m a s s .

Biotechnology has h e l p e d by producing h i g h

-

372

-

INTAKE OF ELEMENT

F i g . 1. The " S u p e r n u t r i t i o n C u r v e " s h o w i n g t h e t h r e e r a n g e s o f i n t a k e v e r s u s h e a l t h b e n e f i t s d e s c r i b e d i n t h e t e x t . Source: Based on P a s s w a t e r , R . 1975. S u p e r n u t r i t i o n . New Y o r k : D i a l P r e s s .

yielding plant varieties, pesticides.

e n v i r o n m e n t a l f r i e n d l y f e r t i l i z e r s and

On t h e o t h e r h a n d b i o t e c h n o l o g y i s b e i n g u s e d t o d e v e l o p

more p e s t r e s i s t e n t p l a n t s .

The h e t e r o t r o p h i c b i o m a s s o f man i s i n -

c r e a s i n g l y d e p l e t i n g t h e a u t o t r o p h i c biomass. q u e s t i o n t h a t i s now a s k e d i s :

One o f t h e m o s t v i t a l

Can t h e p h o t o s y n t h e t i c c a p a c i t y o f

t h e b i o s p h e r e b e e n h a n c e d y i e l d i n g more b i o m a s s ? T h i s c o u l d a l s o c o u n t e r a c t t h e c a r b o n d i o x i d e s u r p l u s and be a r e a l t r i u m p h o f b i o technology.

Some p r o b l e m s a r e a l r e a d y b e i n g a d d r e s s e d s u c c e s f u l l y b y

b i o t e c h n o l o g y . I n t e n s i f i e d a g r i c u l t u r e i s e n a b l i n g us t o f e e d t h e peo p l e l i v i n g on t h e e a r t h ' s

poor surface.

t o i t i s a hugh c a t t l e s t o c k .

A Netherlands contribution

A n i m a l h u s b a n d r y p r o v i d e s h i g h l y ap-

p r e c i a t e d m i l k and meat w i t h e s s e n t i a l p r o t e i n . an h u g h d u n g p r o b l e m . t h e water supplies.

B u t i t has created

T h i s dung has a c i d i f i e d t h e s o i l and p o l l u t e d

Some t e l i e f c a n b e o b t a i n e d b y a n a e r o b i c f e r m e n -

t a t i o n o f t h e dung which p r o d u c e s c o m p o s t a n d g i v e s a s m a l l r e t u r n i n t h e form o f biogas.

I t w o u l d b e a g r e a t i m p r o v e m e n t i f we c o u l d consume l e s s a n i m a l protein.

G r o w i n g s o y b e a n s w o u l d y i e l d 7 t i m e s more e d i b l e p r o t e i n p e r

a c r e t h a n r a i s i n g c a t t l e on t h e same f i e l d . c a n b e made v e r y d i g e s t a b l e

Soybeans and o t h e r beans

f o r man b y a s i m p l e f e r m e n t a t i o n t e c h n i -

que t r a d i t i o n a l l y a p p l i e d i n I n d o n e s i a .

The p r o d u c t i s c a l l e d tempeh.

-

373

-

Soybeans a r e f e r m e n t e d i n s o l i d - s t a t e

w i t h t h e mould Rhizopus.

The

p r o d u c t h a s n o p r o n o u n c e d t a s t e and i t c a n b e c o o k e d o r b a k e d j u s t [ F i g u r e 2.

l i k e meat w i t h s i m i l a r f l a v o u r i n g . o f d e h u l l e d soybeans:

Tempehl.

t y a c i d s and l a c k s c h o l e s t e r o l , [W.

S h u r t l e f f and A .

p e r 8 Row p u b l .

Moreover,

Thisopus fermentation

i t i s low i n saturated fat-

t h u s i t i s good f o r o u r h e a r t s .

A o y a g i 1985.

The b o o k o f t e m p e h ,

2nd ed.

Har-

New Y o r k l .

F i g . 2 . Tempeh: D e h u l l e d s o y b e a n s a r e s o a k e d , i n o c c u l a t e d w i t h R h i zopus spores, wrapped i n p l a s t i c f o i l , i n c u b a t e d a t 30 degrees c e l s i u s f o r 3 6 h o u r s as shown. F o r c o n s u m p t i o n t h i s p r o d u c t is c o o k e d , baked, e t c . l i k e t e n d e r meat.

E N V I R O N M E N T AND POLLUTION The B r u n d t l a n d r e p o r t s t a t e s on t h i s s u b j e c t : development a r e n o t separate challenges.;

" E n v i r o n m e n t and

they are inexorably linked.

Development c a n n o t s u b s i s t upon a d e t e r i o r a t i n g e n v i r o n m e n t a l r e s o u r ce base;

t h e e n v i r o n m e n t c a n n o t b e p r o t e c t e d when g r o w t h d o e s n o t

account f o r t h e cost o f environmental d e s t r u c t i o n .

It i s evident

t h a t these problems cannot be t r e a t e d s e p a r a t e l y by fragmented i n s t i t u t i o n s and p o l i c i e s . s e and e f f e c t ,

They a r e l i n k e d i n a c o m p l e x s y s t e m o f c a u -

[ p 3 2 i n OUR C O M M O N FUTURE,

E n v i r o n m e n t and Development, s i t y P r e s s 19871.

b y G.H.

The W o r l d C o m m i s s i o n on

B r u n d t l a n d e.8.

Oxford Univer-

We b i o t e c h n o l o g i s t s h a v e a g r e a t t a s k t o a c c o m p l i s h

i n t h i s complex and i n t e r l i n k e d w o r l d .

B i o t e c h n o l o g i s t s a r e now t h e

-

374 -

hope f o r s u r v i v a l o f t h e b i o s p h e r e .

B u t how c a n b i o t e c h n o l o g i s t s

work w i t h t h i s " l i n k a g e " ? B i o t e c h n o l o g y c a n b e p o l l u t i n g and i t c a n b e u s e d t o f i g h t p o l lution.

An e x a m p l e :

the Philippines,

I n t h e I n t e r n a t i o n a l R i c e Research I n s t i t u t e i n

h i g h y i e l d i n g r i c e v a r i e t i e s have been developed.

A r i c e h a r v e s t can t h u s be i n c r e a s e d 2 t o 3 t i m e s . a l s o needs

more f e r t i l i z e r .

But t h i s crop

I n t r a d i t i o n a l agriculture the r i c e

c r o p was i n b a l a n c e w i t h t h e a v a i l a b l e n u t r i e n t s .

I t h a s b e e n shown

t h a t i n r i c e f i e l d s a very a c t i v e nitrogen f i x i n g m i c r o f l o r e e x i s t s w h i c h s u p p l i e s t h e g r o w i n g r i c e w i t h i t s need f o r n i t r o g e n . yielding r i c e plant

The h i g h

i s n o t i n b a l a n c e w i t h t h i s m i c r o f l o r e and t h e

requirement f o r a d d i t i o n a l n u t r i e n t s i s great.

A s o l u t i o n would be

t o increase the capacity o f the o r i g i n a l microflore.

Possibly

GEMS

c a n be made w h i c h keep t h e p a c e w i t h t h e r e q u i r e m e n t s o f t h e h i g h yielding rice.Their r y act,

r e l e a s e i n t h e e n v i r o n m e n t w i l l be a b e n e f a c t o -

we assume.

Another example.

P a r t o f t h e t o x i c compounds w h i c h we b r i n g

w a n t i n g l y or u n w a n t i n g l y i n t o t h e environment can be t r e a t e d by b i o remediation.

C h a k r a b a r t y was t h e f i r s t r e s e a r c h e r who was g r a n t e d

a p a t e n t i n t h e U S A on a g e n e t i c a l l y e n g i n e e r e d m i c r o - o r g a n i s m .

The

m i c r o b e s (Pseudomonas s p e c i e s ) were e n g i n e e r e d i n s u c h a way t h a t t h e y p r o d u c e a d e t e r g e n t w h i c h g i v e s them an e n h a n c e d c a p a c i t y t o attack o i l pollution.

R e c e n t l y i t was r e p o r t e d t h a t m i c r o o r g a n i s m s

h a v e b e e n u s e d s u c c e s f u l l y i n p r e v e n t i n g o i l p o l l u t i o n on t h e c o a s t o f Texas. Another example i s t h e development o f h e r b i c i d e - t o l e r a n t

crop

plants.

Tolerance f o r g l y p h o s p h a t e h e r b i c i d e s has been b u i l d i n t o

tomato,

cotton,

t o b a c c o and soybean.

c r o p s can be c u l t i v a t e d w i t h o u t w i l l have h i g h e r h a r v e s t y i e l d s .

The r e s u l t b e i n g t h a t t h o s e

t h e c o m p e t i t i o n f r o m weeds a n d t h u s

I n a d d i t i o n c o s t l y l a b o u r i s saved.

The h e r b i c i d e s and t h e h e r b i c i d e - t o l e r a n t c h e m i c a l companies f o r p r o f i t s sake. the plants.

p l a n t s a r e developed by

They s e l l t h e h e r b i c i d e s a n d

I t h i n k we s h o u l d n o t a l l o w a g r i c u l t u r e t o become s o d e -

p e n d a n t on i n d u s t r i a l c o m p a n i e s .

The b i o l o g i c a l d i v e r s i t y

s a v e d and e x p l o i t e d f o r t h e b e n e f i t o f m a n k i n d . blem.

Companies have n o e n v i r o n m e n t a l

task.

must b e

Here l i e s the pro-

Thus t o s u p p l y s o u n d

i n f o r m a t i o n on t h e r i s k s a n d b e n e f i t s i s a n o t h e r t a s k a n d r e s p o n s i b i l i t y of biologists.

I am n o t p l e a d i n g f o r a more a n d cumbersome

r e g u l a t i o n t h a t c a n b e c i r c u m v e n t e d anyway,

b u t f o r conscience.

P u b l i c awareness i s a v e r y p o w e r f u l t o o l i n t h e s e m a t t e r s .

- 375 -

ECONOMICS The c o m p e t e t i v e n e s s o f t h e E u r o p e a n e n t e r p r i s e s s h o u l d b e p r o -

I t w i l l b e bad i f a l l biotechnology h a s t o be i m p o r t e d . I n

tected.

t h i s c o n n e c t i o n t h e c h e c k l i s t made by t h e O E C D ( O r g a n i s a t i o n f o r Economic C o - o p e r a t i o n

and Development) is still r e l e v a n t . [ T a b l e 1).

TABLE 1 A check list f o r s t r a t e g i c planning i n biotechnology

1. R e s o u r c e s Raw m a t e r i a l s i n c l u d i n g f e e d s t o c k s , w a t e r a n d m i n e r a l s ; energy; land a v a i l a b i l i t y ; competing technol o g i e s ; manpower. 2 . S c i e n t i f i c and t e c h n o l o g i c a l

infrastructure

E d u c a t i o n ; t r a i n i n g ; r e s e a r c h b a s e a n d R II D p r i o r i t i e s ; information transfer. 3 . Climate f o r innovation

I n v e n t i o n - i n n o v a t i o n time l a g ; i n d u s t r i a l b a s e ; competition; finance; regulati3ns; patent laws; social acceotabilitv.

1

4. Trading position

Commodity p r i c e s ; i m p o r t - e x p o r t l a r l v f o r food: markets.

I

balances,

particu-

5. Environmental c o n s i d e r a t i o n s Land use; p o l l u t i o n , on a n d management.

e f f l u e n t and waste,

its l o c a t i -

An e x a m p l e o f a p p l i c a t i o n s o f b i o t e c h n o l o g y w i t h s e v e r e e c o n o m i c a l c o n s e q u e n c e s i s a c t u a l l y t h e c a s e o f High F r u c t o s e Corn S y r u p (HFCS)

[Table 21.

TABL' 2 C o m p o s i t i o n o f HFCS a n d i n v e r t s u g a r HFCS v a r i e t i e s

Components -

[XI

invert sugar [ x j

Fru 3t ose

42

55

90

49

GlLcose

51

40

2

49

5

3

2 (sucrose)

0.03

0.3

0.4

Maltose

4.5

Maltotriose

1

Higher sugars

1.5

Ash

0.04

HCFS i s d e r i v e d f r o m c o r n ( m a i z e ) s t a r c h a n d p r o d u c e d p a r t l y by u s i n g immobilized enzymes.

I t has a well established market.

I t is

-

376

-

u s e d a s a s w e e t e n e r i n many s o f t d r i n k s w h e r e i t r e p l a c e s i n v e r t s u gar.

S u g a r f l a v o u r c a n now a l s o b e p r o d u c e d a s t h a u m a t i n .

T h i s na-

t u r a l compound d e r i v e d f r o m t h e f r u i t o f an A f r i c a n t r e e i s on t h e b a s i s o f weight,1000

t i m e s sweeter

t h a n saccharose.

The g e n e s u s e d

t o produce t a u m a t i n i c a c i d have been s u c c e s f u l l y c l o n e d i n t o y e a s t and b r o u g h t t o e x p r e s s i o n .

[

L. Edens and H. Van d e r Wel, 1 9 8 5 . M i c r o -

b i a l s y n t h e s i s o f t h e sweet t a s t i n g p l a n t p r o t e i n Thaumatin, i n B i o t e c h n o l o g y 3 pp 61-64].

The u s e o f HFCS h a s e f f e c t e d t h e su-

g a r c a n e m a r k e t and t h u s many 3 r d w o r l d c o u n t r i e s . are l e f t over with a surplus o f sugar. ses a r e b e i n g used f o r

c a r combustion.

Trends

These c o u n t r i e s

I n B r a z i l the sugar surplus-

the fermentative production o f ethanol

for

T h i s example o f t h e economic consequences o f b i o -

t e c h n o l o g y s h o u l d however, be a w a r n i n g . may b e p r o d u c e d b y b i o t e c h n o l o g y

Many s p i c e s a n d f l a v o u r s

including coffee,

t e a and cocoa.

I n f a c t a l l t h o s e n a t u r a l p r o d u c t s w h i c h h a v e a l o w mass y i e l d when compared t o t h e b i o m a s s n e e d e d t o p r o d u c e i t , c o u l d t h e o r e t i c a l l y b e grown e c o n o m i c a l l y

i n fermentors.

I t depends on t h e m a r k e t whet-

h e r t h e y w i l l b e p r o d u c e d i n t h i s way o r

not.

Countries producing

t h e s e c o m m o d i t i e s s h o u l d know t h i s . HEALTH S i n c e man h i m s e l f i s a b i o l o g i c a l biotechnology too. diseases.

being,

he may b e s u b j e c t t o

Man h a s l e a r n e d t o c o p e w i t h t h e m o s t i n f e c t i o u s

Many p h a r m a c e u t i c a l s a r e n a t u r a l p r o d u c t s a n d c a n t h u s

be produced by f e r m e n t a t i o n .

A t t e m p t s t o c u r e human b e i n g s w i t h i n -

h e r i t e d a i l m e n t s h a v e a l r e a d y b e e n made b y t h e i m p l a n t a t i o n o f h e a l t h y f o r e i g n c e l l s o r m o d i f i e d human c e l l s .

The n e x t s t e p m i g h t b e

t h a t a p e r s o n would want t o improve c e r t a i n i n t e l l e c t u a l c a p a c i t i e s and t o f i g h t w h a t

i s called eurosclerosis.

May b e we w i s h t o h a v e

human b e i n g s t h a t a r e l e s s g r e e d y s i n c e t h i s p r o p e r t y c a n b e c o n s i dered as t h e f a c t o r r e s p o n s i b l e f o r t h e s e v e r e d e t e r i o r a t i o n ongoing i n t h i s o t h e r w i s e so b e a u t i f u l world. goal of

S h o u l d we a l l o w t h e u l t i m a t e

b i o t e c h n o l o g y t o be t h e improvement o f mankind b y mankind?

T h i s would c e r t a i n l y be t h e s o l u t i o n f o r t h e o v e r p o p u l a t i o n w h i c h

i n t u r n i s thecause o f the shortages.

F o r as l o n g a s m a n k i n d c a n n o t

c o n t r o l h i s own g r o w t h h e i s h e a d i n g f o r h i s own d o w n f a l l .

Do we

need b i o t e c h n o l o g y t o c o r r e c t o u r s e l v e s and c r e a t e A l d o u s H u x l e y ’ s B r a v e New W o r l d ?

-

377

-

T A K I N G RISKS Coming t o t e r m s w i t h t h e b i o s p h e r e a n d t a k i n g i n t o a c c o u n t a l l possible side-effects R i s k Assesment.

i s d e v e l o p i n g i n t o a s c i e n c e on i t s own m e r i t s :

T h i s s c i e n c e s h o u l d n o t become s o d o m i n a n t t h a t i t

w i l l hamper t h e d e v e l o p m e n t o f b i o t e c h n o l o g y ,

though.

Given the f a c t

t h a t . we h a v e c r o s s e d b o r d e r l i n e s we a r e u r g e d t o t a k e r i s k s e v e n i f we d o n ’ t

have enouph knowledge.

I n my p r e v i o u s t a l k i n t h i s same

town I s t r e s s e d t h e p o s s i b l e b l e s s i n g s o f b i o t e c h n o l o g y . t u a l e x e r t i o n s can be used w i t h e v i l i n t e n t i o n s .

m o s t o t h e r human e n t e r p r i s e s w i l l n o t b e p e r f e c t t o o . u n d e s i r a b l e and m a l i c i o u s s i d e e f f e c t s . ned i n t h i s t a l k .

Intellec-

Biotechnology l i k e There w i l l be

Some o f them I h a v e m e n t i o -

O t h e r s we may n o t e v e n h a v e t h o u g h t o f .

I am c o n -

f i d e n t h o w e v e r t h a t w i t h an open e x c h a n g e o f a l l t h e a v a i l a b l e knowl e d g e i n c l u d i n g t h a t on p o s s i b l e n e g a t i v e s i d e e f f e c t s ,

humanity

w i l l be a b l e t o use b i o t e c h n o l o g y w i t h a n e t r e s u l t o f b e i n g a b l e s -

sing.

B i o t e c h n o l o g i s t s m u s t now assume t h e r e s p o n s i b i l i t y

welfare o f our world.

s p h e r e o f w h i c h man i s t h e d o m i n a n t e l e m e n t . might

f o r the

Not o n l y f o r humankind b u t f o r t h e whole b i o This responsibility

t u r n o u t t o b e e v e n h e a v i e r t h a n when n a t i o n s d e v e l o p e d i n t o

n u c l e a r powers.

Therefore

I am p l e a d i n g f o r m o r e r e l i a b l e s c i e n t i f i c

i n f o r m a t i o n a n d f o r more c o n s c i e n c e .

As a g r o u p a n d i n d i v i d u a l l y ,

b i o t e c h n o l o g i s t s s h o u l d be h e l d r e s p o n s i b l e f o r t h e i r works a c t i o n s just

l i k e t h e e n g i n e e r i s r e s p o n s i b l e f o r t h e b r i d g e h e b u i l d s and

t h e medics1 d o c t o r f o r t h e h e a l t h o f h i s p a t i e n t s .

I r e m i n d you t h a t

p o l i t i c i a n s a n d s c i e n t i s t s once a g r e e d on e s t a b l i s h i n g t h e I n t e r n a t i o n a l A t o m i c E n e r g y Agency w i t h t h e o b j e c t i v e of

t o control

t h e misuse

t h e f e a r e d n u c l e a r power and a p p l y i t t o p e a c e f u l developments.

I t a l s o seems t i m e t h a t a p o l i t i c a l b o d y s h o u l d b e c r e a t e d t h a t sup e r v i s e s t h e sound a p p l i c a t i o n o f b i o t e c h n o l o g y t o t h e b e n e f i t o f all.

This Page Intentionally Left TBlank

I M P R O V E M E N T S O F AGRICULTURAL CROPS B Y G E N E T I C E N G I N E E R I N G

J . BOTTERMAN P l a n t G e n e t i c Systems N . V . , 900(1 G e n t , B e l g i u m

J.

Plateaustraat 22,

INTRODUCTION I m p r o v i n g t h e q u a l i t y and y i e l d o f c r o p s t h r o u g h b r e e d i n g has been f o r l o n g t i m e a s t r a i g h t f o r w a r d concept c o u l d o n l y be

U n t i l 1983,

t h i s goal

achieved by s e x u a l r e c o m b i n a t i o n f o l l o w e d by s e l e c -

t i o n o r , t o a l e s s e r degree,

by random o r i n d u c e d m u t a t i o n s .

t r a i t s have been t r a n s f e r r e d t o a g r i c u l t u r a l t e d p l a n t s t h r o u g h t e d i o u s and time-consuming

Useful

crops from non-cultivaprograms.

T h i s approach

has r e s u l t e d i n g e n e t i c improvements y i e l d i n g h i g h e r p r o d u c t i v i t y f o r several major crops i n c l u d i n g corn,

soybean and wheat.

Recent p r o g r e s s i n m o l e c u l a r and c e l l u l a r b i o l o g y has g r e a t l y e x t e n d e d t h e r a n g e o f s o u r c e s f r o m w h i c h new t r a i t s c a n b e o b t a i n e d f o r c r o p improvement.

DNA t e c h n o l o g y ,

U s i n g m o l e c u l a r t e c h n i q u e s b a s e d on r e c o m b i n a n t

i t became p o s s i b l e t o t r a n s f e r g e n e s f r o m any o r g a -

nism i n t o p l a n t s without

sexual crossing.

This technology should

represent a major progress i n our e f f o r t s towards i n c r e a s i n g t h e p r o d u c t i o n and u t i l i t y o f a g r i c u l t u r a l

crops.

The s p e e d a t w h i c h t h e new t e c h n o l o g y a d v a n c e s h a s t o be r e a l i z e d ( T a b l e 1). I n 1983 t h e f i r s t so c a l l e d t r a n s g e n i c p l a n t s were d e v e l o p e d u s i n g a common s o i l b a c t e r i u m ,

Aqrobacterium tumefaciens,

to shuttle a

b a c t e r i a l a n t i b i o t i c u m r e s i s t a n c e gene i n t o t h e m o d e l p l a n t s p e c i e s tobacco or petunia.

Only f i v e y e a r s l a t e r ,

and t i s s u e c u l t u r e t o t r a n s f o r m

i m p o r t a n t c r o p s a n d t h e gene t r a n s f e r

t e c h n o l o g y have grown d r a m a t i c a l l y .

by m i c r o i n j e c t i o n ,

the capacity t o apply c e l l

P l a n t c e l l s can be t r a n s f o r m e d

by use o f Aqrobacterium T i p l a s m i d v e c t o r s o r by

d i r e c t u p t a k e o f e x o g e n o u s DNA

[ l l . T r a n s g e n i c p l a n t s have been

o b t a i n e d f o r more t h a n t w e n t y d i f f e r e n t p l a n t s p e c i e s , p o r t a n t f i e l d c r o p s such as o i l s e e d rape,

sugarbeet,

including im-

cotton,

soybean

and c o r n and i t can be e x p e c t e d t h a t i n t h e n e a r f u t u r e a l l m a j o r

-

380

-

TABLE 1 History o f p l a n t genetic engineering 1911

A q r o b a c t e r i u m t u m e f a c i e n s causes c r o w n - g a l l

1974

Tumour i n d u c i n g p l a s m i d :

tumours

Ti-plasmid

1977

T-DNA

1982

Transformed tobacco p l a n t s

1983

C h i m e r i c gene t r a n s f e r r e d a n d e x p r e s s e d

1984

Versatile

1984

R e g u l a t e d e x p r e s s i o n o f c h i m e r i c gene

transfer t o plant cells

v e c t o r systems

E x p r e s s i o n o f agronomic u s e f u l t r a i t s insect resistance

1985

crop

1986

virus resistance

1986

herbicide resistance

s p e c i e s w i l l b e a c c e s s i b l e t o m o d i f i c a t i o n u s i n g gene t r a n s -

f e r technology. M o l e c u l a r b i o l o g i s t s h a v e made e q u a l l y d r a m a t i c p r o g r e s s i n t h e i d e n t i f i c a t i o n and improvement o f genes e n c o d i n g v a l u a b l e a g r o nomic t r a i t s .

T r a n s f e r o f t h e s e genes i n t o p l a n t s l e a d s t o t h e c r e -

a t i o n o f c r o p s w i t h p r o p e r t i e s w h i c h were u n a c h i e v a b l e by c l a s s i c a l b r e e d i n g approaches. Industry

i s r a p i d l y moving towards p r a c t i c a l a p p l i c a t i o n o f

t h e s e new o p p o r t u n i t i e s .

