Extracellular Microbial Polysaccharides

Extracellular Microbial Polysaccharides In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium...

Author: Paul A. Sandford | Allen Laskin

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Extracellular Microbial Polysaccharides

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

Extracellular Microbial Polysaccharides Paul A . Sandford, EDITOR U. S. Department of Agriculture A l l e n Laskin, EDITOR Exxon Research and Engineering Co.

A symposium co-sponsored by the Division of Carbohydrate Chemistry and the Division of Microbial and Biochemical Technology at the 172nd Meeting of the American Chemical Society, San Francisco, Calif., August 3 0 - 3 1 , 1976

ACS SYMPOSIUM SERIES 45

AMERICAN

CHEMICAL

SOCIETY

WASHINGTON, D. C. 1977

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

Library of Congress CIP Data Extracellular microbial polysaccharides. (ACS symposium series; 45 ISSN 0097-6156) Includes bibliographical references and index. 1. Microbial polysaccharides—Congresses. I. Sandford, Paul Α., 1939- . Π. Laskin, Allen I., 1928. III. American Chemical Society. Division of Carbohydrate Chemistry. IV. American Chemical Society. Division of Microbial and Biochemical Technology. V. Series: American Chemical Society. ACS symposium series; 45. QR92.P6E97 ISBN 0-8412-0372-5

Copyright ©

660'.62 ACSMC 8

77-6368 45 1-326

1977

American Chemical Society All Rights Reserved. N o part of this book may be reproduced or transmitted in any form or by any means—graphic, electronic, including photo copying, recording, taping, or information storage and retrieval systems—without written permission from the American Chemical Society. PRINTED IN T H E UNITED STATES O F AMERICA

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

ACS Symposium Series Robert F. G o u l d , Editor

Advisory Donald G. Crosby Jeremiah P. Freeman E. Desmond Goddard Robert A. Hofstader John L. Margrave Nina I. McClelland John B. Pfeiffer Joseph V. Rodricks Alan C. Sartorelli Raymond B. Seymour Roy L. Whistler Aaron Wold

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

FOREWORD The A C S

S Y M P O S I U M

SERIES

was founded in 1974 to provide

a medium for publishin format of the IN

CHEMISTRY

SERIES

parallels that of the continuing

SERIES

except that in order to save time the

papers are not typeset but are reproduced as they are submitted by the authors in camera-ready form.

As a further

means of saving time, the papers are not edited or reviewed except by the symposium chairman, who becomes editor of the book.

Papers published in the A C S

S Y M P O S I U M

SERIES

are original contributions not published elsewhere in whole or major part and include reports of research as well as reviews since symposia may embrace both types of presentation.

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

PREFACE

A

new fermentation industry, the production of extracellular microbial water-soluble polysaccharides, arose in the late 1950's and early

1960's and is now expanding rapidly. Several factors have

accelerated

the use of microbial polysaccharides as well as the search for new sources of water-soluble polysaccharides. Although hydrocolloids obtained from plants and seaweed have been used successfully for numerous applications in the food, textile, agricultural, paint, and petroleum industries, increasing labor costs, limited sources, adverse climate conditions, and increased demands have several of these traditionally used plant and seaweed gums. Also industry has demands for water-soluble polymers that are not met by the traditional plant and seaweed gums. Extracellular polysaccharide production is a widespread characteristic of microorganisms.

Several of these polymers have proven to be

commercially significant. T h e usefulness of these microbial polysaccharides primarily results from their unique physical and chemical properties which are determined by their individual component sugars and their mode of linkages. Their constant chemical properties and constant supply also increase their desirability. Other reasons for industry's interest in microbial gums are their potentially diverse sources and types. This symposium focuses on the production and properties of extracellular microbial polysaccharides that are currently being used by industry or which have potentially useful industrial properties. Special emphasis is placed on new areas of research that would improve or stimulate industrial production and use of this valuable class of water soluble hydrocolloids. U.S. Department of Agriculture

P A U L A . SANDFORD

Peoria, Ill. 61604 Exxon Research and Engineering C o .

A L L E N I. L A S K I N

Linden, N.J. 07036 January 12, 1977

ix

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

1 Culture Maintenance and

Productivity

DENIS K. KIDBY Department of Soil Science and Plant Nutrition, The University of Western Australia, Nedlands, Western Australia, 6009

Microbial productivity is based upon a very large store of genetic information. I than one million items encoded build-up, i t is a common practice to transfer approximately 10 cells to a fresh medium. To retain the complete genetic identity of such an inoculum, for even a single generation, 10 base pairings must occur with complete f i d e l i t y . However, examination of such a c e l l population would reveal that thousands of errors had occurred. The f i d e l i t y of DNA replication is nevertheless impressive, and given s k i l f u l management, microbes can approach the r e l i a b i l i t y of solution chemistry in terms of product reproducibility. While genetic change may be a disaster when uncontrolled, i t is also the means of improving productivity. Genetic alterations were once achieved more or less by chance. However, the possibility now exists for the deliberate, and specific, alteration of genotype to yield productive chimeras limited only by the imagination. One can envisage the real possibility of producing a bacterial c e l l which could extract i t s energy and growth requirements from a few simple salts, the a i r and sunlight, producing a bacterial product such as Xanthan Gum or, an algal product such as agarose. However, despite such advances in the manipulation of genes, i t seems certain that the inherent genetic i n s t a b i l i t y of microbes w i l l remain an important problem for many years; and it is largely to this type of d i f f i c u l t y that the present paper is addressed. Before discussing i n s t a b i l i t y , the origins of industrial cultures w i l l be briefly considered. 9

16

Sources of Microbes Natural Sources. Many useful microbes are directly obtainable from the s o i l or other natural sources. It is often possible to employ unusual or extreme conditions as selective agents in the search for microbes with special a b i l i t i e s . Bacteria isolated from hot springs, can be grown near the temperature of boiling water (1). Acid mine leachings harbour 1

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

2

EXTRACELLULAR

MICROBIAL

POLYSACCHARIDES

b a c t e r i a able to grow at high concentrations of s u l f u r i c a c i d (2). Microbes f r e e of toxins or e s p e c i a l l y a l l e r g e n i c substances may be sought i n f o o d s t u f f s i n which they are known to r e g u l a r l y occur i n h i g h c o n c e n t r a t i o n s . I s o l a t i o n Procedures. The p r i n c i p l e s employed are those of s e l e c t i v e enrichment or i n h i b i t i o n . The r e q u i r e d , or suspected, n u t r i t i o n a l and p h y s i o l o g i c a l c h a r a c t e r i s t i c s of the organism sought w i l l d i c t a t e and a c t u a l procedure. The o x i d a t i o n , r e d u c t i o n , b i n d i n g , or r e l e a s e , of dyes are p a r t i c u l a r l y adapt a b l e f o r s e r v i c e as i n d i c a t o r s of s p e c i f i c biochemical events. The possession of a p a r t i c u l a r enzyme, or s e r i e s of enzymes, may be l i n k e d to e i t h e r the a b i l i t y , or i n a b i l i t y , to grow on a p a r t i c u l a r medium. B i o l o g i c a l i n d i c a t o r s such as the growth of an i n d i c a t o r organism ar p a r t i c u l a r l s e n s i t i v t h func t i o n s as the e x c r e t i o n o methods have been devise which are by t h e i r nature c r y p t i c and seemingly i n a c c e s s i b l e f o r s e l e c t i o n . For example Okanishi and Gregory Ô ) were able to devise a simple method to r e v e a l yeast c o l o n i e s possessing higher than normal methionine l e v e l s . Protocols f o r the i s o l a t i o n of s p e c i f i c n u t r i t i o n a l types may be sought i n the taxonomic l i t e r a t u r e (4, 5). Specific procedures f o r various groups of organisms are a v a i l a b l e i n the recent l i t e r a t u r e (6>, J7> 8). However, the seeker of d e s i r a b l e microbes must o f t e n r e l y upon h i s own r e s o u r c e f u l n e s s . A fairly thorough biochemical understanding of the event of i n t e r e s t can b e a most u s e f u l guide to i s o l a t i o n procedures. In the case of e x t r a c e l l u l a r products, such as polysaccha r i d e s , there may or may not be c h a r a c t e r i s t i c a l l y mucoid colonies. S e l e c t i v e procedures should, i f p o s s i b l e , e x p l o i t some s p e c i f i c property of the d e s i r e d p o l y s a c c h a r i d e . However, there are p o s s i b i l i t i e s f o r i n d i r e c t s e l e c t i o n using a s s o c i a t e d c h a r a c t e r i s t i c s . For example, many c h a r a c t e r i s t i c s , s u i t e d to r e p l i c a - p l a t i n g methods, are a s s o c i a t e d with polysaccharide producing Xanthomonas campestris ( 9 ) . In the case of mucoid E s c h e r i c h i a c o l i , there appears to be a s s o c i a t e d UV s e n s i t i v i t y (10) . R e p l i c a - p l a t i n g procedures are f r e q u e n t l y the most u s e f u l technique s i n c e one can s e l e c t f o r c e l l s which e i t h e r grow or do not grow. D i a g n o s t i c procedures which are d e s t r u c t i v e may a l s o be used since a l l m a t e r i a l under i n v e s t i g a t i o n i s r e t a i n e d on the r e p l i c a s . The employment of s p e c i f i c enzymes f o r the recogn i t i o n of c e r t a i n types of polysaccharides i s an i n t e r e s t i n g p o s s i b i l i t y f o r the development of screening programmes. In t h i s connection i t i s i n t e r e s t i n g to note that r e c o g n i t i o n systems based upon enzyme s p e c i f i c i t y may already occur i n bacteriophage (11) . C u l t u r e C o l l e c t i o n s . Searching f o r microbes i n e x i s t i n g c u l t u r e s w i l l f r e q u e n t l y be quicker, cheaper and e a s i e r than i s o l a t i o n from nature. As an a i d to such a search, H e s s e l t i n e and Haynes (12) have w r i t t e n a guide to c o l l e c t i o n s containing

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

1.

KiDBY

Culture

Maintenance

and

3

Productivity

i n d u s t r i a l l y u s e f u l microbes. However, there can be no f o r thorough searching of the current l i t e r a t u r e .

substitute

Maintenance of Genotype Nature of the Problem. An i n d u s t r i a l l y u s e f u l microbe i s an asset which may range from being moderately valuable to almost p r i c e l e s s . The p r e s e r v a t i o n of such an asset deserves a p r i o r i t y which i t seldom r e c e i v e s . The greatest b a r r i e r to s u c c e s s f u l p r e s e r v a t i o n of genotype may be a f a i l u r e to appreciate that: ( i ) microbes are i n h e r e n t l y unstable, ( i i ) there i s no method yet devised f o r the complete p r e s e r v a t i o n of genotype. Inherent I n s t a b i l i t y of Microbes. The p o t e n t i a l f o r genotype v a r i a b i l i t y has been i n d i c a t e d i n the i n t r o d u c t o r y remarks. I t i s now necessary to discus th a c t u a l mechanis f chang d how these r e l a t e to phenotype A l l regions of a gen gene mutable than others because they have i n t r a g e n i c regions of high m u t a b i l i t y , are i n f l u e n c e d by some other gene which i s i t s e l f mutable or, are under the c o n t r o l of genes which promote mutation. A l l of these mechanisms are known to occur, i n c l u d i n g some i n which the m u t a b i l i t y i s e f f e c t e d by an extrachromosoma1 element o r , an i n f e c t i o u s agent (13). I t i s these more h i g h l y mutable genes, and e s p e c i a l l y those cases i n v o l v i n g i n f e c t i o u s agents, that are most troublesome. Mutations may be e i t h e r r e p l i c a t i o n dependent or r e p l i c a t i o n - i n d e p e n d e n t . I t i s speculated (14) that replication-dependent mutations r e f l e c t e r r o r s i n DNA r e p l i c a t i o n , and replication-independent mutations r e f l e c t error-prone r e p a i r systems, Mutations may i n v o l v e : ( i ) frame-shift; ( i i ) deletion; ( i i i ) i n s e r t i o n ; ( i v ) base p a i r s u b s t i t u t i o n . The e f f e c t on the code may be e i t h e r the production of missense, nonsense, or a non-code f u n c t i o n may be l o s t . The r e s u l t i n g phenotypes may i n c l u d e : ( i ) a l t e r e d RNA base sequence; ( i i ) a l t e r e d amino a c i d sequence; ( i i i ) premature termination; ( i v ) degenerate s i l e n c e . A c e r t a i n p r o p o r t i o n of these mutants w i l l be c r y p t i c , p a r t i c u l a r l y those i n v o l v i n g missense. Mutations which lead to the i n s e r t i o n of a s i m i l a r amino a c i d o r , because of code degeneracy, the w i l d type amino a c i d , w i l l u s u a l l y not be revealed. I t has been c a l c u l a t e d that 25% of 549 base p a i r s u b s t i t u t i o n s i n v o l v e degeneracy (15) . I t i s a l s o i n t e r e s t i n g to note that there i s a greater than random p r o b a b i l i t y that base p a i r s u b s t i t u t i o n s w i l l lead to s u b s t i t u t i o n of a s i m i l a r r a t h e r than a d i s s i m i l a r amino a c i d (16). L e t h a l mutations w i l l a l s o be c r y p t i c since these w i l l not p e r s i s t , unless they are c o n d i t i o n a l . Intragenic mutations are non-random. S i t e s which are h i g h l y mutable are hot spots (17) . Evidence on the nature of hot spots has been reviewed by Clarke and Johnston (1976) and w i l l be merely summarized here. High M u t a b i l i t y Regions, ( i ) Frameshift mutations tend to occur i n regions of repeated base p a i r s . Runs of e i t h e r AT or GC base p a i r s have been a s s o c i a t e d with f r a m e s h i f t s . ( i i ) Base