P r e s e n t r e s e a r c h and development programs

e m p h a s i z e on c r o p p r o t e c t i o n , and t e c h n i c a l

feasibility.

because o f i t s i n d u s t r i a l r e l e v a n c e

The f i r s t a c h i e v e m e n t s were t o i n t r o d u c e

agronomic t r a i t s such as t o l e r a n c e t o h e r b i c i d e s and r e s i s t a n c e t o i n s e c t s and v i r u s e s . MOLECULAR S T R A T E G I E S FOR C R O P P R O T E C T I O N

Herbicide tolerance H e r b i c i d e s a r e an i n d i s p e n s a b l e t o o l i n m o d e r n a g r i c u l t u r e f o r e c o n o m i c weed c o n t r o l .

A t present,

over a hundred p h y t o t o x i c mole-

c u l e s a r e u s e d as c o m m e r c i a l h e r b i c i d e s .

A number o f new compounds

combine h i g h e f f e c t i v i t y w i t h n o n - t o x i c i t y break-down i n t h e s o i l .

However,

t o a n i m a l s and r a p i d

they o f t e n l a c k s e l e c t i v i t y which

l i m i t s t h e i r use t o preemergence a p p l i c a t i o n s .

Engineering o f plants

t o become r e s i s t a n t t o b r o a d s p e c t r u m h e r b i c i d e s w o u l d a l l o w a a e l e c -

-

381

-

t i v e use o f these chemicals f o r c r o p p r o t e c t i o n . species w i t h b u i l t - i n tolerance,

[21.

ted

To c r e a t e new p l a n t -

two approaches have been i n v e s t i g a -

The f i r s t c o n s i s t o f e i t h e r r e n d e r i n g t h e t a r g e t

for

h e r b i c i d a l a c t i o n i n s e n s i t i v e t o t h e herbicide or overproducing a s e n s i t i v e t a r g e t p r o t e i n a s shown w i t h e n g i n e e r e d t o l e r a n c e t o g l y R p h o s a t e (RoundupR, M o n s a n t o ) , t h e s u l f o n y l u r e a s ( e . 9 . G l e a n R , O u s t , DuPont) and i m i d a z o l i n o n e s (e.9. mid).

S c e p t e r R , A r s e n a l R A m e r i c a n Cyana-

The s e c o n d a p p r o a c h i n v o l v e s t h e i n t r o d u c t i o n i n p l a n t s o f a

pathway w h i c h degrades or d e t o x i f i e s t h e h e r b i c i d e p r i o r t o i t s action.

Two s u c c e s s f u l e x a m p l e s i l l u s t r a t e how g e n e s f o r d e t o x i f y i n g

enzymes w e r e i s o l a t e d f r o m m i c r o o r g a n i s m s a n d p l a n t s e x p r e s s i n g t h e se genes w e r e r e s i s t a n t t o g l u f o s i n a t e ( B a s t a R H o e c h s t )

t o b r o m o x y n i l (Rhone P o u l e n c )

respectively.

131 o r

P l a n t s grown i n t h e

g r e e n h o u s e o r i n o p e n f i e l d showed c o m p l e t e r e s i s t a n c e t o w a r d s h i g h d o s e s o f g l u f o s i n a t e a n d t h e i r p e r f o r m a n c e was i n d e p e n d e n t f r o m t h e p l a n t s p e c i e s used, to,

a s shown w i t h t r a n s g e n i c t o b a c c o ,

potato,

toma-

o i l s e e d r a p e and s u g a r b e e t . The i n t r o d u c t i o n o f e n g i n e e r e d h e r b i c i d e r e s i s t a n t c r o p s w i l l

h a v e c o n s i d e r a b l e i m p a c t on f u t u r e weed c o n t r o l s t r a t e g i e s a n d on the g l o b a l herbicide markets.

F a r m e r s w i l l be a b l e t o a p p l y t h e s e

h e r b i d i c e s f o r p o s t emergence a p p l i c a t i o n s i n m o r e e f f e c t i v e ,

fle-

x i b l e a n d c h e a p e r weed c o n t r o l p r o g r a m s .

Insect resistance I n modern a g r i c u l t u r e ,

chemical i n s e c t i c i d e s are extensively

used f o r i n s e c t p e s t c o n t r o l .

However,

i t i s very d i f f i c u l t t o t a r -

get insecticides s p e c i f i c a l l y a t pest insects, ted ecological side effects.

Moreover,

without

h a v i n g unwan-

i n s e c t s become m o r e a n d m o r e

r e s i s t a n t t o t h e c h e m i c a l s making t h e i r use more e x p e n s i v e or even obsolete. B i o l o g i c a l c o n t r o l o f insect pests,

on t h e o t h e r hand,

combines a h i g h i n s e c t t o x i c i t y w i t h e n v i r o n m e n t a l s a f e t y .

Biological

i n s e c t i c i d e s based on f o r m u l a t i o n s o f t h e m i c r o o r g a n i s m B a c i l l u s t h u r i n q i e n s i s have been used o v e r t h e l a s t t w e n t y years.

Their to-

x i c i t y t o w a r d s i n s e c t s i s due t o t h e p r o d u c t i o n o f a p r o t e i n c a l l e d delta-endotoxin sects,

(Bt),

which i s s e l e c t i v e l y

t o x i c t o larvae o f in-

b u t c o m p l e t e l y h a r m l e s s t o humans a n d a n i m a l s a n d e v e n t o b e -

n e f i c i a l insects,

s u c h as honey bees.

However,

u s i n g t h e s e compouds

i n s p r a y i n g p r o g r a m s p r e s e n t s some p r a c t i c a l p r o b l e m s .

The t o x i n s a r e

r e l a t i v e l y u n s t a b l e i n f i e l d c o n d i t i o n s and t h e y have a l s o no s y s t e m i c a c t i o n on p l a n t s .

Moreover, production c o s t s f o r B.t.

sprayable

- 382

-

p r o d u c t s a r e s t i l l r e l a t i v e l y h i g h compared w i t h competing c h e m i c a l s . R e c e n t l y , a major o b j e c t i v e o f p l a n t b i o t e c h n o l o g y is t o c r e a t e i n sect resistant plants.

In 1986, c a t e r p i l l a r r e s i s t a n t tobacco p l a n t s

w e r e o b t a i n e d by e n g i n e e r i n g p l a n t s w h i c h e x p r e s s a B a c i l l t r s t h u r i n q i e n s i s (Bt) gene which encodes a h i g h l y a c t i v e and s p e c i f i c i n s e c t controlling protein.

These p l a n t s produce t h e B . t .

protein inside

t h e t i s s u e , thereby eliminating the drawbacks of a sprayable product 141. D i f f e r e n t s t r a i n s o f t h e b a c t e r i u m v a r y i n t h e r a n g e o f

i n s e c t s a g a i n s t which t h e i r t o x i n is a c t i v e and i n s e c t c o n t r o l l i n g p r o t e i n s with a c t i v i t y a g a i n s t Lepidoptera, D i p t e r a and Coleoptera h a v e b e e n i d e n t i f i e d a n d i s o l a t e d . Many g r o u p s a r e c u r r e n t l y w o r k i n g on t h e i n s e r t i o n o f t h e e n c o d i n g g e n e s i n d i f f e r e n t p l a n t s p e c i e s . Virus resistance

A l s o i n 1 9 8 6 , t r a n s g e n i c t o b a c c o p l a n t s p r o t e c t e d from v i r a l i n f e c t i o n were o b t a i n e d b y p r o d u c i n g t h e v i r a l c a p s i d p r o t e i n o f t o bacco mosaic v i r u s

[51.

Recently, engineered virus tolerance

h a s been shown w i t h a number o f v i r u s e s s u c h a s a l f a l f a m o s a i c v i -

rus, cucumber mosaic v i r u s , p o t a t o v i r u s X and t o b a c c o r i n g s p o t v i r u s a n d was o b t a i n e d by e x p r e s s i o n o f a c a p s i d p r o t e i n ,

antisense

mRNA s y n t h e s i s or expression of s a t e l l i t e RNA molecules i n d i f f e r e n t

p l a n t s p e c i e s . Although t h e commercial value o f t r a n s g e n i c p l a n t s e x h i b i t i n g v i r u s t o l e r a n c e i s d i f f i c u l t t o e s t i m a t e due t o l a c k of s u p p o r t i n g d a t a from c r o p l o s s e s from v i r u s a t t a c k , t h i s t e c h n o l o g y m i g h t h a v e b r o a d a p p l i c a t i o n s t o r e d u c e t h e y i e l d loss c a u s e d b y s o -

me w i d e s p r e a d v i r u s e s . FIELD TRIALS A N D R E G U L A T O R Y ISSUES These t h r e e examples i l l u s t r a t e t h e molecular s t r a t e g i e s f o l l o wed f o r c r o p p r o t e c t i o n a n d t h e s e a c h i e v e m e n t s h a v e a l r e a d y e n t e r e d i n t o a f u r t h e r phase of development.

I n 1987 s m a l l s c a l e f i e l d t r i a l s

were performed with t h e s e c r o p s and revealed f i e l d performance which was c o m p a r a b l e w i t h g r e e n h o u s e t e s t s . Some o f

t h e s e c r o p s c a n b e d e v e l o p e d i n t o commercial p r o d u c t s

i n l e s s t h a n f o u r y e a r s from now, b u t d i f f e r e n t i m p o r t a n t i s s u e s have s t i l l t o be a d d r e s s e d . Although p l a n t b r e e d i n g p r o d u c t s have a l ways been f r e e l y d i s t r i b u t e d , t r a n s g e n i c p l a n t s r e q u i r e r e g u l a t o r y a p p r o v a l b e f o r e e v e n small s c a l e f i e l d t e s t i n g c a n b e p e r f o r m e d .

The-

r e f o r e , i t is of extreme importance t h a t t h e process f o r e v a l u a t i n g f i e l d t e s t i n g of g e n e t i c a l l y modified crops responds quickly t o t h e

-

383

-

need f o r t e s t i n g p l a n t s a t m u l t i p l e l o c a t i o n s and under normal agronomic p r a c t i c e s , i n c l u d i n g c o m p l e t i o n o f t h e c r o p r e p r o d u c t i o n cycle i n normal production a r e a s .

Such a r e g u l a t o r y p r o c e s s should s a t i s f y

both l e g i t i m a t e concerns r e g a r d i n g environmental impact and h e a l t h and t h e need t o l e t r e s e a r c h and development proceed i n a r a t i o n a l a n d e f f i c i e n t way.

Moreover,

these f i e l d trials with engineered

c r o p s o v e r t h e n e x t t w o t o t h r e e y e a r s w i l l have t o t e a c h u s more about t h e q u a l i t a t i v e and q u a n t i t a t i v e c h a r a c t e r i s t i c s o f engineered c r o p s r e l a t i v e t o c o m p e t i t i v e e x i s t i n g p r o d u c t s , a b o u t t h e i r ecolog i c a l impact such a s p o t e n t i a l r i s k s f o r t h e outcrossing of herbicide r e s i s t a n c e t r a i t s and g e t t i n g i n s e c t r e s i s t a n c e t o B . t . controlling proteins.

insect

Also, it is c r i t i c a l t h a t regulations dealing

w i t h t h e c o m m e r c i a l i z a t i o n o f t h e s e c r o p s be f o r m u l a t e d a n d harmon i z e d i n a way w h i c h d o e s n o t d i s c r i m i n a t e t h e p a r t i c u l a r b i o t e c h nology p r o c e s s used t o improve t h e v a r i e t i e s . Another important i s s u e is t h a t g e t t i n g a c l e a r - c u t

patent protec-

t i o n i s a c o n d i t i o n e s i n e q u a non f o r t h e s u c c e s s o f a p r o d u c t from plant biotechnology.

Recently a tendency i s emerging t o l i b e r a l i z e

t h e p a t e n t p r o t e c t i o n f o r p l a n t s c r e a t e d t h r o u g h t h e use o f g e n e t i c e n g i n e e r i n g , which i n d i c a t e s t h a t p l a n t b i o t e c h p r o d u c t s w i l l g e t a s i m i l a r p r o p r i e t a r y p r o t e c t i o n a s f o r e x a m p l e new p h a r m a c e u t i c a l compounds.

C R O P QUALITY IMPROVEMENT Molecular s t r a t e g i e s a r e a l s o followed t o improve t h e end prod u c t o f t h e c r o p i t s e l f y i e l d i n g more n u t r i t i o u s o r h i g h e r - q u a l i t y c r o p s . A l t e r i n g t h e p r o t e i n q u a l i t y i n s e e d s by t r a n s f e r o f g e n e s t h a t encode p r o t e i n s c o n t a i n i n g l a r g e amounts o f l i m i t i n g amino a c i d s f r o m o n e p l a n t t o a n o t h e r i s r e c e i v i n g w i d e a t t e n t i o n . Work i s i n p r o g r e s s t o i n c r e a s e t h e o v e r a l l s u l f u r amino a c i d composition o f leguminous seeds and soybeans.

S i m i l a r a p p r o a c h e s w i l l be u s e d t o

overcome t h e l y s i n e d e f i c i e n c y o f s e r e a l s e e d s .

Plant genetic engi-

neering is a l s o j o i n i n g f o r c e s with c l a s s i c a l breeding programs t o improve o i l q u a l i t y i n o i l seeds.

The main o b j e c t i v e s a r e t h e con-

t r o l of chain length and t h e degree of unsaturation of f a t t y ? ( i d . PLANT B I O L O G I C A L SYSTEMS Engineering t r a i t s f o r c r o p p r o t e c t i o n h a s been t h e first g o a l m a j o r i l y because o f t h e l i m i t e d knowledge o f p l a n t biology. during the past five years,

However,

f u n d a m e n t a l r e s e a r c h showed a d r a m a t i c

-

384 -

progress i n the unraveling o f the unique reproductive, and p h y s i o l o g i c a l p r o c e s s e s o f p l a n t s

[ 6-7

1.

developmental

Present technology

and t h e d e v e l o p m e n t o f m o d e l o r g a n i s m s s u c h a s A r a b i d o p s i s t h a l i a n a p e r m i t a l m o s t a n y gene t h a t i s a s s o c i a t e d w i t h a n o b s e r v a b l e p h e n o t y p e t o bR i s o l a t e d a n d s t u d i e d .

F o r example,

p l a n t genes w h i c h a r e

s p e c i f i c a l l y i n v o l v e d i n n a t u r a l defense o f c e r t a i n p l a n t s t o fungal i n f e c t i o n and t o e n v i r o n m e n t a l s t r e s s f a c t o r s s u c h as c o l d ,

drought

o r s a l i n i t y h a v e b e e n i s o l a t e d a n d c r e a t e new o p p o r t u n i t i e s f o r c r o p improvement.

These p r o g r e s s e s teamed up w i t h g e n e r a l a d v a n c e s i n mo-

l e c u l a r b i o l o g y w i l l make i t p o s s i b l e t o d i s s e c t t h e m o l e c u l a r a n d c e l l u l a r events responsible f o r c o n t r o l l i n g p l a n t - s p e c i f i c

processes.

The u n d e r s t a n d i n g o f t h e m o l e c u l a r p r o c e s s e s c o n t r o l l i n g gene e x p r e s s i o n d u r i n g p l a n t d e v e l o p m e n t s h o u l d emerge i n t h e n o t t o o d i s t a n t f u t u r e and may p r o v i d e new o p p o r t u n i t i e s a n d may s u g g e s t n o v e l ways t o p r o d u c e s u p e r i o r c r o p s b y gene e n g i n e e r i n g t e c h n o l o g y . CONCLUSION

In general,

t h e p o t e n t i a l economic uses o f p l a n t g e n e t i c e n g i -

neering are i n p r i n c i p l e u n l i m i t e d (Table 2 ) . TABLE 2 Developments i n a g r i c u l t u r a l b i o t e c h n o l o g y

Crop p r o t e c t i o n herbicide tolerance insect resistance virus resistance fungus r e s i s t a n c e bacteria resistance cold resistance stress tolerance Crop q u a l i t y improved y i e l d increased n u t r i t i o n a l value improved processing value

T a r g e t e d m a n i p u l a t i o n s o f t h e p l a n t g e n e t i c m a t e r i a l t e a m e d up w i t h c l a s s i c a l b r e e d i n g techniques w i l l form a p o w e r f u l

new t e c h n o l o g y .

-

385

-

a l l o w i n q t h e i n t r o d u c t i o n o f e n t i r e l y new g e n e t i c t r a i t s i n commerc i a l germplasm.

The i n t e g r a t e d a p p l i c a t i o n o f t r a d i t i o n a l b r e e d i n g

and g e n e t i c e n g i n e e r i n g t e c h n o l o g i e s t h u s p r o m i s e s t o change d r a m a t i c a l l y t h e i n p u t c o s t s t r u c t u r e o f many c r o p s ,

t o r e d u c e and even

e l i m i n a t e t h e use o f e n v i r o n m e n t a l l y h a r m f u l a g r o c h e m i c a l s and t o d e s i g n c r o p s w i t h new i n d u s t r i a l e n d - u s e s .

A l t h o u g h modern a g r i c u l -

t u r e i n d e v e l o p e d c o u n t r i e s is a h i g h l y c o m p e t i t i v e b u s i n e s s e n t e r p r i s e t h a t o p e r a t e s on a s m a l l p r o f i t margin, developments,

i n view o f t h e ongoing

one may c o n c l u d e t h a t b i o t e c h n o l o g y i s c r e a t i n g a n o t -

h e r (green r e v o l u t i o n ,

w h i c h w i l l h a v e t h e same i m p a c t on a g r i c u l t u -

r a l i n d u s t r y as mechanization, development o f h y b r i d seed,

t h e b u r s t o f a g r o c h e m i c a l s and t h e

which occurred e a r l i e r t h i s century.

R E F ER E N C E S

1

R.T.

Fraley,

S.G.

i n higher plants,

R o g e r s , R.B. H o r s c h , G e n e t i c t r a n s f o r m a t i o n CRC C r i t i c a l Reviews i n P l a n t Sciences 4

(1986) 1-42. 2

3

4

5

6

7

J. B o t t e r m a n a n d J. Leemans, E n g i n e e r i n g o f h e r b i c i d e r e s i s t a n c e i n p l a n t s , i n : G . R u s s e l l (Ed.) B i o t e c h n o l o g y and G e n e t i c E n g i n e e r i n g R e v i e w s , V o l 6, I n t e r c e p t Wimborne 1 9 8 8 pp. 3 1 9 - 3 3 8 . M. De B l o c k , J. B o t t e r m a n , M. V a n d e w i e l e , J. D o c k x , C . Thoen, V . G o s s e l e , N. Movva, C . Thompson, M. Van M o n t a g u a n d J. Leemans, Engineering herbicide resistance i n plants by expressing o f a d e t o x i f y i n g enzyme, The EM80 J. 6 ( 1 9 8 7 ) 2 5 1 3 - 2 5 1 8 . M . Vaeck, A . R e y n a e r t s , H. H o f t e , S . J a n s e n s , M. D e B e u c k e l e e r , C . Dean, M. Zabeu, M. Van M o n t a g u a n d J. Leemans, T r a n s g e n i c p l a n t s p r o t e c t e d from i n s e c t a t t a c k , N a t u r e 328 (1987) 33-37. P. A b e l , R . N e l s o n , B. D e , N. H o f f m a n , 5. R o g e r s , R . F r a l e y a n d R . Beachy, D e l a y o f d i s e a s e d e v e l o p m e n t i n t r a n s g e n i c p l a n t s t h a t e x p r e s s t h e t o b a c c o mosaic v i r u s c o a t p r o t e i n gene, S c i e n c e . 232 ( 1 9 8 6 ) 7 3 8 - 7 4 3 . J. S c h e l l , P l a n t s a s t o o l s t o s t u d y t h e m o l e c u l a r o r g a n i s a t i o n o f p l a n t g e n e s , S c i e n c e 237 ( 1 9 8 7 ) 1 1 7 6 - 1 1 8 3 R . B . G o l d b e r g , P l a n t s : n o v e l d e v e l o p m e n t a l p r o c e s s e s , S c i e n c e 240 (1988) 1460-1467.

This Page Intentionally Left TBlank

THE CYCLODEXTRINS AND T H E I R A P P L I C A T I O N I N E N V I R O N M E N T A L BIOTECHNOLOGY

J.

SZEJTLI

C y c l o d e x t r i n Res. L a b o r a t o r y o f C H I N O I N Pharm.-Chem. 1026 B u d a p e s t , E n d r o d i S . 3 8 / 4 0

Works,

SU M M A R Y C y c l o d e x t r i n s and t h e i r d e r i v a t i v e s e n h a n c e t h e s o l u b i l i t y o f c o m p l e x e d s u b s t r a t e s i n a q u e o u s m e d i a , b u t do n o t damage t h e m i c r o b i a l c e l l s o r t h e enzymes. T h e r e f o r e t h e e n z y m a t i c c o n v e r s i o n o f l i p o p h y l i c s u b s t r a t e s can be i n t e n s i f i e d ( a c c e l e r a t e d , o r performed a t h i g h e r s u b s t r a t e c o n c e n t r a t i o n ) . Examples a r e t h e h y d r o l y s i s o f t r i g l y c e r i d e s or the conversion o f hydrocortisone t o prednisolon. I n t h e p r e s e n c e o f an a p p r o p r i a t e C D - d e r i v a t i v e ( e . 9 . 2 , 6 - d i m e t h y l 0 CD) t h e l i p i d l i k e i n h i b i t o r substances a r e complexed, t h e r e f o r e t h e p r o g a t e i o n o f ' B o r d a t e l l a p e r t u s s i s and t h e p r o d u c t i o n o f p e r t u s s i s t o x i n i n c r e a s e s u p t o 1 0 0 - f o l d . The t o l e r a b l e l e v e l o f t o x i c compounds a t b i o l o g i c a l d e t o x i c a t i o n o f sewage o f o r g a n i c c h e m i c a l i n d u s t r y c a n b e e l e v a t e d by a d m i x i n g a s m a l l amount o f R - c y c l o d e x t r i n t o t h e s y s t e m , b e c a u s e t h e c o m p l e x e d t o x i c s u b s t a n c e s do n o t k i l l the d e t o x i c a t i n g microbes.

C H E M I S T R Y OF C Y C L O D E X T R I N S C y c l o d e x t r i n s are c y c l i c ,

non-reducing oligosaccharides.

d i f f e r e n t c y c l o d e x t r i n s a r e known: d.

-,

-

R

Three

and 1 ' - c y c l o d e x t r i n .

A l l o f them e r e p r o d u c e d i n d u s t r i a l l y a s homogeneous ( p u r i t y o v e r 99,5

X)

c r y s t a l l i n e substances.

The s t r u c t u r e o f

(1,Z).

0 - c y c l o d e x t r i n and t h e m o l e c u l a r d i m e n s i o n s

o f t h e t h r e e c y c l o d e x t r i n s a r e shown i n F i g . The c4 - c y c l o d e x t r i n the

y

-cyclodextrin

c o n s i s t s o f 6,

the

8 glucopyranose u n i t s .

1.

R -cyclodextrin

7 and

A l l secondary OH-groups

a r e l o c a t e d o n one edge o f t h e t o r u s - l i k e c y c l o d e x t r i n m o l e c u l e , w h i l e e l l p r i m a r y OH-groups

a r e on t h e o t h e r s i d e .

t h e i n t a r n e l c a v i t y i s f o r m e d b y H-atoms, - b r i d g e atoms,

The " l i n i n g "

of

and g l u c o s i d i c oxygen-

therefore t h i s surface i s s l i g h t l y

apolar

(Fig

2).

The u n i q u e shape a n d p h y s i c a l - c h e m i c a l p r o p e r t i e s o f c y c l o d e x t r i n s e n a b l e s them t o i n c l u d e t h e m o l e c u l e s o f o t h e r s u b s t a n c e s : t h i s i s t h e essence o f t h e " m o l e c u l a r e n c a p s u l a t i o n " .

-

388

-

Ffg. 1. Structure o f 0 -cyclodextrin, and the approximative geometrical dimensions o f the a-,0 - and y -cyclodextrins.

MOLECULAR E N C A P S U L A T I O N W I T H C Y C L O D E X T R I N S A cyclodextrin molecule can be considered a s an empty capsule o f molecular size (Fig. 2 ) .

When it is Filled with the molecule o f another substance, it i s called "inclusion complex".

AFQLAR CAVITY

SECONDARY HYDROXYLS

PRIMARY HYDROXYLS Fig. 2 . Functional schema o f a cyclodextrin "cylinder". Inclusion complexes are entities comprising two or more molecules, in which one o f the molecules, the "host", includes totally o r in part, only by physical f o r c e s , i.e. without covalent bonding a

"guest"

molecule.

389

-

Cyclodextrins are t y p i c a l "host"

m o l e c u l e s and

may i n c l u d e a g r e a t v a r i e t y o f m o l e c u l e s h a v i n g t h e s i z e o f one o r two benzene r i n g s , comparable s i z e ,

o r e v e n l a r g e r ones w h i c h a r e a s i d e c h a i n o f

t o form c r y s t a l l i n e i n c l u s i o n complexes.

I n aqueous s o l u t i o n t h e s l i g h t l y a p o l a r c y c l o d e x t r i n c a v i t y i s

occupied by water m o l e c u l e s which a r e e n e r g e t i c a l l y u n f a v o u r e d (polar-apolar

i n t e r a c t i o n ) and t h e r e f o r e c a n b e r e a d i l y s u b s t i t u t e d

by a p p r o p r i a t e "guest m o l e c u l e s " which a r e l e s s p o l a r t h a n water. The d i s s o l v e d c y c l o d e x t r i n i s t h e " h o s t " force"

o f t h e complex

molecule,

formation i s the s u b s t i t u t i o n o f t h e high-ent-

h a l p y w a t e r m o l e c u l e s b y an a p p r o p r i a t e " g u e s t "

oo

0

Fig.

3.

and t h e " d r i v i n g

molecule (Fig.

3).

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

O

0

J

Inclusion of

p-xylene

by a C D - " c a p s u l e " .

The c y c l o d e x t r i n c o m p l e x e s a r e r e l a t i v e l y s t a b l e .

Their water

s o l u b i l i t y compared t o p u r e c y c l o d e x t r i n s a r e s t r o n g l y r e d u c e d so t h e y r a p i d l y s e p a r a t e from t h e s o l u t i o n i n c r y s t a l l i n e form.

One,

t w o o r t h r e e c y c l o d e x t r i n m o l e c u l e s c o n t a i n one o r m o r e e n t r a p p e d "guest"

t i on"

molecules.

T h i s i s t h e essence o f t h e " m o l e c u l a r

encapsula-

. Though t h e p h y s i c o c h e m i c a l p r o p e r t i e s o f t h e m o l e c u l e s t r a p p e d

i n the c a v i t y are considerably altered,

t h e s e complexes d i s s o c i a t e

e a s i l y u n d e r p h y s i o l o g i c a l c o n d i t i o n s and t h e g u e s t m o l e c u l e s c a n thus exert t h e i r desired effects. A l m o s t a l l i n d u s t r i a l a p p l i c a t i o n s o f c y c l o d e x t r i n s i n v o l v e complexation.

I n many c a s e s c o m p l e x e s a r e s e p a r a t e d i n m o r e o r l e s s pu-

r e f o r m a n d u t i l i z e d as c r y s t a l l i n e s u b s t a n c e s ( d r o g a n d f l a v o u r complexes), state,

catalysis, patway,

w h i l e i n o t h e r cases complexation i s o n l y a t r a n s i e n t

and becomes a p p a r e n t t h r o u g h t h e f i n a l r e s u l t ( c y c l o d e x t r i n separation o f mixtures,

etc.)

(1, 2 ) .

modification o f the reaction-

M I C R O B I A L CONVERSION,

-

390

FERMENTATION

Enzymes ( e x c e p t a m y l o l y t i c o n e s )

o r m i c r o o r g a n i s m s a r e gene-

r a l l y n o t a f f e c t e d b y t h e p r e s e n c e o f CDs. T h e r e f o r e m i c r o b i o l o g i c a l

o r enzymic p r o c e s s e s can be p e r f o r m e d i n CD s o l u t i o n s , ssolved substrate concentrations, inhibitory,

a t higher d i -

b u t a t lower free-substance

or l e s s t o x i c ) c o n c e n t r a t i o n s . F i g u r e 4

(less

i l l u s t r a t e s the

s o l u b i l i t y o f h y d r o c o r t i s o n e i n v a r i o u s CD s o l u t i o n s ( 3 ) . The d i methyl-

R CD i s t o o e x p e n s i v e ( y e t )

f o r i n d u s t r i a l purposes,

but a

v e r y s i g n i f i c a n t improvement o f t h e m i c r o b i o l o g i c a l c o n v e r s i o n o f h y d r o c o r t i s o n e t o p r e d n i s o l o n i s b a s e d on t h e s e e m i n g l y s m a l l s o l u b i l i t y i n c r e a s e i n 0 CD s o l u t i o n . The s o l u b i l i t y o f h y d r o c o r t i s o n e i n w a t e r i s o n l y 0 . 4

mg/ml.

T h e r e f o r e i n l a r g e v o l u m e s o n l y a r e l a t i v e l y s m a l l amount o f t h i s s t e r o i d c o u l d be converted.

The c o n v e r s i o n p r o c e s s was s l o w ,

e n d p r o d u c t was n o t homogeneous,

because o f m i x e d - c r y s t a l

and t h e

formation

(hydrocortisone-prednisolon).

W

5

cn +

20

a

0

0

a

n > I

10

F i g . 4. S o l u b i l i t y o f h y d r o c o r t i s o n e i n v a r i o u s C D - s o l u t i o n s a t 2 5 OC. P e r f o r m i n g t h i s p r o c e s s i n an aqueous c i t y i n c r e a s e d by more t h a n 3 0 0 %. increases,

3-4

converter,

the reaction i s faster,

geneous.

ll CD s o l u t i o n ,

t h e capa-

The s o l u b i l i t y o f h y d r o c o r t i s o n e

t i m e s more h y d r o c o r t i s o n e c a n b e c h a r g e d i n t o t h e a n d t h e e n d p r o d u c t i s more homo-

P r a c t i c a l l y a l l m i c r o b i o l o g i c a l s t e r o i d conversion processes

have b e e n t e s t e d i n a q u e o u s CD s o l u t i o n s w i t h u n a n i m o u s l y p r o m i s i n g

-

391

-

r e s u l t s ( 4 ) . The m e n t i o n e d e x a m p l e i s a l r e a d y p r a c t i c e d i n t h e p h a r maceutical industry.

Recently,

examples have been p u b l i s h e d on t h e

u t i l i z a t i o n o f cyclodextrins i n a n t i b i o t i c fermentation.

The p r o d u c -

t i o n o f L a n k a c i d i n A a n d C b y S t r e p t o m y c e s r o c h e i v o l u b i l i s was enh a n c e d b y a d d i n g 11 mM

mM t o 0 . 5 5

0.04 ce,

SPF-1000

ccus,

0 CD t o t h e f e r m e n t a t i o n b r o t h f r o m 0 . 0 5

and 4 . 6 mM,

respectively.

A new a n t i - t u m o r

and

substan-

i s produced by g r o w i n g a s p e c i f i c s t r a i n o f S t r e p t o -

a c c o r d i n g t o t h e p a t e n t a p p l i c a t i o n t h e c u l t u r e media a l s o

c o n t a i n s CD ( 6 ) .

ENHANCEMENT OF V A C C I N E P R O D U C T I O N Pertussis t o x i n ( = Leukocytosis promoting factor, tinin)

LPF-hemaglu-

i s one o f t h e m a i n p r o t e c t i v e a n t i g e n s a g a i n s t w h o o p i n g c o u g h

infection,

a n d i s a l s o one o f t h e c o m p o n e n t s o f a p e r t u s s i s v a c c i n e .

I t i s produced by B o r d a t e l l a p e r t u s s i s . F o r t h e p r o d u c t i o n o f a l e s s reactogenic vaccine,

a s y n t h e t i c medium i s n e e d e d .

However t h e p r o -

d u c t i o n o f p e r t u s s i s t o x i n was r a t h e r d i f f i c u l t i n s y n t h e t i c medium, e s p e c i a l l y i n shaken c u l t u r e s .

B o r d a t e l l a p e r t u s s i s i s v e r y suscep-

t i b l e t o a number o f i n h i b i t o r s , i c acid)

(5).

f a t t y a c i d s ( p a l m i t i c or o l e -

e.g.

a l r e a d y a t 10 p M c o n c e n t r a t i o n s t o p s t h e c e l l p r o p a g a t i o n

A d d i n g however 0 . 5

0 CD ( o r t r i m e t h y l -

mg/ml d i m e t h y l -

an i n c r e a s e d c e l l g r o w t h was o b s e r v e d , t o x i n p r o d u c t i o n 100 f o l d ( 8 ,

9,

10).

x i n production a t various dimethyl-

0 CD)

w h i c h enhanced t h e p e r t u s s i s F i g u r e 5.

i l l u s t r a t e s the to-

0 CD-concentrations (11).

Howe-

v e r t h e mechanism o f t h e e f f e c t i s n o t y e t f u l l y u n d e r s t o o d ( s e q u e s tration of inhibitory

f a t t y acids,

or s t a b i l i z a t i o n o f reduced glu-

tathione,

o r m o d i f i c a t i o n o f c e l l membrane p e r m e a b i l i t y ) , i t i s an

important

e x a m p l e f o r u t i l i z a t i o n o f CDs i n b i o t e c h n o l o g y ( 1 2 ,

13).

The enhancement o f p r o d u c t i o n o f f i l a m e n t o u s h e m a g l u t i n i n was e v e n higher yet,

w i t h s e v e r a l h u n d r e d t i m e s more b e i n g p r o d u c e d i n t h e

presence o f d i m e t h y l -

0 CD t h a n w i t h o u t i t ( 9 ) .

E N Z Y M I C R E A C T I O N OF L I P I D S H y d r o l y s i s o f t r i g l y c e r i d e s by l i p a s e s i n a q u e o u s s y s t e m s i s a very slow process.

E i t h e r a l i p i d dissolving watermiscible organic

s o l v e n t h a s t o b e added t o t h e s y s t e m

-

which i s t o l e r a t e d only t o

a r e l a t i v e l y low c o n c e n t r a t i o n because o f t h e enzyme-protein turation

-

be present. se w i t h o u t

or an a p p r o p r i a t e d e t e r g e n t

-

e.g.

natural b i l e

-

denahas t o

T a b l e 1 i l l u s t r a t e s t h e h y d r o l y s i s o f o l i v e o i l by l i p a any d e t e r g e n t o f p r e s e n c e o f h o g b i l e ,

or d i m e t h y l - O C D

-

392

-

( 1 4 ) . The h y d r o l y s i s was f o l l o w e d b y t i t r a t i n g t h e l i b e r a t e d f a t t y a c i d s w i t h NaOH.

As i t i s seen,

t h e d i m e t h y l - 0 CD r e m a r k a b l y a c c e -

l e r a t e d the l i p o l y s i s . The o b s e r v a t i o n t h a t p h o s p h a t i d e s ( l i g n o c e r i c a c i d , de,

ceramide)

cerebrosi-

can be s o l u b i l i z e d w i t h COs p r o b a b l y w i l l b e e x p l o i -

t e d i n l i p i d enzymology

(15).

Studying the Lignoceryl-CoA t i o n prepared from r a t b r a i n , d C D was u t i l i z e d ,

l i g a s e a c t i v i t y i n microsomal f r a c

the lignoceric acid,

s o l u b i l i s e d by

b u t t h e one s o l u b i l i s e d by T r i t o n WR 1 3 3 9 was

not u t i l i z e d (16).

-E

\

3

1

DlMEB

1000

800v,

w 1

P

6oo-

3

*oo{

I-

HOURS OF INCUBATION

F i g . 5. E f f e c t o f d i m e t h y l - R - c y c l o d e x t r i n on p e r t u s s i s t o x i n p r o d u c t i o n i n B . p e r t u s s i s Tohama p h a s e I i n s y n t h e t i c medium.

r

Reaction time [h]

Consumed 0 . 0 0 5 a b control bile

n NaOH m l C

dimethyl-

0.5

0.0

0.82

1.74

1

0.36

1.17

2.00

2

0.48

1.37

2.20

19

0.63

2.62

4.77

reaction rate acceleration

- 4 x

-

7.4

x

0 CO

-

393

-

T I S S U E CULTURES The CD c o m p l e x e s o f u n s a t u r a t e d f a t t y a c i d s c a n b e u t i l i z e d as serum s u b s t i t u t e s i n mammalian c e l l c u l t u r e s .

Both o l e i c acid- O C D

a n d l i n o l e i c a c i d - O C D c o m p l e x e s showed a g r o w t h e n h a n c i n g e f f e c t o f human l y m p h o b l a s t c e l l s ,

up t o 1 0 0 m g / l

medium.

A t h i g h e r concen-

t r a t i o n s t h e f a t t y a c i d O C D complex appeared t o be t o x i c , can p r o b a b l y be a t t r i b u t e d t o t h e f a t t y a c i d s . O C D c o m p l e x and 1 0 0 0 mg f r e e effects,

100 mg o f f a t t y a c i d -

O CD t o g e t h e r r e s u l t e d i n n o t o x i c

b u t e x h i b i t e d a s t a b l e and r e p r o d u c i b l e g r o w t h p r o m o t i n g

I n human d i p l o i d f i b r o l a s t c u l t u r e s g r o w t h ,

effect.

but t h i s

i n t h e b o v i n e albumin, OCD-fatty

similar t o that

a s u p p l e m e n t e d medium was o b s e r v e d a f t e r t h e

a c i d complex r e s o l v e d t o a f i n a l c o n c e n t r a t i o n o f 10-20

B o v i n e serum a l b u m i n c a n b e p a r t i a l l y o r c o m p l e t e l y s u b s t i -

mg/ml.

t u t e d by O C D - f a t t y

a c i d c o m p l e x e s i n mammalian c e l l c u l t u r e s

(17,

18)

e . g . , i n human i n t e r f e r o n p r o d u c t i o n ( 1 9 ) .

High density c u l t u r e 0-lymphoblast-like

c e l l s t r a i n s can be

p r e p a r e d i n a s e r u m - f r e e medium w h i c h c o n t a i n s d C D . c a s e o f p r o d u c t i o n o f i n t e r f e r o n s f r o m UMCL c e l l s , t h e p r o d u c e d i n t e r f e r o n s may b e i n c r e a s e d ( 2 0 ) .

E.g.

i n the

t h e amount o f

Mouse mammary t u -

mor c e l l s c a n b e c u l t u r e d u n d e r serum f r e e c o n d i t i o n s when t h e b o v i n e a l b u m i n i s s u b s t i t u t e d b y d CD c o m p l e x o f o l e i c a c i d ( 2 1 ) . The s t e r i l i z a t i o n o f t h e c o m p o n e n t s o f s e r u m l e s s m e d i a f o r c u l t i v a t i n g animal c e l l s i s described.

Such c o m p o s i t i o n may c o n t a i n

a C D u n s a t u r a t e d f a t t y a c i d i n c l u s i o n complexes ( 2 2 ) . O C D i s a u s e f u l m a t e r i a l as a serum s u b s t i t u t e i n i n d u c i n g p r i m a r y a n t i b o d y response i n v i t r o . a t 113

X

antibody

F e t a l c a l f serum must be p r e s e n t

i n t h e c u l t u r e medium f o r o p t i c a l l y e l i c i t i n g t h e p r i m a r y response t o sheep e r y t r o c y t e s i n m u r i n e l y m p h o c y t e s .

The

response can n o t be o b s e r v e d w h e n t h e c o n c e n t r a t i o n o f t h e f e t a l c a l f serum i s l e s s t h a n 1 most e f f e c t i v e , a

,T

X. The a d d i t i o n o f 250-500 p g / m l -CD

O C D was t h e

a n d D I M E B were l e s s e f f e c t i v e ( 2 3 ,

24).

a C D can be used a s a c h o l e s t e r o l c a r r i e r i n a s e r u m - f r e e r i e r p r o t e i n f r e e medium i n t i s s u e c u l t u r e s , p a g a t i o n o f a newborn r a t a d r e n o c o r t i c a l n e a r l y 1:l

complex w i t h c h o l e s t e r o l

The u s e o f a C 0 i n a s e r u m - f r e e

res prolonged c e l l l i f e ,

car-

f o r example i n t h e p r o -

cells.

The a C D f o r m s a

(25). medium o f mammalian c e l l c u l t u -

and i n c r e a s e d t h e p r o d u c t i o n o f m o n o c l o n a l

antibody (26). B e s i d e s t h e s o l u b i l i t y enhancement,

the s t a b i l i z a t i o n i s the

o t h e r i m p o r t a n t consequence o f c o m p l e x a t i o n o f N y s t e t i n

-

a polyene

antibiotic

-

antibiotic, ment.

w i t h

CD.

The N y s t a t i n i s a f r e q u e n t l y u s e d a n t i f u n g a l

w i d e l y u s e d i n human m e d i c i n e f o r l o c a l a n t i f u n g a l t r e a t -

Tissue c u l t u r e

soluble,

-

394

-

an i m p o r t a n t t o o l i n b i o t e c h n o l o g y

a n t i f u n g a l agents,

t u r e medium.

-

needs

w h i c h have t o be d i s s o l v e d i n t h e c u l -

N y s t a t i n would be q u i t e adequate f o r t h i s purpose,

ho-

i t i s p r a c t i c a l l y i n s o l u b l e i n w a t e r a n d r a p i d l y decomposes

wever

by o x y d a t i o n .

The N y s t a t i n

y

CD c o m p l e x i s a r e l a t i v e l y s t a b l e ,

ea-

s i l y s o l u b l e powder, w e l l a p p l i c a b l e f o r such purpose ( 2 7 ) .

SEWAGE D E T O X I C A T I O N

A b i g v a r i e t y o f organic t o x i c substances (hydroxy-halogeno-, nitro-,

amino e t c .

d e r i v a t i v e s o f a r o m a t i c a n d a l i p h a t i c compounds)

o c c u r s i n t h e waste w a t e r s o f t h e o r g a n i c c h e m i c a l and pharmaceutical industry.

The b i o l o g i c a l w a s t e w a t e r t r e a t m e n t means t h a t t h e s e

t o x i c s u b s t a n c e s a r e degraded by c e r t a i n y e a s t s and b a c t e r i a w h i c h are present i n the b i o l o g i c a l sludge.

These m i c r o o r g a n i s m s c a n t o -

l e r a t e t h e t o x i c substances i f t h e i r c o n c e n t r a t i o n does n o t exceed a c r i t i c a l c o n c e n t r a t i o n a n d t r a n s f o r m s them b y m e t a b o l i c p r o c e s s e s t o non-toxic

substances.

By a p p r o p r i a t e a d a p t a t i o n ,

c o n c e n t r a t i o n o f t o x i c s u b s t a n c e s can be enhanced, t h i s c r i t i c a l concentration l i m i t

-

the tolerable but surpassing

even f o r a s h o r t t i m e

i n k i l l i n g the mentioned microorganisms,

i.e.

-

results

irreversible impairing

o f the detoxicating capacity o f the l i v i n g sludge.

t h i s b i o l o g i c a l system i s n o t a r a p i d process.

Regeneration o f

Therefore preserva-

t i o n o f the detoxicating capacity o f the " l i v i n g sludge"

i s a pri-

mary g o a l o f e n v i r o n m e n t a l p r o t e c t i o n . To a v o i d t h e c r i t i c a l

-

i.e.

"killing"

-

c o n c e n t r a t i o n o f waste,

w a t e r s o f s u p e r c r i t i c a l c o n c e n t r a t i o n s h a v e t o b e d i l l u t e d w i t h water,

t o reduce t h e c o n c e n t r a t i o n s t o s u b c r i t i c a l values.

Sometimes

t h i s method would need thousands o f t o x i c s u b s t a n c e f r e e w a t e r ,

which

is n o t always p o s s i b l e . A J d i n g CD,

w h i c h o f c o u r s e m u s t n o t be a p u r e o n e ,

the conver-

s i o n m i x t u r e w i t h about 50 % CD c o n t e n t i s s a t i s f a c t o r y t o t h e waste water,

and a c o n s i d e r a b l e p a r t o f t h e m e n t i o n e d o r g a n i c t o x i c compo-

unds w i l l be complexed.

(Fig.

6 ) . The c o m p l e x e d m o l e c u l e s w h i c h c a n

n o t p e n e t r a t e t h e c e l l membrane,

are not t o x i c

(28).

The c o n c e n t r a t i o n o f t h e f r e e t o x i c s u b s t a n c e s i s s t r o n g l y reduced,

below t h e c r i t i c a l c o n c e n t r a t i o n .

further

by t h e metabolic process,

As i t s c o n c e n t r a t i o n d e c r e a s e s

t h e CD-complex

behaves as a dynamic

depot,

-

395

r e l e a s i n g t h e i n c l u d e d molecules,

ned by t h e d i s s o c i a t i o n e q u i l i b r i u m ( F i g . Low CONC.

NON TOXIC FOR MICROORGANISMUS METABOLISM PROEEDS

and t h i s p r o c e s s i s gover-

7).

HIGH CONC

HIGH CONC

TOXIC FOR MICROORGANISMUS NO METABOLISM

NON TOXK: FOR MICROORGANISMUS METABOLISM PROCEEDS

F i g . 6 . The t o l e r a b l e l e v e l o f t h e t o x i c compounds i s a u g m e n t e d b y a d d i n g CD t o t h e t o x i c sewage.

NON -TOXIC

1

DETOXICATION

NON-TOXIC METABOCITES

F i g . 7. A d d i n g CD t o t h e t o x i c p - n i t r o c h l o r o b e n z e n e c o n t a i n i n g s e wage, a f r a c t i o n o f t h i s compounds w i l l b e c o m p l e x e d b y t h e C D . The CD-complexes b e i n g h y d r o p h y l i c - have l o w e r a f f i n i t y t o t h e l i p o p r o t e i n c e l l membranes o f t h e m i c r o b i a l c e l l s , t h e r e f o r e a r e c o n s i d e r e d t o be l e a s t o x i c . Only t h e non-complexed f r a c t i o n w i l l p e n e t r a t e t h r o u g h t h e c e l l membranes.

-

A m i x e d i n d u s t r i a l and communal w a s t e w a t e r , phenol,

p-chlorophenol,

t r e a t e d b y t h e c o n v e n t i o n a l merhod,

RCD.

t h a t contained

b e n z e n e a n d f u r t h e r o r g a n i c s u b s t a n c e s was

w i t h o r w i t h o u t a d d i n g 40 m g / l

The r e s u l t s a r e s e e n i n T a b l e 2.

Before treatment

Phenol p-Chlorphenol Benzene Oxygen C o n s u m p t i o n

30 10 4 BOO

A f t e r 24 h A f t e r 24 h conventional treatment w i t h treatment a d d i n g 40 m l / l 0 CD

10 5

3 200

1 1 0905 60

- 396

The a d d i t i o n o f s o l u b l e

n

-

CD-polymer

t o p h e n o l c o n t a i n i n g sewa-

ge i n c r e a s e s t h e e f f i c i e n c y o f m i c r o b i o l o g i c a l d e t o x i c a t i o n o f p h e n o l by Candida t r o p i c a l i s . with

0 CO-polymer

The c e l l s f i x e d i n p o l y m e r b e d s t o g e t h e r

c a n b e r e u s e d f o r sewage t r e a t m e n t

(29).