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

4

EXTRACELLULAR

MICROBIAL

POLYSACCHARIDES

p a i r s u b s t i t u t i o n s are i n f l u e n c e d by neighbouring bases. The AT-GC s u b s t i t u t i o n induced by 2-aminopurine at the second p o s i t i o n of a t r i p l e t has been demonstrated to occur 23 times more f r e quently when an AT base p a i r was present i n the t h i r d p o s i t i o n (18). ( i i i ) Mutator polymerase acts p r e f e r e n t i a l l y on s p e c i f i c regions of the gene, ( i v ) The frequency and l o c a t i o n of d e l e t i o n s i s non-random and such s i t e s are considered d e l e t i o n hot spots, (v) U l t r a - v i o l e t induced mutations are most frequent i n t r a c t s of p y r i m i d i n e s . Development of a S t a b l e Mutation. Most mutations are formed from pre-mutational l e s i o n s . The l e s i o n may or may not be r e p a i r e d o r , the r e p a i r process i t s e l f may lead d i r e c t l y to mutat i o n . F a i l i n g r e p a i r , the pre-mutational l e s i o n may be e s t a b l i s h ed as a mutation by DNA development of a mutatio these steps may be subject to the i n f l u e n c e of adjacent base pairs. In the l i g h t of these o b s e r v a t i o n s , one might ask what avenues e x i s t f o r the a m e l i o r a t i o n or removal of hot spots? I f the mutation i s e f f e c t e d by a mutagen, i t may be p o s s i b l e to e i t h e r remove or suppress the c o n d i t i o n l e a d i n g to the presence of the mutagen or n e u t r a l i z e i t s a c t i v i t y with an antimutator. Precedents f o r t h i s l a t t e r approach are now w e l l documented (14). Antimutagenesis. I t has been q u i t e p r o p e r l y s t a t e d (14) that one cannot understand mutagenesis or the r e g u l a t i o n of mutation frequency without c o n s i d e r i n g antimutagenic e f f e c t s . Antimutagenesis may be d e f i n e d as a decrease i n the a c t u a l r a t e of mutation. Decreased apparent rates may be caused by e i t h e r a l t e r e d s u r v i v a l or dose r e d u c t i o n , and these e f f e c t s are termed apparent antimutagenesis. A mutation or premutation may a r i s e by: ( i ) r e a c t i o n between a mutagen and DNA; ( i i ) i n c o r p o r a t i o n of a mutagen-altered precursor or base analogue; ( i i i ) r e p l i c a t i o n e r r o r ; ( i v ) recombination e r r o r ; (v) r e p a i r e r r o r ; ( v i ) t r a n s c r i p t i o n e r r o r ; ( v i i ) t r a n s l a t i o n e r r o r . The l a s t two mechanisms i n v o l v e the p r o d u c t i o n of error-prone RNA or p r o t e i n s which a l t e r the base sequence of DNA e i t h e r d i r e c t l y or i n d i r e c t l y (19, 20, 21). C l a r k e and Shankel (14) have d i s t i n g u i s h e d between genetic antimutagenesis, which i s the antimutagenic e f f e c t of r e p l i c a t i o n genes, r e p a i r genes, or other genetic determinants, and p h y s i o l o g i c a l antimutagenesis which i s achieved by added chemicals or a l t e r e d c e l l c o n d i t i o n s . The p h y s i o l o g i c a l mechanism would appear to o f f e r c o n s i d e r a b l e p o t e n t i a l f o r the r e d u c t i o n of mutation r a t e s f o r c e r t a i n c l a s s e s of mutation. For example, adenosine appears to be capable of v i r t u a l l y a b o l i s h i n g the mutagenicity of purine mutagens (14). Spontaneous mutation rates have a l s o been d r a m a t i c a l l y reduced by the use of a c r i d i n e s (22). An o b s e r v a t i o n of c o n s i d e r a b l e i n t e r e s t i s that genes are more l i k e l y to mutate when being t r a n s c r i b e d (14). Thus the r e p r e s s i o n of gene a c t i v i t y i s antimutagenic. I t might be expected,

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

1. KiDBY

Culture

Maintenance

and

WILD GENOTYPE

WILD PHENOTYP

ι

5

Productivity

LESION REPAIR

PREMUTATION

SUPRESSION SILENT MISSENSE DEGENERACY

REPLICATION

MUTATION SELECTION

Figure 1.