R EF E R E N C E S

1 2 3 4

5 6 7 8 9

10 11 12

13 14 15 16 17 18 19 20 21

22 23 24 25 26 27

J . S z e j t l i , " C y c l o d e x t r i n s a n d T h e i r I n c l u s i o n C o m p l e x e s " , Akad B m i a i K i a d 6 . B u d a p e s t , 1982. J . S z e j t l i , " C y c l o d e x t r i n T e c h n o l o g y " , K l u w e r Academic P u b l . , O o r d r e c h t , 1988. I.Habon, A . S t a d l e r - S z o k e and J. S z e j t l i , M a g y a r K B m i k u s o k L a p j a , 60 231 ( 1 9 8 5 ) . N.E. U d v a r d y , I. B a r t h a , G . H a n t o s , M. T r i n n , 2 s . V i d a , J. S z e j t l i , A . S t a d l e r - S z o k e , I.Habon a n d M. B a l d z s ( R i c h t e r Gedeon), 99:4069). B e l g . P a t . 894, 501 (19831, ( C . A . T . S u z u k i , J. Okada a n d H . Sawada ( T o k e d a C h e m i c a l I n d . ) , E u r . P a t . A o D ~ . E P 91. 7 8 1 ( 1 9 8 3 ) . ( C . A . 100:4792). S h i k i s h ' i m a B o s e k i ( 1 9 8 6 1 , J p n . K o k a i 8 5 , 239, 4 2 4 . Y . S u z u k i , A . I m a i z u m i , H . S a t o and Y . S a t o , J p n . J. Med. S . , 36 111 ( 1 9 8 3 ) . A . I m a i z u m i , Y . S u z u k i , 5 . Ono, H. S a t 0 a n d Y . S a t o , I n f e c t i o n a n d Immun. 1138 ( 1 9 8 3 ) . Y . S u z u k i , A . I m a i z u m i , 5 . Onos, H . S a t o a n d Y . S a t o A b s t r a c t ymp. o n Book o f 3 r d I n t . Symp. C l a t h r a t e Comp. a n d 2nd I n t . C y c l o d e x t r i n s , Tokyo, J u l y 23-27, 1 9 8 4 . T e i j i n KK ( 1 9 8 5 ) , J p n . K o k a i 8 5 , 1 5 5 , 1 2 7 . A . I m a i z u m i , Y . S u z u k i , 5 . Ono, H. S a t o a n d Y . S a t o , J. C l i n . Microbiol. 17 781 (1983). Y . S u z u k i , A . 1maizumi;H. Yamaguchi, M. K a n e s a k i a n d 5 . Ono, Jpn. K o k a i 8 3 , 6 7 , 1 8 2 ( 1 9 8 3 1 , (C.A. 3 3 : 1 3 8 1 9 1 ) . T e i j i n L t d , J p n . K o k a i , 83, 6 7 , 1 8 8 ( 1 9 8 3 1 , (C.A. 3 3 : 1 8 3 2 2 5 ) J. S z e j t l i , L . S z e n t e , K . K d l b i , J. M a r t o n a n d A . G e r l G c z y , Hung. P a t . A p p l . 7 5 / 8 5 1 9 8 5 . I. S i n g h a n d Y . K i s h i m o t o , J. L i p i d Res. 24 6 6 2 ( 1 9 8 3 ) . I. S i n g h , R . S i n g h , A . Bhushan a n d A . K . S G g h ( 1 9 8 5 ) , A r c h - 8 ochem. B i o p h y s . 236 418, ( C . A . 102:91817). I. Yamane, M. Kan, Y . M i n a m o t o a n d Y . A m a t s u j i , P r o c . J p n . Acad., S e r . B. 11 385 ( 1 9 8 1 ) (C.A. 8 6 : 1 0 0 4 8 8 ) . I. Yamane, M. Kan, Y . M i n a m o t o a n d Y . A m a t s u j i , C o l d S p r i n g H a r 97:212006). b o r Conf. C e l l P r o f i f e r a t i o n , 1982. p . 87, ( C . A . 98:124208). A j i n o m o t o Co., J p n . K o k a i , 8 2 , 1 9 4 , 787, ( 1 9 8 2 ) ( C . A . Agency o r I n d . S c i . Tech. ( 1 9 8 6 1 , J p n . K o k a i 86, 6 3 , 279. K . Kawamura, J. E n a m i , K . Kohmoto a n d M. Koga ( 1 9 8 5 1 , O o k k y o J. Med. S c i . 167, ( C . A . 104:164689). Agency o f I n d . S c i . Tech. ( 1 9 8 5 ) , Jpn. K o k a i 8 5 , 8 7 , 7 8 5 ) . H . O h m o r i , I. Yamamoto ( 1 9 8 7 1 , J. Immun. 17 79 ( C . A . 106:174219). H. O h m o r i , M . I e n a g a a n d I. Yamamoto ( 1 9 8 7 1 , Jpn. J. P h a r m a c o l . 44 2 2 5 , ( C . A . 107:51288). M. Hammani, G. Maume a n d B . Maume ( 1 9 8 6 ) , C e l l . B i o l . T o x i c o l . 105:219064). 2 41, ( C . A . Y . Minamoto and K. M i t s u g i , G r o w t h D i f f e r . C e l l s D e f i n e d E n v i r o n . Kodansha T o k y o , 1985. P r o c . I n t . Symp., 1 9 8 4 . (Ed.: M u r a k a m i H . ) , p. 127. J. S z e j t l i , A . S t a d l e r - S z o k e , A . Vikmon, 5. P i u k o v i c h , 1. I n c e f y , C . K u l c s A r a n d G. Z l a t o s , Hung. P a t . A p p l . 4 5 0 8 / 8 3 ( 1 9 8 3 ) .

2

12

28

29

397 -

P. F a r k a s , L . S z e n t e , J. O l a h , J . S z e j t l i and T . C s e r h e t i ( 1 9 8 6 ) 105:84597). Hung. T e l j e s HU 3 7 , 7 2 7 , ( C . A . B . B d n k y , K . Recseg and 8 . Novdk, Magy, K B m . L a p j a 40 1 8 8 ( 1 9 8 5 ) .

This Page Intentionally Left TBlank

BIOTECHNOLOGY OF METALS AND T H E E N V I R O N M E N T

KUSNIEROVA, D . K U P K A , E . P A S T I R f K O V A , J. E E C H O V S K A a n d V . S E P E L A K

F . SPALDON,

M.

Mining I n s t i t u t e o f the Slov.

Biotechnology of nology.

Academy o f Sc.,

ESFR

m e t a l s i s a new f i e l d o f s c i e n t i f i c b i o t e c h -

I t s aim i s t o e x t r a c t m e t a l from o r e s , c o n c e n t r a t e s ,

and s o l u t i o n s u n d e r t h e a c t i o n o f b o l i t s /l/. a)

KoSice,

microorganisms and/or

rocks

t h e i r meta-

I t s sections are:

biohydrometallurgy or b a c t e r i a l metal leaching,

b) ore enrichment, c) b i o s o r p t i o n o f m e t a l s from s o l u t i o n s . C o n s i d e r i n g b i o g e o t e c h n o l o g y p u r e l y f r o m t h e t e c h n o l o g i c a l and e c o n o m i c a l p o i n t o f v i e w we m u s t r e a l i z e t h e s h o r t c o m i n g s a s w e l l as t h e advantages.

As f o r t h e s h o r t c o m i n g s ,

i t i s mainly a tedious

p r o c e s s t h a t i n f l u e n c e s t h e r e l a t i v e l y l a t e r e t u r n o f t h e expended c a p i t a l costs.

However,

on t h e o t h e r h a n d ,

t h e o p e r a t i n g c o s t s can

b e r e d u c e d a n d t h e s t r e s s on t h e e n v i r o n m e n t c a n be d i m i n i s h e d . p o s i t i v e i n f l u e n c e o f b i o g e o t e c h n o l o g y on t h e e n v i r o n m e n t

The

i s the

subject matter o f our r e p o r t .

I t i s g e n e r a l l y known t h a t one o f t h e m o s t i m p o r t a n t p r o b l e m s a f f e c t i n g t h e environment i s the emission o f s u l f u r dioxide.

More

t h a n 100 m i l l i o n t o n s o f c o a l i s e x p l o i t e d annualy i n C z e c h o s l o v a k i a .

I f t h e e s t i m a t i o n o f t h e average c o n t e n t o f s u l f u r i s 1 , 5 X , then t h r e e m i l l i o n t o n s o f s u l f u r d i o x i d e i s r e l e a s e d d u r i n g t h e combust i o n . o f t h a t amount.

I f added t o i t a r e t h e e x h a l a t i o n s f r o m m e t a -

l l u r g i c a l and chemical p r o d u c t i o n and from t r a f f i c ,

we come t o v a -

l u e s which a r e a l a r m i n g and t h e y a r e r i s i n g w i t h i n c r e a s i n g i n d u strialization.

T h e r e f o r e i t i s n a t u r a l t o l o o k f o r methods t o dec-

rease t h i s danger t o a c c e p t a b l e l e v e l s .

The t h i o b a c t e r i a T h i o b a c i l -

l u s t h i o o x i d a n s and m a i n l y T h i o b a c i l l u a f e r r o o x i d a n s d i s c o v e r e d i n mining waters /2/

by Colmer and H i n k l e i n 1947 c o u l d h e l p i n s o l v i n g

t h a t problem.

They a r e a e r o b i c ,

400

-

a u t o t r o p h i c b a c t e r i a f o r which the

source o f carbon f o r t h e i r metabolism i s C02 from a i r , dation reactions o f inorganic substrates o f iron, t h e source o f energy.

and t h e o x i -

sulfur,

etc.

are

The p r a c t i c a l u s e o f phenomena o f b i o c h e m i c a l

oxidation o f sulfides i s investigated intensively f o r the exploitat i o n and t r e a t m e n t o f o r e s .

I t i s n o t w i t h o u t i n t e r e s t t h a t t h e European primacy o f o b t a i n i n g c o p p e r by b i o l e a c h i n g i s c o n c e d e d t o o u r m i n i n g / 3 / .

According

t o h i s t o r i c a l documents a s e a r l y a s 1 5 - t h c e n t u r y i n Smolnfk,

copper

was o b t a i n e d f r o m t h e d e p o s i t o f c h a l c o p y r i t e b y c e m e n t a t i o n r e d u c t i o n from m i n i n g water /4/.

T h i s way o f t r e a t m e n t h a s o u t l a s t e d t h e

c e n t u r i e s t o t h e p r e s e n t day / 5 / . c o p p e r went

into

ferrooxidans.

Our a n c e s t o r s d i d n o t know t h a t

s o l u t i o n by t h e o x i d a t i o n e f f e c t o f T h i o b a c i l l u s

A t p r e s e n t i t i s known a n d t h e r e f o r e i t w o u l d b e p o -

s s i b l e t o m o d i f y and modernize t h e o p e r a t i o n o f b i o l e a c h i n g e x p e d i e n tly.

The a c t i o n o f T h i o b a c i l l u s

f o r m a t i o n o f copper

f e r r o o x i d a n s b a c t e r i a on t h e t r a n s -

from i n s o l u b l e c h a l c o p y r i t e i n t o a s o l u b l e form

o f CuS04 c a n b e e x p r e s s e d by t h e f o l l o w i n g c h e m i c a l e q u a t i o n s :

T.f.

CuFeS2

+ 4 O2

CuS04 + FeS04

T.f.

4 FeS04

+

O2

+

2H2504

2 Fe2(SOLt)3 + 2 H20

Chem.

CuFeS2

+ 2

+

3 0

Fe2(S04)3

-

CuS04

+

5 FeS04

+

2 So

(3)

T.f.

2 So

2

+

H20

2 H2S04

(4)

T.f.

Oxidation reactions 1,2

and 4 t a k e p l a c e u n d e r t h e c o a c t i o n o f bac-

t e r i a l enzymes a n d r e a c t i o n 3 is e x c l u s i v e l y a c h e m i c a l o x i d a t i o n .

A l o w pH v a l u e i s o f i m p o r t a n c e f o r t h e o c c u r a n c e o f t h e a b o v e r e a c t i o n s s i n c e a t pH

=

1.91 formation o f hydroniumjarosite p r e c i p i t a t e

s t a r t s according t o the equation

Fe3+

+ SO, + HS04 + 6 H20

-1

HFe(S04)2(0H)6

+ 6 H+

(5)

- 401

a n d on f u t h e r pH i n c r e a s e s above 2.8

and a p r e c i p i t a t e o f f e r r i c

h y d r o x i d e i s formed Fe3+

whereby

+

-1

3 H20

Fe(OHI3

+

3 H'

t h e o x i d a t i o n a g e n t F e 2 ( S O 4 l 3 v a n i s h e s f r o m t h e l i q u i d me-

dium / 6 / . I n t h e c l o s e n e i g h b o r h o o d o f a c h a l c o p y r i t e o r e m i n e i n t h e Smoln i k l o c a l i t y t h e r e a r e o l d p i t dumps c o n t a i n i n g s e c t i o n s w i t h a c o p p e r c o n t e n t o f up t o 1 % .I t i s f r o m t h e o l d h i s t o r i c i m p e r f e c t o r e dressing technology,

the time p r i o r t o the i n v e n t i o n o f the f l o t a -

tion,

when p o o r e r f i n e l y i n t e r g r o w n o r e s were n o t r e c o v e r e d .

day's

technological feasibilities,

r a l deposits

and,

By t o -

s u c h dumps a r e c o n s i d e r e d n a t u -

consequently a r e p r o t e c t e d under the m i n i n g law

and must n o t be i m p a i r e d , o f e x t r a c t i n g copper

i.e.

nor recultivated.

One o f t h e m e t h o d s

f r o m o l d dumps i s b y b a c t e r i a l l e a c h i n g w h i c h

can be f o l l o w e d by r e g e n e r a t i o n o f t h e e n v i r o n m e n t t h r o u g h r e c u l t i vation.

l o r e c o v e r c o p p e r f r o m t h e S m o l n i k dumps a p l a n t was b u i l t i n t h e s e v e n t i e s f o r w e t t i n g dumps w i t h a s t r o n g a c i d s o l u t i o n o f F e S 0 4 , containing the bacteria o f Thiobacillus ferrooxidans. l h e l e a c h i n g s o l u t i o n s o a k e d t h r o u g h t h e dump a n d l e a c h e d c o p p e r from t h e c h a l c o p y r i t e , t h e mine water

soaked i n t o t h e mine,

and t o g e t h e r w i t h

( a l s o c o n t a i n i n g t h e leached copper

from underground

m i n e s p a c e s ) was pumped i n t o c e m e n t a t i o n f o r p r e c i p i t a t i o n o f c o p p e r

/7/.

1-hanks t o U V R t h e p l a n t was p r e p a r e d and e q u i p p e d w i t h g o o d

technical standards,

i n c l u d i n g copper c e m e n t a t i o n and r e g e n e r a t i o n

o f the bioleaching solution.

A t first,

extensive l a b o r a t o r y rese-

a r c h was c a r r i e d o u t t o f i n d t h e o p t i m a l p a r a m e t e r s f o r g r o w i n g t h e s t r a i n of

T h e o b a c i l l u s f e r r o o x i d a n s w h i l e i s o l a t e d from t h e Smolnik

mine water and under c o n d i t i o n s f o r o p t i m a l i z a t i o n o f a l e a c h i n g r a t e and r e c o v e r y r a t i o o f Cu f r o m t h e Smolnik o r e . the temperature e f f e c t , lues,

Besides studying

t h e g r a n u l o m e t r y o f t h e s o l i d phase,

pH va-

t h e a e r a t i o n e f f e c t and l e a c h i n g under s t a b l e as w e l l as k i -

n e t i c c o n d i t i o n s were a l s o s t u d i e d .

Under o u r c l i m a t i c c o n d i t i o n s

t h e t e m p e r a t u r e e f f e c t was o f g r e a t i m p o r t a n c e s i n c e t h e a c t i v i t y of

t h i o b a c t e r i a i s known t o d e c r e a s e w i t h a t e m p e r a t u r e d r o p a n d i t

was n e c e s s a r y t o f i n d o u t t h e l o w e r t e m p e r a t u r e l i m i t a t w h i c h t h e technological

u t i l i z a t i o n o f b i o l e a c h i n g can s t i l l be p r o f i t a b l e .

The number a n d t h e a c t i v i t y o f b a c t e r i a a r e r e v e a l e d b y t h e o x i d a t i o n

o f Fez+.

402

-

Thus t h e d r o p o f F e 2 + c o n c e n t r a t i o n i n d e p e n d e n c e on t h e

d r o p o f c u l t i v a t i o n t e m p e r a t u r e g a v e us i n f o r m a t i o n o n t h e c u l t i v a -

1). Hence a c o n c l u s i o n was d r a w n

t i o n temperature conditions (Fig.

t h a t t h e l e a c h i n g i n t e n s i f i c a t i o n p r o c e s s can be c o n s i d e r e d t o be s u c c e s s f u l i f t h e ambient t e m p e r a t u r e does n o t d r o p b e l o w 12

OL--

12 14 16 I8 20

OC.

Z 24 T

[TI

F i g . 1. D i a g r a m o f F e z + d e c r e a s e ( b y b a c t e r i s l o x i d a t i o n i n t o F e 3 + ) i n d e p e n d e n c e on T ( O C ) .

This l e d us t o the d e c i s i o n t o r e s t r i c t the a p p l i c a t i o n o f the leac h i n g t e c h n o l o g y t o dumps i n t h e m o n t h s o f A p r i l - O c t o b e r t h e t e m p e r a t u r e c o n d i t i o n s can be met.

i n which

On t h e b a s i s o f l a b o r a t o r y

r e s u l t s a n d m o d e l t e s t s a p i l o t p l a n t was b u i l t f o r t h e l e a c h i n g o f dumps a n d f o r i n t e n s i f i c a t i o n o f c o p p e r l e a c h i n g f r o m dumps a t Smolnik.

The t e c h n o l o g i c a l

scheme i s shown i n F i g .

2.

The w h o l e i n s t a l l a t i o n c o n s i s t s o f t w o s e c t i o n s . presents a modernized cementation.

S e c t i o n Z re-

A p a r t o f waste water i s u t i l i z e d

f o r scavenging t h e p r e c i p i t a t e d cementation copper

from t h e troughs.

The w a a t e w a t e r t h e n f l o w s i n t o t h e n e i g h b o r i n g s e c t i o n R ,

w h i c h 8er

v e s f o r i t s r e g e n e r a t i o n o f pH c o n d i t i o n i n g b y means o f a u l p h u r i c acid,

a d d i t i o n o f f r e s h a c t i v e b a c t e r i a l c u l t u r e and t h e n e c e s a a r y

o x i d a t i o n by a e r a t i n g .

The r e g e n e r a t i o n i s p e r f o r m e d i n t h e r e g e n e -

r a t i o n t a n k t h e volume o f w h i c h a l l o w s r e p l a c e m e n t o f t h e l i q u i d i n

1.8

day /8/. D u r i n g one c a m p a i g n ( A p r i l - O c t o b e r )

l i q u i d p h a s e was f o u n d t o b e 9.8

the lose of

the

%.

A f r e s h a c t i v e b a c t e r i a l c u l t u r e was p r e p a r e d i n t h e c u l t i v a t o r set-up

shown i n F i g .

3.

I t s main p a r t s are a c u l t i v a t o r o f the

shape o f a t h e r m a l i s o l a t e d c y l i n d r i c a l v e s s e l w i t h b u i l t - i n a e r a t i n g t u b e s and a c y l i n d r i c a l m i x i n g t a n k .

arch

-

403

'I-

z

-

R

F i g . 2 . L a y o u t o f c e m e n t a t i o n a n d o f b a c t e r i a l l e a c h i n g i n t h e Smolbiologic leaching; n i k l o c a l i t y 2- cementation; R - r e g e n e r a t i o n . 1 2 , 4 , 6 - e n r i c h e d s o l u t i o n ; 3 - c o l l e c t i n g t a n k f o r t h e e n r i c h e d sol u t i o n ; 5 - c o n i c a l cementator; 7 - trough cementator; 8 - cementat i o n sediment; 9 - d r y i n g o f cementation sediment; 10 - d r y cementaa c i d wast i o n s e d i m e n t f i n a l p r o d u c t ; 11 - d e p l o t e d s o l u t i o n ; 1 2 t e s o l u t i o n ; 13 - c o l l e c t i n g t a n k f o r r e g e n e r a t i o n o f t h e s o l u t i o n ; 14 h o p p e r ti2504; 1 5 - H z S 0 4 ; 1 6 - c u l t i v a t o r ; 1 7 - a i r i n l e t ; 18 - s u p p l y t a n k f o r n u t r i e n t m e d i a ; 1 9 - n u t r i e n t s u b s t a n c e s ; 20 - n u t r i e n t medium; 2 1 - f r e s h l e a c h i n g medium; 2 2 - medium f o r l e a c h i n g a n d c e m e n t a t i o n ; 23 - p a r t i a l f l o w o f w a s t e c e m e n t a t i o n m e d i a f o r r i n s i n g t h e t r o u g h ; 24 - e m e r g e n c y o u t f l o w i n c a s e o f f a i lure i n regeneration s e c t i o n .

-

-

-

-

c u l t i v a t o r ; B - mixingF i g . 3 . R e g e n e r a t i o n o f l e a c h i n g medium A cementing water; 2 - additions to t a n k ; C - h e a t e r ; D - pump; 1 c u l t i v a t i n g medium 9 K ; 3 cultivating substrate 9 K; 4 - air inlet; 5 p e r f o r a t e d t u b e s f o r a i r d i s t r i b u t i o n ; 6 - l e a c h i n g solution of high bacteria concentration.

-

-

-

-

404

-

The d a i l y c o p p e r r e c o v e r y was a b o u t 1 5 0 k g w h i c h a f f o r d e d ab o u t 30 t o f copper.

When m a k i n g a c o m p a r i s o n b e t w e e n t h e p r o d u c t i o n

w i t h t h e i n t r o d u c e d b a c t e r i a l c u l t i v a t i o n and t h e p r o d u c t i o n w i t h o u t i t , t h e former proved t o be 18-20

X h i g h e r . However,

i n the meantine

t h e S m o l n i k d e p o s i t was d e p l e t e d a n d i t was d e c i d e d t o c e a s e t h e m i n i n g o p e r a t i o n s and a l l o w a b l a s t o u t o f t h e abandoned p i l l a r s w h i c h were p o o r i n c o p p e r

( a b o u t 0,15

% Cu) a n d t o r e c o v e r c o p p e r

from

them b y a p p l y i n g t h e m e t h o d o f u n d e r g r o u n d l e a c h i n g . An i m p o r t a n t f i e l d o f u t i l i z i n g t h e a c t i o n o f m i c r o o r g a n i s m s i s i n t h e t r e a t m e n t o f t h e mined o r e and i n b e n e f i c a t i o n o f concent r a t e s where t h e f a c t i s made u s e o f t h a t t h e b i o c h e m i c a l o x i d a t i o n

i n the presence o f several s u l p h i d i c minerals i s c a r r i e d o u t selectively.

K a r a v a j k o /1/ s t a t e s t h a t m i x t u r e s o f d i f f e r e n t p a i r s o f

sulphides forming a galvanic c e l l , wer e l e c t r o d e p o t e n c i a l , t h e anode,

t h e sulphide possessing the l o -

i n the given galvanic p a i r representing

w i l l be o x i d i z e d p r e f e r e n t i a l l y by b a c t e r i a ,

e.g.

p a i r s o f sulphides:

preferential biooxidstion:

chalcopyrite-arsenopyrite

arsenopyrit e

chalcopyrite-sphalerite

s p h a l e r it e

chalcopyrite-molybdenite chalcopyrite-pentlandite

pentlandite

antimonite-cinnabar

antimonite

chalcopyrite

T h i s f a c t may be u s e d i n s e l e c t i n g l e a c h i n g a 0 t h a t t h e p r e f e r e n t i a l l y o x i d i z e d m i n e r a l w i l l pass f i r s t

i n t o the solution,

or

i n s e l e c t i v e f l o t a t i o n o f t h e above p a i r s o f m i n e r a l s s o t h a t t h e p r e f e r e n t i a l l y o x i d i z e d m a t e r i a l w i l l be suppressed. I n an e n d e a v o r t o c o n t r i b u t e t o i m p r o v i n g t h e e n v i r o n m e n t , we h a v e i n v e s t i g a t e d t h e p r o b l e m o f d e a r s e n i s a t i o n o f c h a l c o p y r i t e from

t h e S l o v i n k y l o c a l i t y by m i c r o b i o l o g i c a l l e a c h i n g o f t h e con-

c e n t r a t e c o n t a i n i n g 2,5

up t o 3

X As. F o r t h i s r e a s o n we i s o l a t e d

b a c t e r i a l s t r a i n Thiobacillus ferrooxidans from t h e mining waters o f a number o f d e p o s i t s ( S r n o l n i k , Hora, Moat).

Pezinok, Odbrava,

HodruSa,

KutnA

We h a v e a t u d i e d t h e i r o x i d a t i o n a c t i o n i n l e a c h i n g a n d

c a r r i e d o u t an e x t e n s i v e i n v e s t i g a t i o n o f t h e i r a d a p t i b i l i t y i n t h e presence o f heavy m e t a l s and a r s e n i c

as w e l l as t h e e f f e c t o f t h e

p u l p d e n s i t y on t h e l e a c h i n g p r o c e s s a n d t h e e f f e c t s o f a e r a t i o n and c o n c e n t r a t i o n o f c e l l s on t h e r e s u l t a n t p a r a m e t e r s o f l e a c h i n g . Leaching t e s t s used a Cu-concentrate

c o n t a i n i n g 24,82

X

Cu,

23,67

X

Fe,

34,94

-

405

L S i 0 2 , w h i l e t h e p r e s e n c e o f a r s e n o p y r i t e was

% 5 , 2,74

m a n i f e s t e d b y t h e c o n t e n t o f 2 , 4 ?A As.

According t o P o l k i n e t a1

( 9 ) t h e r e i s an e l e c t r o d e p o t e n t i a l o f a r s e n o p y r i t e V i n an a c i d medium a t pH 2,5

c h a l c o p y r i t e -0,547

a r e p r e s e n t i n t h e medium.

-

V and o f

0,497

i f thiobacteria

The a n o d i c d i s s o l u t i o n o f t h e a r s e n o p y -

r i t e w i l l occur as f o l l o w s :

bact.

FeAsS

o r t h e o x i d a t i o n r e a c t i o n w i t h Fe

+

FeAsS

+

F e z + + As3+

-

So

7 e-

(7)

3+

chem.

Fe3+

+

+

2 Fez+

As3+

+

So

+ 6

s-

..

(8)

u n s t a b l e As3+ r e a c t s w i t h w a t e r i n t o H3As03. H3As03, F e z + , S o s r e t h e r m o d y n a m i c a l l y

unstable t h e r e f o r e t h e reac-

t i o n s continue

H ~ A S O+ ~ 3 H ~ O

-

bact.

Fez+

Fe3+

+

H ~ A ~ O+ ; 3 H+

+

2 e-

e-

bact.

So

+

4 H20

SO:-+

8 H+

+

6 e-

a n d Fe3+ r e a c t s w i t h H2As04 i n t o f e r r i c a r s e n a t e :

chem.

Fe3+

+

H2As04

FeAs04

+

2 H+

(12)

L e a c h i n g was c a r r i e d o u t i n 250 m l f l a s k s on a l a b o r a t o r y s h a k e r r o t a t i n g a t 240 min-'

s h a k e s w i t h t h e a m p l i t u d e o f 5 mm a t t h e tem-

p e r a t u r e o f 28-30

The c h a r a c t e r i s t i c r e s u l t s a r e g i v e n i n F i g ,

OC.

I t i s e v i d e n t f r o m t h e above p i c t u r e 4-F s i t i o n o f a r s e n i c i n t o t h e l i q u i d phase,

that during the tran-

some c o p p e r i n t h e f o r m

o f s o l u b l e s a l t CuS04 g e t s i n t o t h e s o l u t i o n f r o m t h e c o n c e n t r a t e . T h i s c o p p e r c a n b e r e c o v e r e d s o t h a t t h e l e a c h pH v a l u e i s m o d i f i e d t o 3 . 1 by h e l p o f l i m e m i l k .

3 H2Aa0;

+

3 Ca(OH)2 + Fe3+-

FeAs04

+

Ca3(AsO4I2

+

6 H20

(13) I n s o l u b l e a r s e n a t e s a l t s p r e c i p i t a t e and t h e r e m a i n i n g l e a c h c o n t a i n i n g CuS04 i s p a a a e d i n t o c e m e n t a t i o n o r e l e c t r o l y s i s w h e r e c o p p e r

4.

-

-s 70

406 -

0

s

m

I

3 60

80

50 40

60

30 20 10

LO

20

40pz<

20

5

10

l5 t [ d l

2L, 80

u.

u?

60 40 20

0

F i g . 4 . A - A d a p t a t i o n e f f e c t on e x t r a c t i o n o f As f r o m C u - c o n c e n t r a E x t r a c t i o n o f As t e , l... n o t a d a p t e d , 2 . . . a f t e r a d a p t a t i o n ; B b y b a c t e r i a Th. f e r r . from v a r i o u s l o c a l i t i e s : M . . . M o s t , KH... Kutn6 Hora, S . . . S m o l n i k , D... Ddbrava, H . . . HodruSa, P . . . P e z i n o k ; C - S u s p e n s i o n d e n s i t y o n As e x t r a c t i o n ; l... ? = 2 X , Z... ? 4 5, 3... 7 1 0 76, 4 . . . P = 2 0 X ; D - E f f e c t o f i n o c u l a v o l u m e on As e x t r a c t i o n from Cu-concentrate, E - A e r a t i o n e f f e c t on e x t r a c t i o n o f As f r o m C u - c o n c e n t r a t e ; F L e a c h i n g s e l e c t i v i t y o f As a n d Cu f r o m Cu-concentrate.

-

-

.

-

407

-

i s r e c o v e r e d f r o m i t . The a c i d l e a c h e d s o l u t i o n i s t h e n r e c i r c u l a t e d i n t o the bioleaching process. Another method o f u s i n g b a c t e r i a l o x i d a t i o n o f s u l f i d e s i s t h e recovery o f fine-disseminated gold from s u l f i d e minerals.

There a r e

ores i n which g o l d i n s u l f i d e s i s u l t r a f i n e d i s s e m i n a t e d and i t i s n o t p o s s i b l e t o o b t a i n i t by c y a n i z a t i o n a f t e r u s u a l g r i n d i n g . ores are c a l l e d "refractory usually contain free gold, pyrhotine.

gold ores"

(10).

Such

Refractory g o l d ores

submicroscopic gold,

carbon,

heavy m e t a l s ,

By means o f t r a n s m i s s i o n e l e c t r o n m i c r o s c o p y a n d Mtlssba-

uer spectroscopy,

r e f r a c t o r y o r e s have been f o u n d t o c o n t a i n g o l d

a l s o c h e m i c a l l y combined i n s u l p h i d e s .

There a r e i n d i c a t i o n s t h a t

etoms o f g o l d s u b s t i t u t e a r s e n i c i n t h e s u l p h i d i c l a t t i c e

Such s u l -

p h i d e s h a v e t o b e c o m p l e t e l y decomposed s o a s t o f r e e o f t h e g o l d bonded i n t h e l a t t i c e .

T h i o b a c t e r i a a r e a b l e t o decompossite c r y s -

t a l i c l a t t i c e o f s u l f i d e s t o such an e x t e n t t h a t d i s s e m i n a t e d g o l d i s r e v e a l e d a n d opened t o t h e l e a c h i n g e f f e c t o f c y a n i d e , a substantial increase of

by which

A comparison o f c a p i t a l

gold i s achieved.

c o s t s f o r o b t a i n i n g g o l d between c l a s s i c m e t a L l u r g i c a 1 methods and b a c t e r i a l leaching. a) by method o f r o a s t i n g b ) by method o f p r e s s u r e l e a c h i n g c) comes

100

x

134

X X

by method o f b a c t e r i a l l e a c h i n g

81

out i n favor o f b a c t e r i a l leaching,

m a i n l y due t o t h e a d v a n t a -

geous s o l v i n g o f t o x i c e m i s s i o n s .

A t o u r w o r k i n g - p l a c e we a r e s t u d y i n g t h e p r o b l e m o f b a c t e r i a l r e l e a v i n g g o l d from our domestic sources o f m i n e r a l ores.

Sulphidic

a u r i f e r o u s c o n c e n t r a t e f r o m t h e P e z i n o k d e p o s i t was u s e d f o r o u r r e search. As,

The c h e m i c a l c o n c e n t r a t e c o m p o s i t i o n was a s f o l l o w s :

L,58 % Sb, 2 8 , 2 8 X Fe, 31,63 7; S ,

From t h e m i n e r a l o g i c a l p o i n t o f v i e w , c a r r i e r s o f gold are arsenopyrite,

6,12

X

S i 0 2 , 37,82

the main s u l p h i d i c minerals-

a n t i m o n i t e and p y r i t e .

The c o n c e n t r a t e g r a i n s i z e d i s t r i b u t i o n : G r a n u l a r i t y g r a d [mm]

0,07 0,04

0,03

-

-

0,Ol 091 0,07 0,04

0,02 0,03 0 , O l - 0,02 0,005- 0901 - 0,006

14,6

ppm Au.

Grade p o r t i o n

7

9 1

17,7

35,5 10,3 10,4 992 397 6 1 100,o

[X]

X

-

408

-

The i n v e s t i g a t i o n o f t h e c o n c e n t r a t e s a m p l e s by means o f an e l e c t r o n m i c r o s c o p e and by o t h e r i n s t r u m e n t s l e a d s t o t h e c o n c l u s i o n t h a t g o l d i s c h e m i c a l l y bonded i n t h e c a r r i e r s u l p h i d e s .

The p o r t i o n

o f t h e i n t i m a t e l y b o n d e d g o l d i s h i g h e r t h a n 9 5 % a n d t h u s t h e samp l e i s t o be c h a r a c t e r i z e d as r e f r a c t o r y g o l d ore. F o r l e a c h i n g t h e a u r i f e r o u s c o n c e n t r a t e we h a v e c h o s e n b a c t e -

r i a l strains

Thiobacillus

va D and M o s t M.

f e r r o o x i d a n s from two d e p o s i t s a t Dubra-

The s t r a i n D i s a n e f f i c i e n t one a n d comes f r o m t h e

deposit i n which t h e m i n e r a l o g i c a l ore composition i s s i m i l a r t o t h a t f r o m P e z i n o k ; t h e s t r a i n M comes f r o m a c o a l d e p o s i t ,

hovewer i t

p o s s e s s e d a c u l t i v a t e d h i g h r e s i s t a n c e t o a r s e n i c a t a good l e a c h i n g efficiency.

The l e a c h i n q was c a r r i e d o u t p a r a l l e l on s h a k e r s i n

f l a s k s o f 2 5 0 m l w h i l e t h e amount o f p u l p was 1 0 0 m l , d e n s i t y o f t h e s o l i d phase. OC

a t t h e 20 %

The t e m p e r a t u r e was m a i n t a i n e d a t 2 8 - 3 0

a l l the time.

The l e a c h i n g t e s t r e s u l t s a r e p r e s e n t e d i n t h e f o l l o w i n g t a b l e : -

Sequence of samples

Degree o f t h e s t r u c t u r e breakdown Arsenopyrite Pyrite

1x3

[z3

Bact. leaching

Extract. gold leaching

I

Gold r e r n v ~ r y i n the leach

(x 1

1

0

D

0

Tm

2

0

0

0

NaCN

3

55,8

11,3

D

Tm

4

55,8

11,3

D

NaCN

37,15

5

48,5

18,5

M

Tm

35,c)O

3,B6

2,98 41,04

The s u l p h i d e d i s s o c i a t i o n d e g r e e s p r e s e n t e d i n t h e s e c o n d a n d t h e t h i r d c o l u m n s o f t h e t a b l e a b o v e were d e t e r m i n e d by t h e X - r a y f f r a c t i o n method. urea-Tm,

di-

G o l d e x t r a c t i o n l e a c h i n g was p e r f o r m e d w i t h t h i o -

o r w i t h s o d i u m cyanide-NaCN.

The f i r s t t w o s a m p l e s u s e d i n

t h e e x t r a c t i o n l e a c h i n g w i t h o u t any p r e v i o u s d i s s o c i a t i o n o f c r y s t a l l a t t i c e s by b a c t e r i a l o x i d a t i o n ,

afforded a very low gold recovery,

which proved t h a t there i s l e s s than 4 p l e o f a refractory character.

L f r e e g o l d i n t h e g i v e n sam-

B a c t e r i a l l e a c h i n g i n samples 3,

4,5

d i s s o c i a t e s t h e s u l p h i d e l a t t i c e s and t o t h i s corresponds g o l d ext r a c t i o n o f 35 up t o 4 1 %.

These v a l u e s depend on u n e q u a l p y r i t e and

arsenopyrite dissociations, differences of

w h i c h f o l l o w s from t h e above m e n t i o n e d

t h e i r e l e c t r o d e p o t e n t i a l s as w e l l as from t h e arse-

nopyrite p r i o r i t y oxidation.

~

-

409

-

With respect t o a very f i n e gold intergrowth w i t h auriferous sulphides,

i t w i t 1 1 be r e a s o n a b l e t o t r e a t t h e s u l p h i d i c c o n c e n t r a -

t e p r i o r t o b a c t e r i a l l e a c h i n g by m e c h a n i c a l a c t i v a t i o n b y g r i n d i n g w h i c h a l l o w s t h e g o l d b e t t e r c o n t a c t w i t h t h e l e a c h i n g medium and, s t same t i m e ,

i n c r e a s i n g c h e m i c a l r e a c t i v i t y o f t h e s o l i d phase.

The i n v e s t i g a t i o n o f t h e m e c h a n i c a l a c t i v a t i o n e f f e c t on t h e r a t e o f b i o o x i d a t i o n a n d on t h e a r s e n o p y r i t e d e c o m p o s i t i o n h a s b r o u q h t t h e r e s u l t s shown i n F i g .

5.

I t f o l l o w s from t h e d i a g r a m t h a t

the optimal g r i n d i n g time i s f i f t e e n minutes,

a t which t h e r a t e o f

=

a r s e n i c r e l e a s i n g i n t o t h e l e a c h has been r e a c h e d as vo d - l by means o f a d a p t e d b a c t e r i a l r a c e Th. lity,

d e n o t e d a s TF-M

ferr.

0,62

g l

-1

f r o m t h e Most l o c s -

2 i n a r s e n o p y r i t e s u s p e n s i o n o f t h e 20

X solid

phase d e n s i t y .

-1

.d-l/ t o i n c r e a s e d t i m e F i g . 5 . C o u r s e o f As l e a c h i n g r a t e v o / g . l o f mechanochemical a c t i v a t i o n t / m i n / . 1 - c o n t r o l t e s t w i t h o u t bact e r i a ; 2 - TF-S n o t s d a p t e d ; 3 - TF-S ada t e d t o 6 g A s . 1 - 1 a n d TF-M 4 s u s p . 4%; 4 - TF-M d s p t e d t o 6 g As.1-f a n d (I s u s p . 4 X; 5 a n d Y s u s p . 2 0 X. adapted t o 10 q

-

AS.^-^

EIxperiments w i t h b i o o x i d a t i o n d e c o m p o s i t i o n o f a r e f r a c t o r y a u r i f e r o u s c o n c e n t r a t i o n f r o m P e z i n o k h a v e shown t h a t : t r a f i n e l y intergrown with auriferous sulfphides,

g o l d is u l -

and t h a t i t s l i b e -

r a t i o n and i t s g e t t i n g i n t o c o n t s c t w i t h c y a n i d e o r t h i o u r e a c a n b e made p o s s i b l e b y b a c t e r i a l p r e l e s c h i n g .

The g o l d r e c o v e r y w i l l b e

proportional t o the dissociation o f sulphides. r i t e oxidation,

I n view o f slow py-

a b o u t 80 % g o l d r e c o v e r y c a n be a c h i e v e d i n a t e c h -

nologicslly tolerable

time.

As f o r t h e d e s u l f u r i z a t i o n o f c o a l , main bearer o f s u l f u r ,

where t h e p y r i t e i s t h e

t h e r e are the f o l l o w i n g problems:

-

-

410

1. A c c o r d i n g t o p r e s e n t s t a t e o f k n o w l e d g e o n l y p a r t i a l d e s u l f u r i te.

2.

bounded i n c o a l i n t h e f o r m o f s u l f a t e o r o r -

The o t h e r s u l f u r ,

ganic s u l f u r ,

c a n n o t be r e c o v e r e d by t h i o b a c t e r i a .

a ) P y r i t e c a n b e l e a c h e d f r o m b l a c k c o a l by t h i o b a c t e r i a .

c o a l must be f i n e d o n t o g r a n u l a r i t y t o bacterial effect. days),

-

i t i s b a c t e r i a l decomposition o f p y r i -

zation o f coal i s possible,-

The

u n d e r 1 0 0 tan t o open t h e s u r f a c e

S i n c e l e a c h i n g is a l o n g p r o c e s s ( a t l e a s t 1 0

t h e necessary l e a c h i n g d e v i c e s would achieve g i a n t dimensi-

ons ( 1 1 ) . b) A short-time

b a c t e r i a l o x i d a t i o n o f p y r i t e and t h e s u c c e s s i v e

f l o t a t i o n a p p e a r more h o p e f u l . /12,13/

the o x i d a t i o n o f 10

-

A c c o r d i n g t o some f o r e i g n s o u r c e s

120 m i n u t e s w i l l do.

The p r o b l e m

of

r e s e a r c h under Czechoslovak c o n d i t i o n s i s b e f o r e us. C) D e p i r i t i z a t i o n o f

b r o w n c o a l i s a u n i q u e p r o b l e m w h i c h was r e -

s e a r c h e d b y t h e I n s t i t u t e o f G e o t e c h n i c s o f C z e c h o s l o v a k Academy o f Sciences i n Prague /14/. r a c t e r i z e d as f o l l o w s :

The a c h i e v e d r e m a r k a b l e r e s u l t s c a n b e c h a While i t i s necessary t o g r i n d t h e batch o f

black coal t h a t i s fine-disseminated i n i n g 30

by p y r i t e ,

t h e brown c o a l c o n t a -

X o f w a t e r w i l l do t o b e c r u h e d t o - 1 5 0

mm.

Coal has a w e l l -

developed p o r o s i t y t h a t enables a s u f f i c i e n t f a s t t r a n s i t i o n o f t h e l e a c h i n g o x i d a t i o n agent t o t h e s u l f i d e p a r t i c l e s i n s i d e c o a l g r a i n s and t h e d r a w i n g o f o x i d a t i o n p r o d u c t . d e t e r m i n e d t o be a d e c i s i v e p a r a m e t e r ,

The d i f f u s i o n r a t e o f i o n s was on w h i c h d e p e n d t h e r a t e o f

t r a n s i t i o n o f oxygen t o c o a l g r a i n s and d r a w i n g o f o x i d a t i o n p r o ducts,

A t t h e same t i m e ,

a substantial p a r t o f arsenopyrite present

i n c o a l is l e a c h e d t o g e t h e r w i t h p y r i t e u n d e r t h e e f f e c t o f t h i o bacteria. I n an e f f o r t t o a p p l y t h e r e s u l t s o f t h e b a s i c r e s e a r c h t o p r a c tice, from

a r e a l i z g t i o n s t u d y has been e l a b o r a t e d d e m o n s t r a t i n g t h a t t e c h n i c a l and e c o n o m i c a l p o i n t s o f v i e w t h e most f a v o r a b l e w o u l d

be t h e l e a c h i n g o f p y r i t i c s u l p h u r f r o m c o a l i n t h e head o f t h e c o a l c u t t h a t i s p r e p a r e d by s h a k i n g b l a s t and t h e c o a l i s showered w i t h m i n i n g w a t e r t h a t c o n t a i n s a s u f f i c i e n t number o f b a c t e r i a a s w e l l as t h e n u t r i e n t s u b s t a n c e s n e c e s s a r y f o r them.

A large-scale

expe-

r i m e n t a t t h e CSA l a r g e mine a f f o r d e d t h e f o l l o w i n g r e s u l t s presented i n the table. Another troublesome m i n i n g and b e n e f i c a t i o n problem i s t h e a t t e n d a n ce t o p u r i f i c a t i o n o f waste w a t e r s which, creased content of

as a r u l e ,

c o n t a i n an i n -

i o n s o f heavy o r r a d i o a c t i v e m e t a l s .

Heavy m e t a l s

s u p p r e s s k h e c o u r s e o f m e t a b o l i c p r o c e s s e s o f h i g h e r aa w e l l a s l o w e r

-

Days o f leaching

Content o f S ( i n d r y mate: X rial)

411

-

Decrease o f sulphur 0, 0

Consumption o f l e a c h i n g s o l u t i o n m3 p e r 1 t o f coal

0 66,24

1,07

69,62

2,48

organisms by b l o c k i n q t h e i r e n z y m a t i c systems so t h a t t h e y r e a c t

w i t h s u l p h i d i c g r o u p s o f k e y enzymes a n d d e s t r o y t h e i n t e g r i t y o f the c e l l walls /15/.

The d e g r e e o f h a r m f u l n e s s o f e l e m e n t s f o u n d i n

w a t e r may depend o n t h e i r p h y s i c a l o r c h e m i c a l s t a t e . F o r e x a m p l e , 6+ . chromium C r is h i g h l y p o i s o n o u s b u t i n t h e f o r m C r 3 + i t i s a l m o s t 5+ . is l e s s t o x i c t h a n n e u t r a l b i o l o g i c a l l y , a r s e n i c p e n t a v a l e n t As As3+.

Thus one o f t h e ways o f d e c r e a s i n g w a t e r t o x i c i t y may b e b i o -

chemical reduction o r oxidation.

F o r example,

b a c t e r i a o f t h e spe-

c i e s Aeromonas d e c h r o m a t i c u m r e d u c e p o i s o n o u s C r 6 + t e r i a B a c i l l u s a r a e n i c o x i d a n s o x i d i z e As3+ t o As5+.

t o Cr3+,

o r bac-

Selen reducing

b a c t e r i a o f t h e space C l o a t r i d i u m r e d u c e s e l e n i d e s and s e l e n a t e s f r o m t h e a o l u t i o n and a r e d m e t a l s e l e n e p r e c i p i t a t e i s p r e c i p i t a ted.

S u l p h a t e s r e d u c i n g b a c t e r i a r e d u c e d i s s o l v e d s u l p h a t e s o f some

m e t a l s w h i l e p r e c i p i t a t e s o f t h e i r s u l p h i d e s a r e b e i n g formed.

Cya-

n i d e - o x i d i z i n g b a c t e r i a a r e c a p a b l e o f decomposing and n e u t r a l i z i n g c y a n i d e s and t h i o c y a n i d e s . O f g r e a t e r i m p o r t a n c e f o r t h e p u r i f i c a t i o n o f waste w a t e r

metal ions is biosorptin. fungi,

from

A g r e a t number o f m i c r o o r g a n i s m s b a c t e r i a ,

a n d a l g a e a r e known t o b e a b l e t o a d s o r b a n d c u m u l a t e on t h e i r

or i n s i d e t h e i r c e l l s ,

surfaces,

s u c h m e t a l s a s Ag,

Cd,

Cu,

Pb,

Zn,

Au r a d i o a c t i v e m e t a l s a n d o t h e r s .

An e x t e n s i v e r e s e a r c h i n t o t h e p u r i f i c a t i o n o f w a s t e w a t e r s b y means o f s e l e c t e d r a c e s o f a l g a e h a s b e e n c a r r i e d o u t i n t h e l a b o r a t o r i e s o f t h e C S A V i n Prague /16/.

Nevertheless,

there are l o t s o f

p r o b l e m s ahead o f us c o n c e r n i n g t h e i n d i v i d u a l c h a r a c t e r o f p a r t i c u l a r c o n t a m i n a t i o n s o f i n d u s t r i a l waste.

R E F ER E N C E S

1

G.I. kian:

K a r a v a i k o , G . R o s s i , A.D. A g a t e , S . N . G r u d e v and Z . A . AvaB i o g e o t e c h n o l o g y o f M e t a l s - M a n u a l , UNEP, Moskva 1 9 8 8 , p.8,15.