Sequences of events in mutation and selec tion

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

6

EXTRACELLULAR

MICROBIAL

POLYSACCHARIDES

t h e r e f o r e , that i n maintenance and inoculum build-up c u l t u r e s , the r e p r e s s i o n of the productive f u n c t i o n would help to a r r e s t v a r i a b i l i t y by decreasing the r a t e of mutation. I t may a l s o be the case that r e p r e s s i o n of product formation w i l l help prevent s e l e c t i o n against producer c e l l s . There i s some evidence (23^ 24) that product r e p r e s s i o n may be of use i n reducing v a r i a b i l i t y i n Xanthomonas campestris. There seems l i t t l e reason to doubt that DNA which i s not being t r a n s c r i b e d should be r e l a t i v e l y s t a b l e . I t would be of considerable i n t e r e s t to see i f mutations i n derepressed genes are i n f a c t p r o p o r t i o n a l to t r a n s c r i p t i o n r a t e s . I t may w e l l be that c e r t a i n microbes with high product y i e l d s are i n h e r e n t l y unstable because of high t r a n s c r i p t i o n a l a c t i v i t y . L i m i t i n g the Opportunit f o Mutation Mutatio be a f u n c t i o n of r e p a i r mutagen or antimutagen , physica such as r a i s e d temperature, low water a c t i v i t y , or i c e c r y s t a l s . Whatever the c o n d i t i o n l e a d i n g to mutation, the most e f f e c t i v e p r o t e c t i o n i s to minimise the exposure of the c u l t u r e to the conducive c o n d i t i o n . The growth i n mutant numbers i s a f u n c t i o n of the number of r e p l i c a t i o n s (Table I ) . I t f o l l o w s , t h e r e f o r e , that the t o t a l number of r e p l i c a t i o n s should be minimized. If r e p l i c a t i o n - i n d e p e n d e n t mutations are taken i n t o account, then i t a l s o follows that the t o t a l residence time i n c u l t u r e should be minimized. I f , as seems to be the general case, mutation i s p r o p o r t i o n a l to t r a n s l a t i o n a l a c t i v i t y , then the productive f u n c t i o n should be repressed u n t i l needed. The e x c l u s i o n or r e d u c t i o n of potent mutagens may seem too obvious to r e q u i r e f u r t h e r comment. However, many commonly o c c u r r i n g mutagens such as metal i o n s , adenine, c a f f e i n e , ozone, to name a few, seem o f t e n to escape a t t e n t i o n . The number of base analogues generated by chemical, or high temperature, treatment of concentrated sources of purine and pyrimidine bases must o f t e n be c o n s i d e r a b l e . The frequent proximity of c u l t u r e s to e l e c t r i c motors and, i n p a r t i c u l a r , atmospheres r e c e n t l y i r r a d i a t e d with u l t r a - v i o l e t l i g h t must s u r e l y produce l a r g e numbers of ozone-induced mutants. Extremely high l e v e l s of mutation have been observed i n E. c o l i exposed to as l i t t l e as 0.1 ppm ozone f o r 60«minutes (10). The question of l i m i t i n g the opportunity f o r mutation w i l l be f u r t h e r discussed i n connection w i t h p r e s e r v a t i o n techniques. L i m i t i n g the Opportunity f o r S e l e c t i o n . The s e l e c t i o n of a mutant, i n the present context, may be taken to mean the increase of any given mutant to a s i g n i f i c a n t p r o p o r t i o n of the t o t a l p o p u l a t i o n . The extent of t h i s s e l e c t i o n w i l l be a f u n c t i o n of the c u l t u r e c o n d i t i o n s and the number of generations of c u l t u r e growth permitted. S e l e c t i v e media may be employed to remove p a r t i c u l a r c l a s s e s of mutant. N u t r i t i o n a l l y r i c h media w i l l tend to preserve and o f t e n concentrate auxotrophs while a poorer medium may s e l e c t f a i r l y e f f i c i e n t l y against auxotrophs, unless

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

a

m = mutation r a t e

Mutant: T o t a l

Mutant C e l l s

64mN 4m

24mN 3m

8mN 2m

2mN m

0

0

16N

8N

4N

2N

Ν

Total

Cells

4

3

2

1

0

THE PROPORTION OF MUTANTS IN A GROWING CULTURE

Generations

TABLE I

8

EXTRACELLULAR

MICROBIAL

POLYSACCHARIDES

high r a t e s of c r o s s - f e e d i n g occur. Short-term P r e s e r v a t i o n . The p r e s e r v a t i o n of c e l l v i a b i l i t y f o r periods of l e s s than a few months might a r b i t r a r i l y be termed short-term p r e s e r v a t i o n . While there can be no doubt as to the d e s i r a b i l i t y of long-term p r e s e r v a t i o n , methods of a c h i e v i n g t h i s u s u a l l y provide r e l a t i v e l y i n a c c e s s i b l e i n o c u l a and, i n some cases, may be of l i m i t e d success. In order to be u s e f u l , a short -term p r e s e r v a t i o n method must provide a high recovery of v i a b l e c e l l s which grow with a minimum l a g phase. The inoculum should be e a s i l y a c c e s s i b l e and of a standard and s u i t a b l e s i z e . Subc u l t u r e to achieve v i g o r o u s l y growing and r e p r o d u c i b l e c u l t u r e s should not be necessary. I f these c r i t e r i a cannot be met, i t may be b e t t e r to consider the r o u t i n e use of i n o c u l a preserved by long-term methods. U s e f u l short-term p r e s e r v a t i o t i o n s of d r y i n g procedures. A p a r t i c u l a r l y s u i t a b l e method i s the d r y i n g of c u l t u r e s onto paper (2_5, 26). Paper s t r i p s have the advantage of being e a s i l y handled and are r e a d i l y adjusted i n s i z e to y i e l d an appropriate inoculum s i z e . The method has been used w i t h success f o r X. campestris NRRL B1459 ( 9 ) . Other s h o r t term p r e s e r v a t i o n methods have been reviewed elsewhere (26). The repeated t r a n s f e r of c u l t u r e s f o r r o u t i n e maintenance must be considered an unwise p r a c t i c e and i s d i f f i c u l t to j u s t i f y where a l t e r n a t i v e non-propagative methods e x i s t . Long-term P r e s e r v a t i o n . Storage of l y o p h i l i z e d , frozen, or L - d r i e d c e l l s are the p r i n c i p l e means of long-term p r e s e r v a t i o n (26). There i s an extremely widespread b e l i e f that the method of choice i s l y o p h i l i z a t i o n . This b e l i e f i s not j u s t i f i e d by e i t h e r f a c t or theory. The reasons f o r the widespread preference f o r l y o p h i l i z a t i o n are: ( i ) t h i s was the f i r s t g e n e r a l l y s u c c e s s f u l method of longterm p r e s e r v a t i o n ; ( i i ) the product has an " a t t r a c t i v e " appearance; ( i i i ) i n j u r y from concentrated solutes i n the l i q u i d s t a t e seemed a reasonable s u p p o s i t i o n ; ( i v ) p r o t e c t i o n against i n j u r y by d r y i n g at f r e e z i n g temperatures seemed an a t t r a c t i v e advantage. I t i s now c l e a r that h i g h l y concentrated solutes are not as i n j u r i o u s as has been formerly supposed and may i n f a c t exert s i g n i f i c a n t p r o t e c t i o n (27). In the l i g h t of extensive i n v e s t i g a t i o n s of the L-drying methods of Annear (28-33) by other workers (26, 34, 35), i t seems that t h i s procedure i s to be p r e f e r r e d since recovery of many d i f f i c u l t to preserve organisms i s t y p i c a l l y 10 to 100 times higher than i s achieved with l y o p h i l i z a t i o n . I t has a l s o been observed that l a r g e increases i n mutants can accompany l y o p h i l i z a t i o n (36, 3_7, 38) . While no proper comparison appears to have been made between mutant y i e l d s from l y o p h i l i z a t i o n and L - d r y i n g , i t seems reasonable to expect that the higher r e c o v e r i e s obtained by L-drying would be accompanied by l e s s damage and t h e r e f o r e fewer mutants. There are a number of steps i n p r e s e r v a t i o n and subsequent recovery procedures which may cause genetic damage ( F i g u r e 2 ) .