-

6

7

412

-

A.R. C o l m e r a n d M.E. H i n k l e : The r o l e o f m i c r o o r g a n i s m s i n a c i d m i n e d r a i n a g e . i n S c i e n c e 1 0 6 ( 1 9 4 7 ) p. 253-256. T . P o d a n y i : I n P r o c e e d i n g s o f t h e I n t e r n a t i o n a l C o n f e r e n c e on Use o f M i c r o o r g a n i s m u a i n H y d r o m e t a l l u r g y . PBcs 1 9 8 0 p . 1 0 9 . R.Y. Hajnbczy: A s z e p e s i banyavarosok t b r t 6 n e t e . L e v o r a 1903. F . S p a l d o n a n d M. K u g n i e r o v b : Anwendung v o n M i k r o o r g a n i s m e n i n d e r A u f b e r e i t u n g b e r g b a u l i c h e r R o h s t o f f e . i n : P r o c e e d i n g s 2. I n t e r n a t i o n a l e F a c h t a g u n g - F o r t s c h r i t t e i n T h e o r i e und P r a x i s d e r A u f b e r e i t u n g a t e c h n i k . F I A F r e i b e r g 1 9 8 9 p. 21. L . Ahonen, P. H i l t u n e n a n d O.H. T u o v i n e n : The r o l e o f p y r r h o t i t e and p y r i t e i n t h e b a c t e r i a l l e a c h i n g o f c h a l c o p y r i t e o r e s . i n : F u n d e m e n t e l a n d a .D.D l i e d b i o h y d r o m e t a l l u r q- y_,- E l s e v i e r , Amsterdam 1 9 8 6 p. 20. V. SDaEek. J. B a r b n e k , J. TombBek a n d MarBblek: Proces bakter i o l o g i c k k h o l o u f e n f r u d s problemy p r i j e h o a p l i k a c i na l o f i s ko Smolnik. i n : Proceedingsof t h e Conference Poznatky a skds e n o s t i z cementbcie a m i k r o b i o l o g i c k d h o l d h o v a n i a h a l d na l o 2 i s k u S m o l n f k , 1 9 7 6 p p . 55-81. J . Tombgek a n d V. SpaEek: E x p e r i e n c e i n R e g e n e r a t i o n o f B i o l o g i c a l L e a c h i n g S o l u t i o n s u s i n g S u r f a c e A e r a t i o n . i n : G. R o s s i , A.E. Torma: R e c e n t P r o g r e s s i n B i o h y d r o m e t a l l u r g y ( C o n g r e s , S a r d i n i a 1983). S . J . P o l k i n , V.V. Psnin, E.V. Adamov, G . I . K a r a v a i k o a n d A.S. E e r n i a k : Theory and p r a c t i c e o f u t i l i z i n g m i c r o o r g a n i s m s i n p r o c e s s i n g d i f f i c u l t - t o - d r e s s o r e s and c o n c e n t r a t e s . i n : P r o c e e d i n g s o f XI. Min. P r a p . c o n g r e s , C a g l i a r i 1 9 7 5 p p . 901-923. P.M. Svash: A m i n e r a l o g i c a l i n v e s t i g a t i o n o f r e f r a c t o r y g o l d o r e s and t h e i r b e n e f i c a t i o n w i t h s p e c i a l r e f e r e n c e t o a r s e n i c a l ores. i n : J o u r n s l o f t h e S o u t h A f r i c a n I n s t i t u t o f M i n i n g and M e t a l l u r g y . Vol. 8-5/1988. P. B o s , T.F. H u b e r , C h . Koa, C . Ras a n d J . G . Kuenen: A D u t c h f e a s i b i l i t y s t u d y on m i c r o b i a l c o a l d e s u l p h u r i z a t i o n . i n : Fundamental and A p p l i e d B i o h y d r o r n e t a l l u r g y , E l s e v i e r , Amsterdam 1 9 8 6 . Z.M. Dogan, G. O z b a y o g l u , C. H i c y i l m a z , M. S a r i k a y a a n d G. Ozceng i z : B a c t e r i a l l e a c h i n g v e r s u s b a c t e r i a l c o n d i t i o n i n g and f l o t a t i o n i n desulphurization o f three d i f f e r e n t coals. i n : Fundamental and a p p l i e d B i o h y d r o m e t a l l u r g y , E l s e v i e r , Amsterdam 1 9 8 5 . P.R. Dugan: M i c r o b i a l d e s u l f u r i a a t i o n o f c o a l a n d i t s i n c r e a s e d m o n e t a r y v a l u e . i n : B i o t e c h n o l o g y a n d B i o e n g i n e e r i n g Symp. No. 6(1986). Z . V o l S i c k f : o p r a v a o b s a h u p o p o l o v i n a s f r y v hngd6m u h l i SHR. D o c t o r a l t h e s i s , P r a h a 1985. A.N. I l j a l e t d i n o v : MikrobiologiEeskije prevraBEenija metsllov. I z d a t e l s t v o "Nauka" K a z . S S R , A l m a - A t a 1 9 8 4 . K. N e b e r a n d J. Z b h r a d n i k : D o E i i 3 t o v b n f v o d a u t o t r o f n f m i m i k r o o r g a n i z m y a v y B B i m i r a e t l i n a m i . i n : S t u d i e E S A V 2 4 1986, Academia Praha.

.

-

9

10

11

12

13 14 15 16

A SMALL SIMULATION SYSTEM A N D E C O L O G I C A L FORECASTING

DEGERMENDZKY

A.G.

I n s t i t u t e o f B i o p h y s i c s o f t h e S i b e r i a n B r a n c h o f t h e USSR Academy o f S c i e n c e s , 660036, USSR INTRODUCTION

Water q u a l i t y p r e d i c t i o n a n d t h e f o r e c a s t i n g o f t h e s t s t e o f a q u a t i c e c o s y s t e m s becomes a v i t a l problem g i v e n an i n c r e a s i n g s h o r t a g e of f r e s h water.

The d e v e l o p m e n t o f m e t h o d o l o g y f o r c r e a t i n g

t h e a d e q u a t e m a t h e m a t i c a l m o d e l s o f a q u a t i c e c o s y s t e m s a n d t h e rel a t i v e s e l f p u r i f i c a t i o n model i s t h e n e c e s s a r y s t a g e i n p r o g n o s t i c m o d e l i n g i n b i o c h e m i c a l e c o l o g y . The p r i n c i p l e s o f s u c h m o d e l b u i l d i n g c a n b e e l a b o r a t e d b y m o d e l i n g t h e d y n a m i c s o f l a b o r a t o r y homogeneous ecosystems o f continuous t y p e f o r c o n t r o l l e d b i o p h y s i c a l e x p e r i m e n t c o n d i t i o n s [ l l . I n t h i s p a p e r , we c o n s i d e r t h e n o n - c o n v e n t i o n a l a p p r o a c h t o water q u a l i t y m o d e l l i n g ,

b a s e d on t h e "biophy-

s i c a l " c h a i n o f t h e o r e t i c a l and e x p e r i m e n t a l r e s e a r c h s t e p s :

inves-

t i g a t i n g t h e l a w s g o v e r n i n g t h e o r g a n i z a t i o n , s t a b i l i t y and cont r o l a b i l i t y o f "ideal" laboratory ecosystems - - * i d e n t i f y i n g t h e

-

means t o d e t e r m i n e c h e m i c a l and o t h e r d e n s i t y - d e p e n d e n t ( a u t o s t a b i l i z a t i o n phenomenon)

--

-

factors

f i e l d experiments t o determi-

ne t h e l i m i t i n g f a c t o r s i n t h e n a t u r a l ecosystem

-

--

carrying

e x p e r i m e n t s w i t h i d e n t i f i e d c h e m i c a l f a c t o r s a n d some o t h e r h y d r o bi0nt.s t o meaaure k i n e t i c parameters

-

--

synthesizing t h e ecosys-

tem m o d e l , i n c o r p o r a t i n g t h e o b t a i n e d k i n e t i c c h a r a c t e r i s t i c

--

(i.e.,

-

-

--

v e r i f y i n g t h e model on t h e b a s i s o f t h e e x i s t i n g e v i d e n c e r e l a t i n g t o a s i m i l a r water b o d y )

--

computations of t h e

f o r e c a s t and o v e r a l l c o a t o f e c o l o g i c a l i m p l i c a t i o n a o f t h e p r o j e c t . LAWS OF A N I D E A L " H O M O G E N E I T Y "

COMMUNITIES A N D ECOSYSTEMS

1. S t a b i l i t y o f m i x e d c u l t u r e ; p r i n c i p l e s o f i n t e r a c t i n g populations coexistence Criterion.

An i d e a l c o m m u n i t y i s a c o l l e c t i o n o f p o p u l a t i o n s

w i t h t h e d e n s i t y d y n a m i c s g o v e r n e d e x c l u s i v e l y by c e r t a i n e n v i r o n m e n -

-

- 414

t a l chemical f a c t o r s ,

which,

a c t u a l p o p u l a t i o n numbers.

i n turn,

m i g h t be i n f l u e n c e d by t h e

Some o f t h e s e f a c t o r s may o r i g i n a t e o u t s i -

A s u f f i c i e n t degree o f

de t h e s y s t e m and b e s u p p l i e d f r o m w i t h o u t . e n v i r o n m e n t a l h o m o g e n e i t y i s a l s o assumed.

I n o r d e r t o o b t a i n a ge-

n e r a l c o n d i t i o n r e l a t i n g t h e number o f c o e x i s t i n g s p e c i e s i n t h e c o m m u n i t y a n d t h e number o f d e n s i t y - d e p e n d e n t t h e environment,

chemical f a c t o r s i n

c o n s i d e r a mixed c u l t u r e w i t h no p r e d a t i o n .

It i s

convenient t o s t a r t a t h e o r e t i c a l analysis o f i n t e r a c t i n g populations (e.g.,

b a c t e r i a l ) w i t h t h e model d e s c r i b i n g t h e d e n s i t y dynamics o f

m s p e c i e s i n an open s y s t e m o f t h e c h e m o s t a t t y p e

t h e SGR

[ 2 , 3 1 . We assume

( s p e c i f i c g r o w t h r a t e ) o f e a c h s p e c i e s t o b e d e p e n d e n t on o r

c o n t r o l l e d by a few n u m b e r s ( n ) o f medium f a c t o r s ,

i n turn,

which,

a r e c o n t r o l l e d by t h e a c t u a l d e n s i t i e s o f t h e s e s p e c i e s .

We a r r i v e

a t t h e f o l l o w i n g system o f d i f f e r e n t i a l e q u a t i o n s ,

X.=

...,

[gi(A1,

-

An)

D1

.

Xi,

-

i=l,m,

m

D ( A OJ -

A .

J

where gi

(A1,

A,)

J

...,

+ t

..., An) .

ajkfkj(Al,

-

A ) i s t h e SGR f o r t h e i - t h species,

A 0 and A are, respectively, J j trations o f the j - t h growth-influencing

z e r o ) . The t e r m a j k f k j X k

D i s the

t h e i n p u t a n d medium c o n c e n factor

(some o f A 0 c a n b e

J

stands f o r the production or u t i l i z a t i o n

s u b s t a n c e by t h e k - t h s p e c i e s .

I t i s o b v i o u s l y v e r y d i f f i c u l t t o s u g g e s t any " v e r s a t i l e " o f g.

...,

a s a f u n c t i o n o f A1,

system o f e q u a t i o n s ( l ) , i n steady-state

o f these species

l o g i c a l theorem"

An.

However,

i t c a n b e shown t h a t t h e number o f s p e c i e s

(i.e.,

factors,

m L n).

determined by the d e n s i t i e s ,

I n the sequel,

cept o f the c o n t r o l l i n g factor i.e.,

t h i s "eco-

f o r mixed c u l t u r e s w i l l be c a l l e d t h e main s t a t e -

ment o r t h e e x t e n s i o n o f t h e Gause p r i n c i p l e . factor,

form

u s i n g t h e form o f t h e

c o e x i s t e n c e w i l l n o t p o s s i b l y e x c e e d t h e number o f

independent q r o w t h - c o n t r o l l i n q

Xi,

(1)

j=l,n,

k=l

flow rate,

rate o f the j - t h

Xk,

I f we e x t e n d t h e c o n -

t o include also the "biotechnological"

t h e c o n t r o l p e r f o r m e d by an e x t e r n a l d e v i c e a n d d i -

r e c t e d t o w a r d s t h e s t a b i l i z a t i o n o f a c e r t a i n f u n c t i o n o f t h e dens i t i e s o f the species

(turbidistat,

chlorophyllostat,

pH-stat,

t h e n t a k i n g i n t o a c c o u n t t h e number o f " b i o t e c h n o l o g i c a l "

t

,

does n o t c h a n g e t h e c r i t e r i o n o f c o - e x i s t e n c e : One o f t h e r e a l m e c h a n i s m s o f c o e x i s t e n c e .

etc.),

factors,

m ,< n t t .

The e x p e r i m e n t s w e r e

-

415

w i t h m i x t u r e o f two k i n d s o f yeast. cerevisiae

-

Two k i n d s o f y e a s t S a c c h a r o m y c e s

t h e d i p l o i d and h a p l o i d forms

j e c t s o f experiments.

-

w e r e c h o s e n a s t h e ob-

G r o w t h was p e r f o r m e d i n a c h e m o s t a t a t s e v e r a l

v a l u e s o f t h e d i l u t i o n r a t e (D)

r a n g i n g from 0.05

h-l

t o 0.4

h-'.

I n t h e z o n e o f g l u c o s e l i m i t a t i o n S G R t h e e x p e r i m e n t showed s t a b i l i z a t o n o f t h e t o t a l c o u n t and s t a b l e c o e x i s t e n c e o f t h e two forms

1, c ) .

o f y e a s t w i t h 40 % b e i n g h a p l o i d a n d 60 % d i p l o i d ( F i g .

The

s t e a d y - s t a t e c o m p o s i t i o n o f t h e m i x e d c u l t u r e was i n d e p e n d e n t f r o m t h e i n i t i a l p r o p o r t i o n o f t h e two forms [ l ] .

c

10 "PH

F i g . 1. The d y n a m i c s o f t h e c o m p o a i t i o n o f t h e m i x e d c u l t u r e a n d i n t e r a c t i o n c o e f f i c i e n t s 6 i n d i l u t i o n e x p e r i m e n t s (50-5 9/11: a , 0 - 0 . 2 h - 1 pH0=6.3, K-4.06; b , D = 0 . 1 2 h-1, pHo= 6 . 3 , K=4.06; c , D-0.05 h - i , pHo= 6 . 3 , K-4.06; d, 0 ~ 0 . 0 5 h-1, pHOz5.5, Kz3.7. E x p e r i m e n t a l d a t a : ooo t h e percentage o f t h e h a p l o i d form; rn t h e t o t a l biomass; A - r e s i d u a l s u g a r ; 0.0 - pH. C a l c u l a t i o n w i t h t h e m o d e l (1): - t h e p r o p o r t i o n o f t h e h a p l o i d form (Xh); -'''-' - t h e t o t a l biomass; -0-0-0- - pH; - glucose.

-

-

- - -

I n t h i s connection,

according t o the coexistence p r i n c i p l e ,

a

s u p p o s i t i o n was p r o p o u n d e d s u g g e s t i n g t h e e x i s t e n c e o f one more dens i t y dependent g r o w t h - c o n t r o l l i n g f a c t o r

(DOGF):

n >, m-2.

t o r was f o u n d t o b e t h e c o n c e n t r a t i o n o f h y d r o g e n i o n s , n = 2 ) d e t e r m i n i n g t h e a c i d i t y o f t h e medium,

H+

This fac(i.e.,

because i n t h o s e e x p e r i -

m e n t s t h e pH o f t h e c r o p i n t h e f e r m e n t e r was l o w e r e d down t o a l e v e l

-

- 416

of 3.2

I n t h e acute experiments,

( t h e i n l e t pH was 6 . 3 ) .

pH depen-

The t w o DDGF ( h y d r o g e n

d e n c e s o f t h e maximum S G R s were m e a s u r e d .

i o n s and g l u c o s e ) were i n t r o d u c e d t o t h e model. N u m e r i c a l a n d a n a l y t i c a l c a l c u l a t i o n s p e r f o r m e d w i t h a comput e r using independently-determined

parameters have demonstrated t h e

s p p l i c a b i l i t y o f t h e model t o t h e d e s c r i p t i o n and e x p l a n a t i o n o f experimental r e s u l t s (see Fig.

1; t h e c o m p u t a t i o n a l d a t a a r e r e p r e s e n -

t e d by c u r v e s ) .

2 . A u t o s t a b i l i z a t i o n o f DDGF I 4 1 P a r t i c u l a r property.

The e s s e n c e o f t h i s phenomenon c o n s i s t s

o f a l a c k o f dependence o f t h e s t e a d y - s t a t e t i o n o f the growth-limiting

supply r a t e s o f these factors.

Theoretically,

f r o m an a n a l y s i s o f t h e r e l a t i o n s h i p s

x J. i n

tions

factors,

-

A , J

= r

.(D)

J

t h i s result follows

f o r the steady-state

concentra-

m=n,

f(Ao),

#

J

I n o t h e r words,

-

X.

. . . , A:);

w . (A;,

j =

p; .i

1,m.

t h e r e i s a c o r r e l a t i o n between p o p u l a t i o n biomasses

and i n p u t c o n c e n t r a t i o n s o f t h e l i m i t a n t s ,

and no c o r r e l a t i o n w i t h

t h e background l e v e l o f t h e l i m i t i n g f a c t o r s ( F i g . Hence,

l e v e l o f concentra-

s y s t e m (1) a s s u m i n g a n e q u a l number o f s p e c i e s a n d

eq.

i.e.,

(mean)

f a c t o r s i n t h e ecosystem upon t h e i n p u t

2;

(51).

no c o r r e l a t i o n s h o u l d be observed between t h e l i m i t e d - p o p u l a -

t i o n biomass and t h e b a c k g r o u n d l i m i t a n t c o n c e n t r a t i o n . f i r s t i d e a f o r a m e t h o d o f DDGF d e t e r m i n a t i o n ,

i.e.

I t i s the

independence o f

t h e r e q u l a t o r c o n c e n t r a t i o n i n t h e medium a t t h e i n p u t l e v e l . The n e x t i d e a

-

the analysis o f steady-state

a t an a c c i d e n t a l p e r t u r b a t i o n o f

level of

input levels.

regulators

B o t h methods a l -

l o w u s t o f i n d t h e number o f c o e x i s t i n g p o p u l a t i o n s ( m )

as w e l l as

using only t h e t h e o r e t i c a l l y developed i n v a r i a n t : n

m

= n

-

1 j=1

Kinetic characteristics (SGR).

-

dA./dAo

J

dence o f SGR ( g )

( f o r example:

(Fig.

1).

(5.1.

[3l

A f t e r d e t e r m i n a t i o n some DDGF

( a c c o r d i n g a u t o s t a b i l i z a t i o n phenomenon)

glucose concentration

J

may b e r e c e i v e d t h e depen-

h a p l o i d and d i p l o i d ,

f o r Fig.

3 ) on

The S G R s a r e u s e d by m a t h e m a t i c a l m o d e l

-

= I

417

I

a

0,

I

b

f

/

1s

/ f

I f

/

0

2

4

6 So

8

1

0

1

2

METANOL. g l I 1

F i g . 2. S t e a d y - s t a t e o f b i o m a s s c o n c e n t r a t i o n M e t h y l o m o n a s ap. (Ti) a n d r e s i d u a l m e t h a n o l c o n c e n t r a t i o n ( S ) vs. i n p u t m e t h a n o l ( S O ) : D = 0 . 2 4 h-l [ S l .

O

P

0

-10

0

10

20

1.2

2.4

s

[g/l1

Fig. 3. D e p e n d e n c e o f S G R ( g ) for t h e h a p l o i d ( h ) a n d d i p l o i d (d) f o r m s o f y e a s t S. c e r e v i s i a e o n g l u c o s e c o n c e n t r a t i o n ( S ) (a); t h e s a m e o n s r e c i p r o c a l s c a l e (b). E x p e r i m e n t a l values: 0 , x - f o r t h e haploid and diploid forms respectively. 3. T o t a l e f f i c i e n c y o f p o p u l a t i o n a u t o r e g u l a t i o n The c o n t r o l o f t h e S G R o f m i c r o b i a l a n d o t h e r p o p u l a t i o n s p r o c e e d s via b i o c h e m i c a l a n d p h y s i c a l , a s a r u l e u n k n o w n , c o n t r o l l i n g

factors,

DDGF.

From t h e p h y s i c a l p o i n t o f v i e w ,

t i o n and t o t a l " f o r c e " o f t h e system's

response t o a p e r t u r b a t i o n o f biomass c o n c e n t r a t i o n

for the i - t h population,

population,

i n response t o t h e p e r t u r b a t i o n ,

1-th

t h a t must be c o n s i d e r e d t h e c r i t e r i o n o f i n t e r a c t i o n :

-

=

Bil

dgi/dt

I

P

-

dgi/dt

t*

I

U

Coefficients

Eil

,

T-*.

[Eil]

t*

where P s t a n d s f o r t h e p e r t u r b e d s t a t e , te.

the type o f interac-

o f r e g u l a t i o n c a n b e j u d g e d by t h e c h a r a c t e r

I t i s t h e increment o f the time d e r i v a t i v e o f the growth r a t e ,

A X1. e.g.

-

418

and U ,

t h e unperturbed sta-

have t h e n e x t a n a l y t i c a l view: n

A X1

Bli

1

-

l,i=I,m.

(dgi/dAj).(dAj/dt),

j=l Hence,

t h e e x p e r i m e n t a l l y - d e t e r m i n e d c h a r a c t e r and i n t e n s i t y

t e r a c t i o n s expressed i n terms o f i n t e r a c t i o n c o e f f i c i e n t s

Eil

f

i

l-

can

serve as a b a s i s f o r comparison w i t h t h e c o e f f i c i e n t s "synthesized" theoretically

f o r t h e c o n s i d e r e d ecosystem model.

The c o m p a r i s o n

c a n b e made f o r s p a c e a s w e l l a s f o r t i m e .

APPLICATION:

K R A S N O Y A R S K R E S E R V O I R MODELLING

C o n s t r u c t i o n o f ecomodel: E x p e r i m e n t s .

The a b o v e r e s u l t s h a v e

been used as a b a s i s i n d e v e l o p i n g w a t e r q u a l i t y p r e d i c t i o n models f o r the worst blooming p a r t o f the Krasnoyarsk r e s e r v o i r ,

t h e Sydin-

s k i bay.

The w o r k was c o n d u c t e d w i t h i n t h e s c i e n t i f i c p r o j e c t " C l e a n

Enisei".

Necesssry e x p e r i m e n t s and measurements o f t h e a l g a e k i n e t i c

c h a r a c t e r i s t i c s were c a r r i e d o u t a f t e r a n a l y s i n g f i e l d e v i d e n c e p r o v i d e d by f i e l d s t a t i o n s , I n 1984,

PO,,'

b a s e d on t h e a u t o s t a b i l i z a t i o n phenomenon.

t h e common d y n a m i c s o f t h e m i n e r a l p h o s p h o r u s c o n c e n t r a t i o n ,

and t h e n e t a l g a e biomass i n t h e bay had been m o n i t o r e d (Fig.4)

P h o s p h o r u s was s e l e c t e d a s a p o s s i b l e f a c t o r t o l i m i t t h e p r o d u c t i o n process. ve,

We h a v e made s n a t t e m t t o t e s t t h e t h e o r e t i c a l r e s u l t s abo-

implying a

week

c o r r e l a t i o n between t h e background l e v e l o f t h e

l i m i t i n g f a c t o r a n d t h e amount o f l i m i t e d b i o m a s s . Actually,

i n s t e a d o f t h e a l g a e biomass,

t i o n o f t h e c h l o r o p h y l l 'la",

Cch,

i t was t h e c o n c e n t r a -

t h a t was m e a s u r e d ,

p e r i o d s o f t i m e i s d i r e c t l y r e l a t e d t o t h e biomass.

which over small The v a l u e o f

C c h c a n b e t r a n s l a t e d i n t o t h e e q u i v a l e n t amount o f p h o s p h o r u s ,

Cph,

-

-

419

. ... . a

a

ma

. . .

..

.:**

1

2.5

POL [ m k g l l l F i g . 4. O b s e r v a t i o n d a t a on s j o i n t o c c u r e n c e o f t h e c h l o r o p h y l l II a II C and t h e l e v e l o f phosphorus c o n c e n t r a t i o n from 24/08/84 9 t o 29 / 6 B j 8 4 i n t h e S y d i n s k y bay.

which p r o v i d e d t h e a l g a e g r o w t h and t h e f o r m a t i o n o f t h e g i v e n qusnt i t y o f the chlorophyll.

I t i s known t h a t C

v a r i a t i o n range o f t h e " i n p u t "

--

0.325.Cch Then, t h e Ph p h o s p h o r u s c o n c e n t r a t i o n , A C;h, co-

rreaponding t o t h e observed c h l o r o p h y l l content v a r i a t i o n ,

50 m c g / l ,

(Fig.

=

4 ) e s t i m a t e s t o A Co

16 mcg/l.

w

A Cch

The v a r i a t i o n r a n -

Ph ge o f t h e b a c k g r o u n d p h o s p h o r u s c o n c e n t r a t i o n c a n b e a p p r o x i m a t e l y estimated from Fig.

4 a t 1.6 mcg/l,

concentration (no c o r r e l a t i o n " i n p u t

i.e.

-

one-tenth o f the "input"

phone ( p h o s p h o r u s ) " ) .

This

f a c t p r o v i d e s a s e r i o u s b a s i s f o r an e x p e r i m e n t a l t e a t o f t h e l i m i t i n g a c t i o n o f phosphorus, work season.

a n d t h i s was done, d u r i n g t h e 1 9 8 5 f i e l d

We o b t a i n e d t h e SGR ( F i g .

(Aphanizomenon f l o s - a q u a e )

5 ) f o r blue-green algae,

gBGA,

as a f u n c t i o n o f t h e m i n e r a l phosphorus

concentration,

P.

obtained (Fig.

6 ) , w h i c h i a t h e SGR o f a g g r e g a t e d b a c t e r i o p l a n k t o n ,

Similarly,

t h e f o l l o w i n g k i n e t i c c u r v e s have been

-

420

-

gB, v e r s u s d i s s o l v e d o r g a n i c m a t t e r (COD). d i a t o m a c e o u s a l g a e gDA ( E ,

The c h a r a c t e r i s t i c s f o r

t o ) ( E i s i l l u m i n a t i o n and t o i s tempe-

r a t u r e ) have been t a k e n from l i t e r a t u r e

[61.

I

2 m

01

0.35 -

0.1

1

0

0,12

0.24 P Irnglll

F i g . 5 . Dependence o f gBGA f o r t h e Aphanizomenon f l o s - a q u a e n e r a l phosphorus.

0

50

on m i -

100 SCOD [ mg I L 1

Fig.

6.

D e p e n d e n c e o f gB f o r t h e a g g r e g a t e d b a c t e r i o p l a n k t o n on COD.

-

421

-

U s i n g f i e l d d a t a on e c o s y s t e m c o m p o n e n t s a n d t h e o b t a i n e d e x perimental characteristics,

t h e m o d e l o f t h e e c o s y s t e m was f o r m u l a -

t e d ( b l o c k d i a g r a m shown i n F i g .

I

Fig.

7.

7).

DIATOMS

B l o c k - d i a g r a m f o r t h e Sydinsky bay ecosystem model.

The s m a l l s i m u l a t i o n p r e d i c t i o n s y s t e m ( S S P S )

and c a l c u l a t i o n .

The e c o l o g i c a l m o d e l was i n c o r p o r a t e d i n t o a s p e c i a l l y d e s i g n e d s i m u l a t i o n p r e d i c t i o n system i n t e n d e d f o r p e r s o n a l computers. tem f e a t u r e s t h e f o l l o w i n g m a i n u n i t s : menting g r a v i t a t i o n a l , morphometry u n i t ,

w i n d and r e s i d u a l c u r r e n t s ) ,

the ecological unit,

The s y s -

a hydrological u n i t (imple

etc.

t h e w a t e r body

The S S P S i a i n t e n d e d

t o be used i n s i m u l a t i o n e x p e r i m e n t s based on a m a t h e m a t i c a l model o f an a q u a t i c e c o s y s t e m ( a l a k e ,

a water reservoir,

a bay,

A s e q u e n t i a l c o m p u t a t i o n a l f l o w c h a r t i s implemented.

a river). First,

h y d r o l o g i c a l parameters a r e c a l c u l a t e d by t h e E u l e u r method, then a f t e r a hydrological mixing, ecosystem model a r e computed. j u s t i t t o a new w a t e r v o d y , i d e n t i f y s u r f a c e chambers,

t h e e c o l o g i c a l parameters o f the

The m o d e l i s q u i t e v e r s a t i l e .

To ad-

one h a s t o s p e c i f y t h e d e p t h map,

to

t o set the appropriate input aeries ( e i t -

h e r r e t r o s p e c t i v e o f p r e d i c t e d ones),

and t o d e s c r i b e t h e c o r r e s p o n -

d i n g hydro-meteorological c o n d i t i o n s (winds, r a t i o n , solar radiation,

the

and

water temperature,

precipitation, and e t c . ) .

evapo-

-

-

422

The e c o l o q i c a l u n i t o f t h e m o d e l ( E C O S )

d e s c r i b e s t h e ecosystem

processes i n water l a y e r s and i s designed u s i n g r e s u l t s o f s p e c i a l e x p e r i m e n t a l t e s t s and a v a i l a b l e e v i d e n c e f r o m l i t e r a t u r e a s w e l l aa pertinent hypothesis. series unit (ISU)

The i n p u t ( t i m e )

provides i n f o r m a t i o n about

t h e known e x t e r n a l p a r a m e t e r s f o r t h e g i v e n e c o s y s t e m d y n a m i c s c a l c u l a t i o n scenario.

The I S U i s a l o c a l d a t a b a n k o f s p a t i a l a n d tem-

poral characteristics. Multi-variant

c a l c u l a t i o n s t a k i n g i n t o account h y d r o l o g i c a l ,

m e t e o r o l o g i c a l and o t h e r i n f o r m a t i o n r e l a t i n g t o t h e model have been c a r r i e d o u t f o r t h e f o l l o w i n g e c o s y s t e m s t r u c t u r e s ( d e n o t e d b y 1,2,

...,

8 i n Fig.

1

8):

cesses a r e " f r o z e n " ; l e organic matter, are present: n i c matter, mations;

4

-

h y d r o l o g i c a l model alone,

-

2

-

-

3

n o o t h e r components;

diatomaceous and blue-green, phosphorus,

b i o l o g i c a l pro-

b a c t e r i o p l a n k t o n i a l i m i t e d by t h e a v a i l a b -

nitrogen,

t h e f o l l o w i n g components

bacteria,

protozoa,

no zooplankton ( Z )

orga-

and no t r a n s f o r -

i t i s t h e 3 r d supplemented w i t h t r a n s f o r m a t i o n s ;

l i k e t h e 3 r d e x c e p t t h a t gDA i s i n c r e a s e d a n d Z i s i n t r o d u c e d ; l i k e the 5 t h but with transformations, gDA i s s t i l l f u r t h e r i n c r e a s e d ;

8

-

7

6

goA i s somewhat

smaller.

+ 3

3 -.-.4 ====== 5 6 = = = =

46

7-

I

1.4

1

+

7

8

I

0

20

1

I

1

40

60 DAYS

Fig

8.

-

l i k e the 6 t h except t h a t

close t o 3rd but also includes

Z c a n n i b a l i s m as w e l l as t r a n s f o r m a t i o n s ,

2.t

-

-

5

D y n a m i c s o f t h e t o t a l m i n e r a l n i t r o g e n (g/m 3 1.

-

423

-

Component d y n a m i c s due t o h y d r o l o q i c a l p r o c e s s e s .

Shown i n

8 t o I1 are part o f c a l c u l a t i o n r e s u l t s for the e i g h t variants

Figs.

a l o n g w i t h t h e o b s e r v a t i o n d a t a ( + ) . An a n a l y s i s o f h y d r o l o g i c a l c a l c u l a t i o n s ( v s r i a n t No. t h e season,

1) shows t h a t on t h e a v e r a g e t h r o u g h o u t

and e x c l u d i n g t h e b l o o m i n g p e r i o d s ,

s u l t s are q u a l i t a t i v e l y , quantitatively,

the s i m u l a t i o n re-

a n d f o r some c h a m b e r s a n d c o m p o n e n t s a l s o

close t o the observations.

The d y n a m i c s o f a q q r e a q a t e d b a c t e r i o p l a n k t o n

of

B

(Fig.

growth-limiting clines,

The d y n a m i c s

o r g a n i c s i n t o t h e chambers.

W h e r e v e r t h i s i n f l o w de-

t h e d y i n g p r o c e s s becomes p r e d o m i n a n t a n d t h e d e n s i t y o f B

g o e s down ( e s p e c i a l l y flow).

(B).

9) i s related t o the variations i n the inflow o f the

i n chambers,

which l a c k strong s u b s t r a t e i n -

An i m p o r t a n t i m p l i c a t i o n o f t h i s r e s u l t i s t h a t t h e i n f l o w

o f t h e a l l o c h t h o n i c o r g a n i c m a t t e r cannot by i t s e l f a d e q u a t e l y exp l a i n t h e observed B dynamics,

and t h e f o r m a t i o n o f a d d i t i o n a l a u t o -

c h t h o n i c m a t t e r h a s t o b e assumed.

T h i s was t h e r e a s o n t o a d v a n c e

t o more c o m p l e x e c o s y s t e m m o d e l s . The d y n a m i c s o f t h e e c o s y s t e m w i t h t h e t u r n - o v e r account.

taken i n t o

I n t h i s a n d t h e n e x t c a l c u l a t i o n o f v a r i a n t s we s h a l l d i s -

c u s s o n l y t h e d y n a m i c s o f some s p e c i f i c b i o l o g i c a l c o i a p o n e n t s o f a n d we h a v e t o a d m i t i t , t h e d y n a m i c s o f h y d -

t h e ecosystem because, ro-chemical

components (COD,

phosphorus ( F i g .

t o t a l mineral nitrogen (Fig.

8 ) and

1 0 ) ) d o e s n o t whow much c h a n g e w i t h t h e e c o s y s t e m

variant.

. . _ . . .. . ' 19

. 0

'

20

I

LO

1

I

60

75 DAYS

Fig.

9. B a c t e r i a l d y n a m i c s .

-

424

-

0,:

0.1'

I

Or

Fig.

40

20

6o

DAYS

10. Dynamics o f phosphorus.

Diatoms.

The o b t a i n e d DA d y n a m i c s i s q u a n t i t a t i v e l y q u i t e c l o -

se t o the experiment i n the flow-through regions w i t h t h e calculat i o n according t o variant 8 ( w i t h the Z cannibalism). ce o f the pressure o f 2 ,

I n t h e absen-

t h e number o f DA r i s e s t o v e r y h i g h l e v e l s .

I t f o l l o w s t h a t t h e Z g r o w t h k i n e t i c s and t h e n u t r i t i o n range a r e bou n d t o p l a y a n i m p o r t a n t p a r t i n t h e DA " b l o o m i n g "

kinetics (parti-

c u l a r l y so i n t h e f a l l ) . The b l u e - q r e e n

alqae.

The c a l c u l a t e d BGA d y n a m i c s ( F i g .

l e s s c l o s e t o o b s e r v a t i o n s t h a n i n t h e c a s e o f DA,

11) i s

a l t h o u g h t h e a-

utumn e x p l o s i o n phase i s r e a s o n a b l y w e l l p r e d i c t e d i n v a r i a n t s 7 and B ( i n c r e a s e d SGR o f DA o r c a n n i b a l i s m ,

Z).

b r i n g i n g down t h e n u m b e r s o f

We a r e a s y e t u n a b l e t o a d e q u a t e l y r e p r o d u c e t h e f a l l - o f f

phase

i n BGA n u m b e r s b e c a u s e o f t h e o b v i o u s l a c k o f i n f o r m a t i o n a b o u t t h e r e a l b a s i c mechanisms o f t h i s p r o c e s s . Bacterioplankton.

The d y n a m i c s w e r e c a l c u l a t e d f o r t h e s e a s o -

n a l c h a n g e o f t h e f e e d b a c k c o e f f i c i e n t Ebb f o r s e v e r a l e c o s y s t e m t y -

pe variants, etc.

t a k i n g i n t o a c c o u n t t h e d y n a m i c s o f COD,

temperature,

We c a n s e e i n t r a s e a s o n a l p e a k s i n t h e amount o f n e g a t i v e f e e d -

back i n the B c o n t r o l ,

r e a c h i n g u p t h e l e v e l Ebb= 0.005 h-*.

If t h e

-

425

40

20

0

-

60 DAYS

F i q . 11. B l u e - g r e e n a l g a e d y n a m i c s ; i s i n c r e a s e d b y a f a c t o r o f 10.

COD l e v e l ,

a B l i m i t i n g factor,

h a v e Bbb=-0.004

h-

t h e o r d e r o f -0.07

2

. The

h-2,

was as a c t u a l l y o b s e r v e d ,

"natural" i.e.

for variants 5 t o 8 the scale

some 20 t o 2 0 0 t i m e s g r e a t e r i n t h e

a b s o l u t e v a l u e t h a n one o b t a i n e d t h e o r e t i c s l l y .

The s i g n o f t h e

feedback ( i t i s n e g a t i v e ) c o i n c i d e s w i t h t h e p r e d i c t i o n . t h e n a t u r a l "amount"

we w o u l d

e x p e r i m e n t a l l e v e l o f Ebb is o f

Therefore,

o f f e e d b a c k i n t h e 8 r e g u l a t i o n is s u b s t a n t i a l l y

larger than i n the theory.

There a r e s e v e r a l hypotheses t h a t c o u l d

e x p l a i n t h e above d i s c r e p a n c y .

The p r o p o s e d p a r a m e t e r s B

i.i a r e

an

e s s e n t i a l l y new c r i t e r i o n t o t e a t t h e m o d e l s v a l i d i t y a n d t o o b t a i n new k n o w l e d g e a b o u t t h e n a t u r e o f i n t r a e c o s y s t e m r e l a t i o n s (Bbb i s j u s t one o f s u c h p a r a m e t e r s ) . CONCLUSION

1. A n o n - c o n v e n t i o n a l

approach i s proposed t o develop p r e d i c -

t i o n models f o r water q u a l i t y .

Thi;

a p p r o a c h i s b a s e d on a t h e o r e t i -

c a l and e x p e r i m e n t a l a n a l y s i s o f l a b o r a t o r y ecosystems and t h e " i d e alization"

o f aome h y d r o p h y s i c a l p a r a m e t e r s ( i d e a l m i x i n g ,

thermo-

stabilization). 2.

The a n a l y s i a o f s t a b l e l a b o r a t o r y c o m m u n i t i e s h a s l e d t o t h e

f o l l o w i n g conclusions:

(a)

426

-

t h e number o f c o e x i s t i n g s p e c i e s i s n o t

g r e a t e r t h a n t h e number o f e n v i r o n m e n t a l t h e Gause p r i n c i p l e ) ;

(b)

p a r t t o play i n species coexistence, weak e x c h a n g e f l o w s ; effect;

(c)

f a c t o r s (an extension o f

s p a t i a l h e t e r o g e n e i t y has a v e r y l i t t l e even under t h e c o n d i t i o n s o f

the importance o f the a u t o s t a b i l i z a t i o n

( d ) t h e method f o r d e t e r m i n i n g t h e t o t a l e f f i c i e n c y o f po-

pulation autoregulation.

These p r i n c i p l e s w e r e t h e b a s i s f o r t h e

ecosystem model o f t h e S y d i n s k y bay o f t h e K r a s n o y a r s k r e s e r v o i r ( t h e s m a l l s i m u l a t i o n p r e d i c t i o n system).

3 . M u l t i v a r i a n t c a l c u l a t i o n s o f t h e ecosystem dynamics were i n r e a s o n a b l e agreement w i t h t h e f i e l d o b s e r v a t i o n data.

For the f i r s t

t i m e t h e t h e o r e t i c a l and e x p e r i m e n t a l e s t i m a t e s have corresponded t o t h e t y p e and t h e s t r e n g t h o f n a t u r a l i n t r a p o p u l a t i o n r e l a t i o n s i n t h e a g g r e g a t e d b a c t e r i o p l a n k t o n component.

On t h e w h o l e ,

the

accuracy o b t a i n e d by t h e model i s n o worse t h a n t h a t o b t a i n e d by t h e c o n v e n t i o n a l approach. REFERENCES M i x e d c o n t i n u o u s c u l t u r e s o f m i c r o o r g a n i s m s , N o v o s i b i r s k , "Nauka" (Russ.), 1 9 8 1 , p p . 26-106. A.G. D e g e r m e n d z k y , On t h e P r o b l e m o f c o - e x i s t e n c e o f i n t e r a c t i n g c o n t i n u o u s p o p u l a t i o n s , 1981, aee r e f . I l l . A.G D e g e r m e n d z k y , V . A . A d a m o v i c h a n d V.N. P o z d y a y e v , On t h e c y b e r n e t i c s o f b a c t e r i a l communities: o b s e r v a t i o n s , experiments, 1 9 8 9 , 2 ( 6 1 , pp. 437-477. and t h e o r y . " C y b e r n e t i c s and Systems A . G . Degermendzk'y,N.S. P e c h u r k i n a n d A.N. S h k i d c h e n k o , A u t o s t a b i l i z a t i o n o f f a c t o r s c o n t r o l l i n g t h e g r o w t h i n b i o l o g i c a l systems, N o v o s i b i r s k , "Nauka" ( R u s s . ) , 1 9 7 9 , 1 4 1 pp. J.H. K i m a n d D . Y . Ryu, O p t i m i z a t i o n o f medium a n d m a x i m i z a t i o n o f biomass p r o d u c t i v i t y i n p r o d u c t i o n o f s i n g l e - c e l l p r o t e i n from J. F e r m e n t . T e c h n o l . " , 1 9 7 6 , ( 6 ) , pp. 4 2 7 - 4 3 6 . methanol. D. T i l m a n , E c o l o g i c a l c o m p e t i t i o n b e t w e e n a l g a e : e x p e r i m e n t a l c o n f i r m a t i o n o f r e s o u r c e - based c o m p e t i t i o n t h e o r y . "Science", 1 9 7 6 , 192 ( 4 2 3 8 1 , pp. 463-4651,

,

A STERILIZABLE CENTRIFUGAL S E P A R A T I O N S Y S T E M FOR A S E P T I C AND C O N T A I N E D CELL H A R V E S T AND RECYCLE C.

KRODK,

H.

AXELSSON a n d C.

Alfa-Lava1 Separation AB,

THORSSON

Tumba,

Sweden

ABSTRACT Many new p r o c e s s e s i n t h e b i o t e c h n o l o g i c a l i n d u s t r y r e q u i r e c o m p l e t e l y c l o s e d equipment because o f t h e concern f o r t h e s a f e t y o f process operators.

I n s o l v i n g t h e containment problem,

s o l u t i o n s a r e employed,

technical

which are also suitable i n solving also the

T h i s i s t h e case w i t h c e n t r i f u g a l s e p a r a t o r s .

a s e p s i s problem.

c e n t l y developed separator, i t s complete i n s t a l l a t i o n .

t h e BTUX 5 1 0 ,

A re-

i s p r e s e n t e d as w e l l as

Performance d a t a f o r b a c t e r i a s e p a r a t i o n

are alao given. C e n t r i f u g a l s e p a r a t o r s a r e f r e q u e n t l y used f o r down-stream processing i n biochemical processes.

The s t a n d a r d m a c h i n e s r e q u i r e ,

however, a i r f r o m t h e o u t s i d e f o r v e n t i l a t i o n and c o o l i n g t o reduce f r i c t i o n heating,

g e n e r a l l y m a k i n g them u n a c c e p t a b l e f o r

aseptic pro-

cessing. The o u t g o i n g a i r s t r e a m w i l l a l s o i n many c a s e s c a r r y w i t h i t a e r o s o l a p r o d u c e d i n t h e machine.

These m i g h t c a u s e p r o b l e m s i f a

biosafe installation i s required.It

has been s u g g e s t e d t h a t separa-

t o r s s h o u l d be p l a c e d i n c l o s e d boxes,

but this i s only practical i n

caae o f v e r y s m a l l a e p a r a t o r s . There a r e ,

however,

s p e c i a l l y designed s e p a r a t o r s which can be

u s e d i n c l o s e d s y s t e m s a n d w h i c h c a n be s t e r i l i z e d b y steam. SPECIAL FEATURES FOR S E P A R A T O R S I N CLOSED S Y S T E M S The m a i n d i f f e r e n c e s b e t w e e n a n o r m a l b i o p r o c e s s i n g s e p a r a t o r (e.g.

f o r bakers’ yeaat)

and a s p e c i a l s e p a r a t o r d e s i g n e d f o r con-

t a i n m e n t and a s e p s i s and s t e r i l i z a t i o n are:

A.

The n o r m a l l a b y r i n t h s p i n d l e s e a l i n g i s r e p l a c e d b y a m e c h a n i c a l sealing (Fig,

1). I f t h e r e q u i r e m e n t s a r e n o t t o o h i g h ,

a single

-

428

m e c h a n i c a l s e a l m i g h t be u s e d ,

-

b u t normally a double s e a l i n g

w i t h some b a r r i e r l i q u i d b e t w e e n t h e s e a l s i s u s e d .

During ste-

r i l i z a t i o n t h e b a r r i e r l i q u i d i s r e p l a c e d b y s t e a m w h i c h means t h a t t h e p r o d u c t c o n t a c t e d p a r t o f t h e s e a l between t h e gear hous i n g and t h e b o w l c a s i n g w i l l b e s t e r i l i z e d f r o m b o t h s i d e s a t t h e same t i m e .

B.

I f t h e s e p a r a t o r i s t o be s t e r i l i z e d by s t e a m ,

t h e n t h e bowl co-

v e r and s o l i d s r e c e i v i n g c y c l o n e must be d e s i g n e d as p r e s s u r e vessels.

S e a l i n g s a n d o t h e r m a t e r i a l m u s t be o f t y p e s t h a t c a n

be used a t t h e s t e r i l i z a t i o n t e m p e r a t u r e ( n o r m a l l y 1 2 1 C ) .

C.

I f i t i s a separator w i t h i n t e r m i t t e n t s o l i d s discharge a t the p e r i p h e r y o f t h e bowl,

t h e n p r e c a u t i o n s must be t a k e n t h a t t h e

a i r s h o c k wave p r o d u c e d when d i s c h a r g i n g i n t o a c l o s e d s y s t e m ,

w i l l n o t c a u s e any p r o b l e m s .

Normally,

t h i s i s a c h i e v e d by f i t -

t i n g an a i r r e c i r c u l a t i o n t u b e b e t w e e n t h e c y c l o n e a n d t h e b o w l c o v e r t o d e c r e a s e t h e s h o c k wave ( s e e F i g .

1).

ADVANTAGES W I T H C O N T I N U O U S DISCHARGE T h e r e a r e a number o f a d v a n t a g e s w i t h c o n t i n u o u s p r e s s u r i s e d discharge o f solids,

-

namely

r e c y c l i n g t o t h e f e r m e n t e r o f c e l l s o r s u p e r n a t a n t w i t h o u t need o f e x t r a equipment.

I t i s very valuable i f i t i s performed i n s i d e

the s t e r i l e barrier

-

l e s s c e l l breakage and l e a s t e m p e r a t u r e i n c r e a s e s i n c e sudden l a r ge p r e s s u r e d r o p s , c h a r g i n g machine,

-

a s a t t h e d i s c h a r g e o f an i n t e r m i t t e n t l y d i s are avoided

l o w e r m e c h a n i c a l c o m p l e x i t y o f t h e n o z z l e s e p a r a t o r means l e s s mec h a n i c a l wear and m a i n t e n a n c e

-

n o d i s t u r b a n c e o f t h e s e p a r a t i o n p r o c e s s a s when d i s c h a r g i n g an i n t e r m i t t e n t l y d i s c h a r g i n g machine.

C O N C E N T R A T I O N OF BIOMAS'S The B T U X 510 c e n t r i f u g a l s e p a r a t o r i s a n o z z l e - t y p e m a c h i n e . N o z z l e machines d i s c h a r g e c o n t i n u o u s l y a s o l i d s c o n c e n t r a t e a t a cons t a n t f l o w r a t e t h r o u g h n o z z l e s n e a r o r a t t h e max.

bowl diameter.

The f l o w r a t e i s p r o p o r t i o n a l t o t h e o p e n i n g a r e a o f t h e n o z z l e , number o f n o z z l e s , t h e b o w l speed.

t h e i r d i s t a n c e f r o m t h e c e n t e r o f r o t a t i o n and

the

-

A.

IMPROVED S P I N D L E S E A L I N G

8.

BOWL COVER D E S I G N E D AS PRESSURE V E S S E L

C.

A I R R E C I R C U L A T I O N LOOP

429

-

1. C e n t r i f u g e d e s i g n d e t a i l s f o r b i o s a f e t y

Fig.

I n p r a c t i c e there i s a lower l i m i t o f the nozzle diameter, 0.6

mm d e p e n d i n g on t h e r i s k o f b l o c k a g e b y o v e r s i z e d p a r t i c l e s .

0.3This

means o f c o u r s e t h a t t h e r e i s a l o w e r l i m i t o f t h e c o n c e n t r a t e F l o w rate. When t h e s u s p e n s i o n c o n s i s t s o f c o m p a r a t i v e l y l a r g e p a r t i c l e s , that settle quickly,

t h e r e s i d e n c e t i m e i n t h e b o w l can be s h o r t t o

achieve' t h e s e p a r a t i o n . t h e r e f o r e be l a r g e ,

The p o s s i b l e f l o w r a t e t o t h e s e p a r a t o r w i l l

and t h e n o z z l e Flow can e a s i l y be c o n t r o l l e d t o

-

430

-

achieve the r i g h t concentration i n the nozzle concentrate.

This i s

t h e case w i t h yeast suspensions. When,

however,

t h e p a r t i c l e s t o be s e p a r a t e d a r e s m a l l ,

the

p o s s i b l e f e e d f l o w r a t e w i l l b e o f t h e same o r d e r o f m a g n i t u d e a s t h e minimum p o s s i b l e n o z z l e f l o w r a t e a n d t h e c o n c e n t r a t e o b t a i n e d n o t much m o r e c o n c e n t r a t e d t h a n t h e f e e d m a t e r i a l . case w i t h b a c t e r i a .