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

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9

F r e e z i n g i s i n i t s e l f i n j u r i o u s (39) . The extent of d r y i n g a l s o appears to i n f l u e n c e the y i e l d of mutations (40, 4l)· Prophage may a l s o be induced by d e s i c c a t i o n (42, 43). The r e h y d r a t i o n procedure i s a l s o of importance and there appears to be some evidence of c e l l leakage l e a d i n g to poor recovery (27). The r e covery medium i s an important s e l e c t i v e agent and can c l e a r l y i n f l u e n c e the recovery of c e r t a i n types of mutants. For example, some medium components can i n h i b i t recovery of nonsense suppressors i n Saccharomyces c e r e v i s i a e , w h i l e other components can r e l i e v e t h i s i n h i b i t i o n (44). Storage i n the f r o z e n s t a t e has l i t t l e to recommend i t except convenience. Storage i t s e l f i s not considered to be i n j u r i o u s provided that i c e c r y s t a l damage i s precluded by h o l d i n g the temperature below -130°C (45) I t i s suggested tha procedures f o r both long requirement y particularly successful. I t i s not c l e a r how low a temperature should be employed f o r storage of d r i e d m a t e r i a l , but i n the absence of evidence to the c o n t r a r y , as low a temperature as i s a v a i l a b l e would seem des i r a b l e . For long-term p r e s e r v a t i o n , the m a t e r i a l i s normally h e l d under vacuum while f o r short-term p r e s e r v a t i o n , l e s s s t r i n gent, and t h e r e f o r e more convenient, c o n d i t i o n s may be employed. When r e h y d r a t i n g , a low c e l l r c u l t u r e volume r a t i o should be employed. The c u l t u r e medium should be as n u t r i t i o n a l l y r i c h as i s c o n s i s t e n t w i t h good growth. This procedure w i l l to some degree s e l e c t f o r auxotrophs. However, i t i s p o s s i b l e to screen these out i n subsequent c u l t u r e i f necessary. No c e l l population i s g e n e t i c a l l y i d e n t i c a l to i t s parent c u l t u r e . The change i n i d e n t i t y can, however, be minimized by the use of methods which lead to high recovery r a t e s . The p r e s e r v a t i o n of f r e s h i s o l a t e s should not be delayed and i t i s worth adopting a standard p r o t o c o l to deal with t h i s s i t u a t i o n ( F i g u r e 3 ) . Improvement of Genotype. C o n t r o l Mutants. One of the most u s e f u l types of mutant i s the c o n t r o l mutant where feed-back i n h i b i t i o n or r e p r e s s i o n i s absent. In the case of p o l y s a c c h a r i d e production such mutants are most l i k e l y to be recognized by t h e i r production of l a r g e mucoid c o l o n i e s . C o n d i t i o n a l mutants. The c o n d i t i o n a l mutant has great potent i a l f o r c o n t r o l l i n g complex c e l l f u n c t i o n s by such simple means as r a i s i n g or lowering of temperature. Such mutants are r e l a t i v e l y easy to o b t a i n . For example, p o l y s a c c h a r i d e production which i s c o n d i t i o n a l may be switched on and o f f or, c o n d i t i o n a l growth may be switched o f f to permit polysaccharide production i n the absence of growth. C o n d i t i o n a l l y s i s i s a l s o of c o n s i d e r a b l e a p p l i c a t i o n where i t i s d e s i r a b l e , and i t u s u a l l y i s , to remove the c e l l s from the completed fermentation. L y s i s may be achieved by the i n d u c t i o n of bacteriophage. B a c t e r i o c i n s a l s o

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

EXTRACELLULAR

10

LYOPHILIZATION

F.EEEZJ_N6

[FREEZING!

IFREEZINGI

MICROBIAL

POLYSACCHARIDES

DRYING STORAGE

STORAGE

L-DRYING

DRYING I STORAGE

I

REHYDRATION ITHAWINGI • i GROWTH GROWTH GROWTH Figure 2. Comparisons between sequences of events involved in preservation of cells and their subsequent recovery REHYDRATION

ENRICHMENT SELECTION

PURIFICATION

CHARACTERIZATION

REPEATED TRANSFER

VIABLE COUNT

L-DRYING

CHARACTERIZATION

Figure 3. Selection and preservation of microbes. The scheme described incorporates tests of irdried cultures to determine viability and any alteration of characteristics as a result of the preservation procedure.

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

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o f f e r great p o t e n t i a l f o r l y s i n g of c u l t u r e s . S t a b i l i z e d Genes. The p o t e n t i a l f o r s t a b i l i z a t i o n v a r i e s according to the o r i g i n of the i n s t a b i l i t y . . In the case of hot spots, the breaking up of runs of base p a i r s might be expected to be e f f e c t i v e . An increase i n the number of genes may be e f f e c t i v e and may, i f t r a n s l a t i o n i s the r a t e - l i m i t i n g step i n production, a l s o lead to higher production l e v e l s . I t may be p o s s i b l e to t r a n s f e r genes from a r e l a t e d organism e x h i b i t i n g a more s t a b l e genotype. Stable genotypes may be f a i r l y r e a d i l y revealed by employing the s e l e c t i v e pressure of chemostat c u l t u r e (46). Methods f o r Genotype A l t e r a t i o n . Genotypes are a l t e r e d by: ( i ) induced mutation; ( i i ) spontaneous mutation; ( i i i ) t r a n s f e r of e x i s t i n g genes. The first.method i s r a p i d and some degree of s p e c i f i c i t y i s p o s s i b l e as f o r example i n the case of ozone and UV induced mutants (10) wanted mutations may a l s are, of course, slower, but are capable of producing the r e q u i r e d mutants i n a s u r p r i s i n g l y short time. The s e l e c t i o n pressure to o b t a i n p a r t i c u l a r types of spontaneous mutants should be a p p l i e d i n a continuous, r a t h e r than a discontinuous, manner. This permits a more complete range of p o s s i b i l i t i e s to be expressed and i s l i k e l y to lead to a more s t a b l e mutant s i n c e the d e s i r e d character can be acquired by a s e r i e s of small steps rather than one l a r g e step which could, f o r example, be due to a s i n g l e point mutation. For example, s t a b l e and high l e v e l a n t i b i o t i c r e s i s t a n c e has been achieved i n Xanthomonas by u s i n g gradient p l a t e s but was not r e a d i l y achieved when using d i s c r e t e steps (24). A p a r t i c u l a r l y h e l p f u l account of methods of mutant i s o l a t i o n i s given by Hopwood (47). Perhaps the most a t t r a c t i v e methods of genotype improvement i n v o l v e t r a n s f e r of genetic m a t e r i a l . The advantage of t h i s method i s s p e c i f i c i t y , s t a b i l i t y , and r e l a t i v e freedom from unwanted changes i n other genes. Some very e x c i t i n g a l t e r a t i o n s can be attempted by t h i s means. I t i s d e s i r a b l e f o r the organisms to be c l o s e l y r e l a t e d because the t r a n s f e r r e d gene i s more l i k e l y to behave c h a r a c t e r i s t i c a l l y i n the r e c i p i e n t . However, genes c e r t a i n l y are t r a n s f e r a b l e between d i s t a n t l y r e l a t e d species and genetic engineering may be expected to r e v o l u t i o n i z e the synthesis of n a t u r a l products. The methods of genetic t r a n s f e r among b a c t e r i a are: ( i ) conjugation; ( i i ) t r a n s d u c t i o n ; ( i i i ) t r a n s f e c t i o n ; ( i v ) t r a n s formation, and (v) i n v i t r o recombination and t r a n s f e r from divergent species or genetic engineering. The f i r s t four methods are conventional and are e x t e n s i v e l y described (48). However, genetic engineering i s a combination of methodologies and the t o t a l procedure may be v a r i e d c o n s i d e r a b l y . One r e c e n t l y described method (49) c o n s i s t s of i s o l a t i o n of the gene as i t s RNA t r a n s c r i p t i o n product, r e t r a n s c r i p t i o n back to DNA and synt h e s i s of a complementary s t r a n d . These strands are elongated w i t h homopolymer t a i l s of o l i g o - ( d G ) . This double stranded gene

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

12

EXTRACELLULAR

MICROBIAL

POLYSACCHARIDES

i s then mixed w i t h a plasmid which has been prepared as f o l l o w s . A n i c k i s placed i n the c i r c u l a r plasmid to provide l i n e a r DNA which i s r e p a i r e d then extended w i t h a homopoIyer t a i l o f o l i g o (dC) which i s , o f course, complementary to the a r t i f i c i a l t a i l on the copied gene. The plasmid p i c k s up the gene by the complementary t a i l s e c t i o n s and, i n doing so, becomes c i r c u l a r and thus i n f e c t i v e . F o l l o w i n g i n f e c t i o n , the plasmid i s c o v a l e n t l y l i n k e d to the copied gene by host enzymes. This gene may be t r a n s f e r a b l e to a wide range o f b a c t e r i a . Furthermore, i n t h i s p a r t i c u l a r example, the gene may be removed again from the plasmid, using a s p e c i f i c r e s t r i c t i o n nuclease, and t r a n s f e r r e d to some other plasmid. Thus i t i s p o s s i b l e to conceive o f n a t u r a l products which are e i t h e r i n a c c e s s i b l fermenters w i t h i n hours o n l y f o r production cost production volumes can be r e g u l a t e d .

Abstract Sources of microbes and procedures for their selection, isolation and maintenance are discussed. Maintenance of genotype is considered in terms of the nature of genetic variability, antimutagenesis, inoculation schedules, growth media and preservation methods. The improvement of genotype is discussed in terms of control mutants, conditional mutants, and methods of genotype alteration. Some common practices which may be conducive to culture degeneration are discussed and suggestions are made as to alternative procedures. Literature Cited 1. Brock, T . D . , Ann. Rev. Ecology System (1970) 1, 191. 2. Lundgren, D . , et al., "Water Pollution Microbiology", John Wiley, New York (1972) 69-88. 3. Okanishi, M . , Gregory, K.F., Canad. J. Microbiol. (1970) 16, 1139. 4. "Bergey's Manual of Determinative Bacteriology" Williams and Wilkins. 5. "Abstracts of Microbiological Methods", John Wiley, New York (1969). 6. "Methods i n Microbiology" 3A, Academic Press, New York (1970) 7. "Methods in Microbiology" 3B, Academic Press, New York (1970) 8. "Methods i n Microbiology" 4, Academic Press, New York (1971) 9. Kidby, D . K . , et al., unpublished. 10. Hamelin, C., Chung, Y . S . , Mutat. Res. (1975) 28, 131. 11. Sutherland, I.W., J. gen. Microbiol. (1976) 94, 211. 12. Hesseltine, C.W., Haynes, W.C., Progress in Industrial Microbiology (1973) 12, 3. 13. Clarke, C.H., Johnston, A.W.B., Mutat. Res. (1976) 36, 147. 14. Clarke, C.H., Shankel, C.M., Bacteriol. Rev. (1975) 39, 33.

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

1.

KiDBY

15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49.