Therefore,

T h i s is o f t e n t h e

t h e u s u a l t y p e o f s e p a r a t o r f o r bac-

t e r i a i s the i n t e r m i t t e n t l y discharging centrifuge.

N O Z Z L E M A C H I N E FOR B A C T E R I A S E P A R A T I O N H a v i n g d e t e r m i n e d t h a t t h e b e s t s e p a r a t o r t y p e i s a n o z z l e machine,

t h e m o s t i m p o r t a n t t e c h n i c a l p r o b l e m t o s o l v e i s how t o r e d u -

ce t h e f l o w r a t e t h r o u g h t h e nozzles.

By c r e a t i n g a f l o w - l i m i t i n g

w h i r l i n a chamber i n f r o n t o f t h e n o z z l e o p e n i n g , r e d u c e d by a f a c t o r o f 5

-

t h e f l o w can be

10, w h i c h i n p r a c t i c e i s t h e n e c e s s a r y

r e d u c t i o n t o concentrate b a c t e r i a s u c c e s s f u l l y (see F i g 2).

1. BODY 2 . TANGENTIAL INLET 3. SPIN CHAMBER 4. COVER 5. OUTLET HOLE Fig.

2.

V o r t e x chamber a n d n o z z l e .

-

431

-

These s o c a l l e d V O R T E X n o z z l e s h a v e a l s o a n o t h e r c h a r a c t e r i s t i c feature;

Fig.

t h e f l o w r a t e i s d e p e n d e n t on t h e v i s c o s i t y ( s e e F i g 3 ) .

3 . The dependence o f v i s c o s i t y on V O R T E X n o z z l e f l o w r a t e

The v i s c o s i t y e f f e c t i s shown i n F i g s 4 a n d 5.

They i l l u s t r a -

t e t h e i n f l u e n c e o f s o l i d s c o n t e n t a n d v o r t e x n o z z l e a n d chamber d i m e n s i o n s on t h e n o z z l e f l o w r a t e f o r b a k e r s ’

y e a s t a n d E.

c o l i KlZ,,

respectively. The w h i r l a c t s a s a r e s i s t a n c e chamber.

With a t h i n c o n c e n t r a t e ,

f o r the flow through t h e vortex

t h e w h i r l r u n s a t h i g h speed,

which

c r e a t e s a h i g h c o u n t e r - a c t i n g p r e s a u r e g r a d i e n t and a h i g h r e s i s t a n ce t o the flow,

so t h a t t h e f l o w r a t e t h r o u g h t h e system i s b e i n g

reduced.

I f s o l i d s concentration,

and t h e r e f o r e a l s o v i s c o s i t y i n c r e a s e s ,

t h e speed o f t h e w h i r l w i l l be l o w e r and t h e c o u n t e r a c t i n g p r e s s u r e g r a d i e n t w i l l a l s o be l o w e r .

T h i s causes a h i g h e r f l o w

f r o m t h e noz-

zle. The a d v a n t a g e o f t h i s phenomenon i s t h a t t h e s e p a r a t i o n r e s u l t i s stabilized.

The s o l i d s c o n t e n t o f

the discharged concentrate i s

k e p t a t a r e l a t i v e l y h i g h and even l e v e l i r r e s p e c t i v e o f f l u c t u a t i o n s

i n feed flow r a t e or feed concentration.

This regulation takes place

a u t o m a t i c a l l y within t h e v o r t e x system d u r i n g separation.

-

432

-

f 2200 Y 0, I

4

2000

U W

t 5 a

VORTEX :

1800

CHPMElER1WTLET HOLE

+ V

1

[ mrnlrnm

1600

V

1012

1400

1200

. 0 l

/

1

&a

1000

1011.4

800

10/1,0

600

51 1.0 511.4

400

200

5 /0,6

0 2

4

6

0

u) 12

14 16 18 20 22

t

0

10

1

20

30

2

LO

50

5

10

60 50

70

I

80

lo001 100 5000

-p

'10WEIGHT 1 WEIGHT o/o

VOL / VOL

VISCOSITY mPas (15.C)

F i g . 4. B T U X 510 w i t h c o u n t e r p r e s s u r e ( 6 0 0 k P a ) . C o n c e n t r a t e f l o w a s a f u n c t i o n o f c o n c e n t r a t e c o n c e n t r a t i o n f o r b a k e r s y e a s t a t 15 O C

T H E CENTRIFUGAL S E P A R A T O R TYPE BTUX 510 An e x a m p l e o f a n o z z l e s e p a r a t o r d e s i g n e d a c c o r d i n g t o t h e r e q u i r e m e n t s o f a s e p s i s and c o n t a i n m e n t and s u i t a b l e for b a c t e r i a separation i s the Alfa-Lava1 T h i s machine,

s e p a r a t o r BTUX 510.

see F i g 6 , i s a d i s k s t a c k s e p a r a t o r where t h e

c o n c e n t r a t e o f s o l i d p a r t i c l e s is d i s c h a r g e d b y VORTEX n o z z l e s t o wards t h e c e n t e r o f t h e bowl,

w h e r e i t i s p i c k e d up b y a p a r i n g t u b e ,

which

-

a c t i n g a s a pump

-

433

-

discharges t h e c o n c e n t r a t e under pressu-

re. l h e s e p a r a t o r h a s a l s o an i n t e r m i t t e n t d i s c h a r g e s y s t e m a t t h e periphery o f t h e bowl,

o p e r a t e d by p r e s s u r i z e d a i r .

T h i s system i s

however n o r m a l l y o n l y employed d u r i n g c l e a n i n g - i n - p l a c e

(CIP).

A n advantage i n h a v i n g a pneumatic d i s c h a r g e system,

which i s

is t h a t t h e r e i s n o r i s k f o r c o n t a m i n a t i o n b e t w e e n

r a t h e r unusual,

p r o d u c t o r any o p e r a t i n g l i q u i d . S e p a r a t o r has a d o u b l e m e c h a n i c a l s e a l as s p i n d l e s e a l i n g and there i s also a seal a t the bottom o f the spindle f o r supply o f d i s charge a i r .

The b o w l c o v e r i s d i m e n s i o n e d a s a p r e s s u r e v e s s e l a n d

t h e r e i s an a i r r e c i r c u l a t i o n t u b e f r o m t h e c y c l o n e .

0

20

40

60

100

80 x Iv l v

F i g , 5.

V i s c o s i t y e f f e c t on E .

c o l i K12

O h 1

- 434 -

F i g . 6 . B T U X 5 1 0 B o w l a n d I n - and O u t l e t . P r e s s u r i z e d d i s c h a r g e o f b o t h c o n c e n t r a t e and e f f l u e n t = N O n e e d f o r d o w n s t r e a m pump. A - F e e d i n l e t , B Concentrate o u t l e t , C - C e n t r i f u g a t e o u t l e t ; 1 - P a r i n g d i s c , 2 - D i s c s t a c k , 3 - Par i n g tube, 4 - Concentrate tubes, 5 - C I P v a l v e plugs, 6 - C I P valve s l i d e , 7 Vortex nozzles

-

-

-

435

-

The b o w l c o v e r h a s a j a c k e t f o r c o o l i n g . can be used.

Tap w a t e r o r g l y c o l

The f i l l e d c o o l i n g j a c k e t r e d u c e s s o u n d l e v e l s u b s t a n -

tially. The c a p a c i t y o f t h i s s e p a r a t o r i s u p t o 10 c u b i c m e t e r p e r h o u r

A s l i g h t l y modified version,

suitable f o r yeast separation,

has a

c a p a c i t y o f 60 c u b i c m e t e r p e r h o u r .

BIOSAFETY AND ASEPSIS

R u l e s and g u i d e l i n e s about b i o s a f e t y c o n s i d e r o n l y complete systems.

As t h e s e p a r a t o r i s o n l y o n e c o m p o n e n t i n s i d e s u c h a s y s t e m ,

i t i s i m p o s s i b l e t o say t h a t a b i o p r o c e s s i n g s e p a r a t o r i s designed according t o a c e r t a i n biosafety l e v e l

because,

d e p e n d i n g o n how

t h e s e p a r a t o r i s i n s t a l l e d a n d how t h e c o m p l e t e s y s t e m i s u s e d , f e r e n t l e v e l s can be reached.

F o r BL-1,2

dif-

and 3 t h e s e p a r a t o r has t o

be equipped w i t h mechanical s e a l s p r e s s u r e r e s i s t a n t cover and a i r r e c i r c u l a t i o n t u b e a s d e s c r i b e d above. The i n s t a l l a t i o n p r i n c i p l e s f o r o b t a i n i n g g o o d a s e p s i s a r e i n c a s e s t h e same a s t h o s e u s e d f o r c o n t a i n m e n t . inevitable,

Small deviations are

b u t some o f t h e d i f f e r e n c e s c o n c e r n o n l y t h e way o f o p e -

r a t i n g the unit. THE CLOSED STERILE BTUX 5 1 0 S Y S T E M I n o r d e r t o e x p l a i n how t h e BTUX i s i n s t a l l e d i n a c l o s e d a n d s t e r i l i z a b l e s y s t e m a n u m b e r o f f l o w c h a r t s ( f i g s 8 - 181, e a c h s h o wing a p a r t o f t h e system,

w i l l be used.

PRODUCT

CIP LIQUID WAlER (STERILE)

CONCENTRATE DRAIN OR CIP RETURN CENTRIFUGATE

a

SOLIDS

Fig.

7.

BTUX 5 1 0 P r o c e s s L i n e $ .

-

436

-

P R O C E S S LINES Fig.

7 shows t h e p r o c e s s l i n e s f o r a n o r m a l ,

installation.

not sterilizable

These l i n e s a r e a r r a n g e d i n s u c h a way t h a t t h e s e p a -

r a t o r c a n e i t h e r be f e d w i t h p r o d u c t o r w i t h s t e r i l e w a t e r .

The o u t -

l e t l i n e s f o r c o n c e n t r a t e and c e n t r i f u g a t e can e i t h e r be connected t o the process or t o drain.

Often,

t h e d r a i n l i n e must c o n t a i n a

k i l l tank i n a contained i n s t a l l a t i o n . Fig.

8 shows how t h e p r o c e s s l i n e s c a n b e s t e r i l i z e d .

f e d t o t h e e n d o f one o f t h e s e l i n e s , trifuge line.

Steam i s

w h i c h i n t h i s case i s t h e cen-

Condensate i s removed a t t h e end o f a l l o t h e r l i n e s .

A t a l l these p o i n t s there i s a pneumatic s h u t o f f valve,

a temperatu-

r e t r a n s m i t t e r and a steam t r a p .

PRODUCT

CONCENTRATE

CIP LIQUID

DRAIN OR CIP RETURN CENTRIFUGATE

WATER (STERILE]

CONDENSATE STEAM

0

CONDENSATE

Fig.

8.

BTUX 510 P r o c e s s L i n e s S t e r i l i z a t i o n

FLUSHING S Y S T E M The f l u s h i n g s y s t e m makes i t p o s s i b l e t o c l e a n t h e b o w l c o v e r , the cyclone, Fig.

t h e a i r r e c i r c u l a t i o n t u b e and t h e o u t s i d e o f t h e bowl.

9 shows t h e s y s t e m i n a n o r m a l i n s t a l l a t i o n .

The e x t e n t t o

w h i c h t h e system i s used depends on t h e r e q u i r e m e n t s o f t h e p r o d u c t and t h e process.

-

437

-

FLUSHING WATE ( STERILE 1 CIP LIQUID

DRAIN

Fig.

9 . BTUX 510 F l u s h i n g System

Fig.

1 0 shows how t h e s y s t e m i s s t e r i l i z e d .

Steam f r o m t h e b o w l

c o v e r goes backwards t h r o u g h t h e f l u s h i n g l i n e s t o a steam t r a p . Condensate i s a l s o removed f r o m t h e b o w l c o v e r d r a i n o u t l e t .

During

o p e r a t i o n t h i s o u t l e t s h o u l d be c o n n e c t e d e i t h e r t o an open d r a i n , a k i l l t a n k o r t o a n o t h e r c l o s e d s y s t e m d e p e n d i n g on t h e r e q u i r e d containment l e v e l .

FLUSHING WATER ISTERILE) CIP LIQUID

DRAIN CONOENSATE

Fig.

10.

B T U X 510 F l u s h i n g S y s t e m S t e r i l i z a t i o n

- 438

-

PNEUMATIC DISCHARGE SYSTEM Fig.

11 s h o w s t h i s s y s t e m i n i t s c o n t a i n e d v e r s i o n .

f i l t e r s a r e p l a c e d on t h e i n -

Sterilizing

and o u t l e t l i n e s f o r d i s c h a r g e a i r .

S m a l l t a n k s a r e p l a c e d on t h e s e l i n e s i n o r d e r t o l i m i t t h e p r e s s u r e s h o c k s t o t h e f i l t e r s upon d i s c h a r g e .

AIR

Fig.

TILATION

11. B T U X 5 1 0 P n e u m a t i c D i s c h a r g e S y s t e m

The s t e r i l i z a t i o n i s shown i n F i g .

12.

The i n l e t l i n e i s s t e r i -

l i z e d by a s e p a r a t e s t e a m s u p p l y a n d t h e o u t l e t l i n e by s t e a m c o m i n g from t h e bowl c o v e r .

STEAM1 -VENTILATION

CONDENSATE Fig.

1 2 . B T U X 510 P n e u m a t i c D i s c h a r g e S y s t e m S t e r i l i z a t i o n

-

439

-

SEALING L I Q U I D SYSTEM Fig.

13 shows t h e s e a l i n g l i q u i d system which i n c l u d e s t h e flow

c o n t r o l and s u p e r v i s i o n o f t h e b a r r i e r l i q u i d t o t h e mechanical sealings.

T h i s s y s t e m i s s t e r i l i z e d b y r u n n i n g steam i n s t e a d o f b a r r i e r

l i q u i d through t h e s e a l s (see F i g .

14).

SEALING LIQUID(STERILE )

USED SEALING WATER

Fig.

13. BTUX 510 S e a l i n g L i q u i d S y s t e m .

SEALING LIQUID STEAM S fERlLE

f

I

Fig.

1 4 . B T U X 510 S e a l i n g L i q u i d s y s t e m S t e r i l i z a t i o n .

CONDENSATE AND USED SEALNG WATER

-

440

-

V E N T I L A T I O N SYSTEM

The v e n t i l a t i o n s y s t e m i n a c l o s e d i n s t a l l a t i o n c o n s i s t s o f a sterilizing 15.

f i l t e r p l a c e d i n t h e v e n t i l a t i o n l i n e a s shown i n ~ i g .

T h i s s y s t e m i s s t e r i l i z e d a s shown i n ~ i g .1 6 by p l a c i n g a s h u t -

o f f v a l v e a f t e r t h e f i l t e r a n d r e m o v i n g c o n d e n s a t e . As t h e s y s t e m a b u r s t i n g d i s c has been

i s completely closed during s t e r i l i z a t i o n ,

placed i n the v e n t i l a t i o n l i n e t o p r o t e c t the separator against overpressure.

T h i s l i n e has t o be c o n n e c t e d a c c o r d i n g t o t h e r u l e s t h a t

a r e a p p l i c a b l e f o r each s p e c i f i c p l a n t .

VENTILATION

Fig.

1 5 . BTUX 510 V e n t i l a t i o n S y s t e m

TI1.ATION

0

CONDENSATE WATER

Fig,

1 6 . B T U X 510 V e n t i l a t i o n s y s t e m S t e r i l i z a t i o n

C O O L I N G SYSTEM F i n a l l y t h e c o o l i n g s y s t e m i s shown i n F i g .

17.

Both bowl cover

and c y c l o n e have c o o l i n g j a c k e t s w h i c h s h o u l d b e s u p p l i e d w i t h c o o l i n g

liquid.

441

-

T h i s s y s t e m i s o u t s i d e t h e s t e r i l i z a t i o n a r e a b u t h a s t o be

drained during s t e r i l i z a t i o n .

THE TOTAL S Y S T E M The c o m b i n e d f l o w c h a r t f o r a l l s y s t e m s c o m b i n e d i s shown i n Fig.

18.

VALIDATION OF T H E STERILIZATION PROCEDURE The s t e r i l i z a t i o n t i m e a n d t e m p e r a t u r e a r e f r e e l y p r o g r a m m a b l e . The s t e r i l i z a t i o n c a n n o t s t a r t u n t i l a few v a l v e s h a v e b e e n s e t i n the r i g h t position

-

they are i n t e r l o c k e d v i a p o s i t i o n switches.

Ot-

h e r v a l v e s w i t h a c t u a t o r s a r e s e t i n c o r r e c t p o s i t i o n by t h e comput e r programme. There a r e 10 t e m p e r a t u r e t r a n s m i t t e r s i n t h e system m o n i t o r i n g t h a t t h e d e s i r e d t e m p e r a t u r e has been reached.

The s t e r i l i z a t i o n t i -

me i s c o u n t e d f r o m t h e moment when a l l t h e t r a n s m i t t e r s show t h e pre-set degr.

temperature.

I t w i l l t a k e a b o u t 20 m i n u t e s t o r e a c h 1 2 1

C. I n one' v a l i d a t i o n

test,

8 s p o r e bags w i t h 8.

stearothermophilus

were p l a c e d o n d i f f e r e n t p o s i t i o n s i n t h e s e p a r a t o r . a n d i n t h e p e r i p h e r a l equipment.

A f t e r 60 mins a t 121 degr.

C t h e y were a l l s t e r i l e .

SOME PROCESS D A T A W I T H B T U X 510 I n T a b l e 1 some d a t a c a n be f o u n d on t h e s e p a r a t i o n o f l i v e c e l l s o f E.

c o l i K12 g r q w n on a s y n t h e t i c medium.

I t i s t o be n o t e d

that

-

t h e DS c o n t e n t o f

-

t h e c o n c e n t r a t e f l o w i n c r e a s e s w i t h f e e d f l o w r a t e and f e e d con-

the separation efficiency i s excellent the concentrate i s v i r t u a l l y constant

centration. The BTUX i s a l s o u s e d f o r t h e c o n c e n t r a t i o n o f b a c t e r i a f o r t h e p r o d u c t i o n o f amino a c i d s , rDNA p r o d u c t s .

starter cultures,

enzymes a n d v a r i o u s

I t has a l s o been t e s t e d w i t h good r e s u l t s i n b l o o d

fractionation. I n aseptic processing,

t h e BTUX 510 s y s t e m h a s b e e n p r o v e n t o

k e e p s t e r i l e f o r a t l e a a t 72 h o u r s i n an a s e p t i c c e l l r e c y c l e s y s t e m . This,

however,

i s n o t t h e upper time l i m i t .

-

442

-

COOLING LIQUID

LIQUID Fig.

1 7 . BTUX 5 1 0 C o o l i n g S y s t e m

TABLE 1 T e s t r u n s w i t h B T U X 510

Feed

[m3/hl 05 [g/11 1st batch

2nd b a t c h

Concentrate

Supernatant

I m 3 / h l D S 19/11

DS [ g / l l

I

Separation efficiency

I %I

2

22

[

0.31

143

3

24

0.43

168

3.6

23

I I

0.52

159

I I

2.1

39

1

0.44

138

I

3.0

39

0.61

185

1.04

97.9

3.5

39

0.68

153

5.98

87.8

2

18

0.37

97

3

17

4

17

I I I

0.41

124

0.52

130

I I I

0.04

99.8

0.09

99.7

0.13

I

99.5

I

0.07

I

99.8

I

0.18

I

99.2

0.17

I

99.1

0.26

I

98.7

I I I

PRODUCT

CIP LIQUID

WATER (STERILE) FLUSHING WATER ISTERILEI

3

CIP LIQUID

STEAM COOLING LIQUID SEALING LlOUlD AIR

I

c c W I

Fig.

18.

BTUX 5 1 0 T o t a l S y s t e m

-

444

-

CONCLUSION

By l o o k i n g a t s e p a r a b i l i t y d a t a f o r b a c t e r i a , t h e c o n c e n t r a t i o n r e q u i r e m e n t s , i t c o u l d , u n t i l r e c e n t l y , e a s i l y b e found t h a t i n most cases an i n t e r m i t t e n t l y d i s c h a r g i n g h i g h s p e e d c e n t r i f u g a l s e p a r a t o r i s t h e most s u i t a b l e t y p e o f machine. However, w i t h t h e i n t r o d u c t i o n of vortex n o z z l e s , a l s o t r u l y continuously o p e r a t i n g nozzle machine c a n be employed.

The m a c h i n e c a n a l s o b e s o d e s i g n e d t h a t steam s t e -

rilization is possible,

which o p e n s t h e way t o a p p l i c a t i o n s where

an i n c r e a s e d b i o s a f e t y l e v e l i s n e e d e d , e.g.

c e l l recycle.

or t o a s e p t i c processing,

Other volumes in this series

1 Atmospheric Pollution 1978 edited by M. M. Benarie 2 Air Pollution Reference Measurement Methods and Systems edited by T. Schneider, H. W. de Koning and L. J. Brasser 3 Biogeochemical Cycling o f Mineral-Forming Elements edited by P. A. Trudinger and D. J. Swaine 4 Potential Industrial Carcinogens and Mutagens by L. Fishbein 5 Industrial Waste Management by S. E. Jsrgensen 6 Trade and Environment: A Theoretical Enquiry by H. Siebert, J. Eichberger, R . Gronych and R . Pethig 7 Field Worker Exposure during Pesticide Application edited by W. F. Tordoir and E. A. H. van Heemstra-Lequin 8 Atmospheric Pollution 1980 edited by M . M . Benarie 9 Energetics and Technology of Biological Elimination of Wastes edited by G. Milazzo 10 Bioengineering, Thermal Physiology and Comfort edited by K. Cena and J. A. Clark 11 Atmospheric Chemistry. Fundamental Aspects by E. MBszaros 12 Water Supply and Health edited by H. van Lelyveld and B. C. J. Zoeteman 13 Man under Vibration, Suffering and Protection edited by G. Bianchi, K. V. Frolov and A. Oledzki 14 Principles o f Environmental Science and Technology by S. E. Jsrgensen and I. J o hnsen 15 Disposal of Radioactive Wastes by 2. Dlouhg 16 Mankind and Energy edited by A. Blanc-Lapierre 17 Quality of Groundwater edited by W. van Duijvenbooden, P. Glasbergen and H. van LeIyveld 18 Education and Safe Handling i n Pesticide Application edited by E. A. H. van Heemstra-Lequin and W. F. Tordoir 19 Physicochemical Methods for Waste and Wastewater Treatment edited by L. Pawlowski 20 Atmospheric Pollution 1982 edited by M . M. Benarie 21 Air Pollution by Nitrogen Oxides edited by T. Schneider and L. Grant 22 Environmental Radioanalysis by H. A. Das, A. Faanhof and H. A. van der Sloot 23 Chemistry for Protection of the Environment edited by L. Pawlowski, A. J. Verdier and W. J. Lacy 24 Determination and Assessment of Pesticide Exposure edited by M. Siewierski 25 The Biosphere: Problems and Solutions edited by T. N. Veziroglu 26 Chemical Events i n the Atmosphere and their Impact on the Environment edited by G. B. Marini-Bettdo 27 Fluoride Research 1985 edited by H. Tsunoda and M - H . Yu 28 Algal Biofouling edited by L. V. Evans and K. D. Hoagland 29 Chemistry for Protection of the Environment 1985 edited by L. Pawlowski, G. Alaerts and W. J. Lacy 30 Acidification and its Policy Implications edited by T. Schneider 31 Teratogens. Chemicals which cause birth defects edited by V. M. Kolb Meyers 32 Pesticide Chemistry by G. Matolcsy, M. Niidasy and V. Andriska 33 Principles o f Environmental Science and Technology by S. E. Jsrgensen (second revised edition)

34 Chemistry for Protection of the Environment 1987 edited by L. Pawlowski, E. Mentasti, C. Sarzanini and W. J. Lacy 35 Atmospheric Ozone Research and its Policy Implications edited by T. Schneider, S. D. Lee, G. J. R . Wolters and L. D. Grant 36 Valuation Methods and Policy Making in Environmental Economics edited by H. Folmer 37 Asbestos in the Natural Environment by H. Schreier 38 How t o Conquer Air Pollution. A Japanese Experience edited by H. Nishimura 39 Aquatic Bioenvironmental Studies by C. D. Becker 40 Radon in the Environment by M. Wilkening 41 Evaluation o f Environmental Data for Regulatory and Impact Assessment by S. Ramamoorthy and E. Baddaloo 42 Environmental Biotechnology edited by A. Blaiej and V. Privarova