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Drake,J.W.,"TheMolecular Basis ofMutation",Holden-Day, San Francisco, 1970. Vogel, F., J. Molec. Evoln. (1972) 1, 334. Benzer, S., Proc. Natl. Acad. Sci. (1961) 47, 403. Koch, R.E., Proc. Natl. Acad. Sci. (1971) 68, 773. Lewis,C.M.,Tarrant,G.M.,Mutat. Res. (1971) 12, 349. McBride, A.C., Gowans, C.S., Genet. Res. (1969) 14, 121. Talmud, P., Lewis, D., Nature (1974) 249, 563. Puglisi, P.P., Mutat. Res. (1967) 4, 289. Cadmus,M.C.,et al., Can. J. Microbiol. (1976) in press. Kidby, D.K., unpublished. Coe, A.W., Clark, S.P., Mon. Bull. Minist. Hlth. (1966) 25, 97. Lapage, S.P. et a l . "Method i Microbiology" 3A Academi Press, New York, (1970 Leach, R.H., Scott, W.J., J . gen. Microbiol. (1959) 21, 295. Annear, D.I., Nature (1954) 174, 359. Annear, D.I., J. Hyg. Camb. (1956) 54, 487. Annear, D.I., J. Path. Bact. (1956) 72, 322. Annear, D.I., J. Appl. Bact. (1957) 20, 17. Annear, D.I., Aust. J . exp. Biol. med. Sci. (1958) 36, 1. Annear, D.I., Aust. J . exp. Biol. med. Sci. (1962) 40, 1. Hopwood, D.A., Ferguson, H.M., J . appl. Bact. (1969) 32, 434. Muggleton, P.W., Progr. Ind. Microbiol. (1962) 4, 191. Hieda, Κ., Ito, T., "Freeze-drying of biological Materials" International Institute of Refrigeration, Paris (1973) 71. Webb, S.J., Tai,C.C.,Canad. J . Microbiol. (1968) 14, 727. "Cryobiology", Academic Press, N.Y. (1966) 213. Mazur, P., Science (1970) 168, 939. Webb, S.J., Nature (1967) 213, 1137. Webb, S.J. and Dumasia, M.D., Canad. J . Microbiol. (1968) 14, 841. Webb, S.J. and Dumasia, M.D., Canad. J . Microbiol. (1967) 13, 33. Webb, S.J. and Dumasia, M.D., Canad. J . Microbiol. (1967) 13, 303. Queiroz, C., Biochem. Genet. (1973) 8, 85. Martin, S.M., Ann. Rev. Microbiol. (1964) 18, 1. Veldkamp, H., "Methods in Microbiology" 3A Academic Press, New York (1970) 305. Hopwood, D.A., "Methods in Microbiology" 3A Academic Press, New York (1970) 363. Hayes, W., "The Genetics of Bacteria and their Viruses" Blackwell, Oxford (1968). Rougeon, F . , Kourilsky, P., Mach, B., Nucleic Acids Res. (1975) 2, 2365.

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

2

The

P r o d u c t i o n of

vinelandii

Alginic

Acid

by

Azotobacter

in Batch and Continuous Culture

L. DEAVIN, T. R. JARMAN, C. J. LAWSON, R. C. RIGHELATO, and S. SLOCOMBE Tate & Lyle Ltd., Group Research and Development, Philip Lyle Memorial Research Laboratory, P.O. Box 68, Reading, Berks., RG6 2BX, U.K.

The production of polysaccharides by fermentation has been heralded by some of the more optimisti fermentation area. It is no meetings to that offered to single cell protein some years ago. This optimism is based on the undoubted success of the one major product, xanthan gum, which has raised the tantalising prospect of a whole range of microbial gums which would not only reflect and improve upon the available plant gums, but also introduce novel properties for exploitation in existing and as yet undeveloped applications. About a dozen companies are thought to be developing on a large scale the production of microbial polysaccharides; some of them are already in the fermentation industry but others, like our own, are newcomers to this technology. Despite this enormous research and development effort the state of the technology, as judged from patents and the scientific literature, is relatively poorly advanced. There is little public literature on the production technologies used by industry and academic microbiology has for the most part ignored the physiology of exocellular polysaccharide synthesis and excretion. For this reason, we, along with other groups,have been studying the physiology of polysaccharide synthesis as a basis for developing production processes. In order to gain a greater understanding of the effects of individual environmental parameters on cell growth and polysaccharide synthesis continuous flow cultures(l) have been used wherever possible. For those unfamiliar with the methods of mass cultivation of microbes, the time honoured industrial and laboratory method is to inoculate a small amount of the microbe into a medium containing all of the necessary nutrients for growth and product formation. The microbes then grow until one or other substrate is exhausted and then growth stops. This is a simple batch culture system. In continuous flow culture, by contrast, the nutrient medium is continuously added to the culture and the culture continuously harvested. 14

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

2.

DEAVIN

E T AL.

Production

of Alginic

Acid

in

15

Culture

T h e r a t i o of the flow rate of the medium to the c u l t u r e v o l u m e is c a l l e d the d i l u t i o n r a t e , a n d e x c e p t at the maximum growth rate of the m i c r o b e , the c o n c e n t r a t i o n o f o n e o f the substances i n the medium determines the c o n c e n t r a t i o n o f the m i c r o b e s . It is

This is c a l l e d the g r o w t h - l i m i t i n g substrate.

w e l l established that c h a n g e s in g r o w t h - l i m i t i n g substrate c a n

c o n s i d e r a b l y a f f e c t the p h y s i o l o g y of m i c r o b e s .

So too c a n changes i n the

d i l u t i o n r a t e , w h i c h in a steady state is e q u a l to the s p e c i f i c growth r a t e . In continuous cultures steady states c a n b e m a i n t a i n e d i n d e f i n i t e l y a n d changes i n i n d i v i d u a l parameters c a n r e a d i l y b e s t u d i e d .

By contrast

i n b a t c h c u l t u r e s , c o n c e n t r a t i o n of nutrients, c e l l s a n d p r o d u c t s , a n d a l l of these w i t h respect to c e l l a g e , c h a n g e c o n t i n u o u s l y , w h i c h makes the study o f c e l l p h y s i o l o g y and b i o c h e m i s t r y e x t r e m e l y c o m p l i c a t e d

This is

i l l u s t r a t e d b y some b a t c h fermentatio T h e best known is of course x a n t h a n p r o d u c t i o n b y Xanthomonas

campestris.

In the simplest fermentation d e s c r i b e d b y M o r a i n e a n d R o g o v i n (2), the c o n c e n t r a t i o n s of the major substrates c h a n g e throughout the f e r m e n t a t i o n . So too do the main products:

bacterial cells and polysaccharide.

Analysis

o f several b a t c h cultures l e d M o r a i n e & R o g o v i n (2) to c o n c l u d e that several f a c t o r s , i n c l u d i n g x a n t h a n c o n c e n t r a t i o n , a f f e c t e d the rate o f x a n t h a n p r o d u c t i o n , though the d e t a i l s o f the r e l a t i o n s h i p were not c l e a r . T h e c o m p l i c a t e d k i n e t i c pattern that emerged from these studies has b e e n of c o n s i d e r a b l e v a l u e in understanding the b a t c h fermentation process for x a n t h a n gum but does not e n h a n c e the understanding o f the control o f b i o synthesis, as it n e c e s s a r i l y deals p r i m a r i l y w i t h the e f f e c t of the c h a n g i n g fermentation parameters o n the environment of the c e l l s rather than d i r e c t l y w i t h the e f f e c t o f the environment on the c e l l s . In b a t c h cultures of a Pseudomonas sp . w h i c h produces an e x o p o l y s a c c h a r i d e composed of g l u c o s e and g a l a c t o s e i n the r a t i o 7 : 1 a n d contains both a c e t a t e and p y r u v a t e (3) p o l y m e r synthesis was d e t e c t a b l e in the later part of the e x p o n e n t i a l growth phase (Figure 1) a n d c o n t i n u e d m a x i m a l l y d u r i n g the p e r i o d of z e r o s p e c i f i c growth r a t e , the s o - c a l l e d stationary phase (4).

The

l i m i t i n g substrate, that is the substrate w h i c h

determined the c e l l mass that was f i n a l l y o b t a i n e d , was not established i n these c u l t u r e s . A n o t h e r e x a m p l e of b a t c h c u l t i v a t i o n for a n e x o p o l y s a c c h a r i d e is that of a l g i n i c a c i d production by Azotobacter v i n e l a n d i i .

W h e n the organism

was grown under p h o s p h a t e - d e f i c i e n t c o n d i t i o n s p o l y s a c c h a r i d e synthesis c o n t i n u e d throughout the growth phase but in contrast to the last e x a m p l e ceased when the microbes stopped g r o w i n g (Figure 2 ) . From the studies of b a t c h cultures of the types discussed it is d i f f i c u l t to draw a n y conclusions o n the w a y in w h i c h b a c t e r i a control the synthesis o f these e x o p o l y s a c c h a r i d e s .

It has b e e n supposed b y many

microbiologists

that such products w o u l d b e formed w h e n a c e l l has a n excess o f c a r b o h y d r a t e

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

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4

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

2.

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Production

E T AL.

of Alginic

Acid

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17

Culture

substrate a n d its growth îs r e s t r i c t e d b y some other p a r a m e t e r .

N e i j s s e l and

Tempest (5) h a v e r e c e n t l y suggested from studies of A e r o b a c t e r aerogenes that t h e y a c t as A T P sinks and a r e p r o d u c e d m a x i m a l l y under c o n d i t i o n s w h i c h w o u l d cause the c e l l s to o v e r p r o d u c e A T P , c o n d i t i o n s such as nitrogen l i m i t a t i o n .

T h e observations o n A z o t o b a c t e r v i n e l a n d i i w o u l d

perhaps c o n t r a d i c t that p a r t i c u l a r hypothesis s i n c e h i g h p r o d u c t i o n rates w e r e observed under p h o s p h a t e - d e f i c i e n t c o n d i t i o n s (Figure 2 ) .

Measurement

o f the rates of synthesis under a v a r i e t y of e n v i r o n m e n t a l c o n d i t i o n s might shed some light o n the c e l l u l a r control a n d the r o l e of e x o p o l y s a c c h a r i d e production.

T h e major rate c o n t r o l l i n g process in a c e l l is its s p e c i f i c

growth r a t e .

A c o m p l e x network o f control mechanisms exist w h i c h permit

the m i c r o b e to assimilate substrates

synthesis

intermediate

d for

polymers ( i . e . p r o t e i n s , n u c l e i p r o d u c e more c e l l u l a r material of the same t y p e a n d i n similar ratios in the f a c e of enormous e n v i r o n m e n t a l changes.

It seems l o g i c a l , t h e n , to look

first at the e f f e c t of growth rate on e x o p o l y s a c c h a r i d e synthesis i n continuous c u l t u r e systems. S i l m a n a n d R o g o v i n (6) studied continuous cultures of Xanthomonas campestris

in cultures thought to b e l i m i t e d b y the nitrogenous component

i n the m e d i u m .

p H was not c o n t r o l l e d in these experiments so the d a t a has

b e e n redrawn t a k i n g o n l y the c o n d i t i o n s i n w h i c h the p H was b e t w e e n 6 . 3 and 7 . 2 , a range i n w h i c h it has b e e n found that p H has l i t t l e e f f e c t on x a n t h a n p r o d u c t i o n (Figure 3 ) .

A t growth rates b e t w e e n 0 . 0 5 a n d 0 . 2 0 h " ^

i . e . d o u b l i n g times between 14 a n d 3 . 5 h , there was l i t t l e c h a n g e in the s p e c i f i c rate of synthesis o f x a n t h a n .

T h e c o n c e n t r a t i o n o f x a n t h a n therefore

increased with decreasing dilution rate.

It is interesting to note that the

x a n t h a n p r o d u c t i o n rate i n these cultures v a r i e d o n l y 1 5 % e i t h e r side of the mean v a l u e .

This is q u i t e d i f f e r e n t from the b a t c h c u l t u r e analysis w h i c h

showed a t h r e e f o l d c h a n g e i n s p e c i f i c rate o f xanthan p r o d u c t i o n o v e r a similar c o n c e n t r a t i o n range

(2).

A similar i n d e p e n d e n c e o f the r a t e of e x o p o l y m e r synthesis on s p e c i f i c growth rate was found both w i t h the Pseudomonas p o l y s a c c h a r i d e (4) a n d a l g i n i c a c i d synthesis b y A z o t o b a c t e r v i n e l a n d i i .

O v e r an even wider

growth rate range the s p e c i f i c rate of synthesis o f Pseudomonas e x o p o l y m e r v a r i e d o n l y 2 5 % about the mean (Figure 4 ) , whilst the p o l y s a c c h a r i d e c o n c e n t r a t i o n i n c r e a s e d i n p r o p o r t i o n to the r e s i d e n c e time o f the c u l t u r e (the r e s i d e n c e time is the r e c i p r o c a l of the d i l u t i o n r a t e ) . l i m i t e d continuous cultures o f A z o t o b a c t e r v i n e l a n d i i

In p h o s p h a t e -

the rate of a l g i n a t e

synthesis was i n d e p e n d e n t of s p e c i f i c growth rate (Figure 5 ) . there was an i n c r e a s e i n biomass at lower d i l u t i o n rates.

In this case

This was almost

e n t i r e l y d u e to the i n t r a c e l l u l a r a c c u m u l a t i o n o f the storage compound poly-B-hydrosybutyrate.

W i t h these three p o l y s a c c h a r i d e s , then the rate

o f synthesis appears to be i n d e p e n d e n t of the rate of growth a n d h e n c e i n d e p e n d e n t o f the rate of most of the other i n t r a c e l l u l a r biosyntheses.

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

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Exopolymer

Ο mg/ml

>mg/OD/h χ 100

Biotechnology and Bioengineering

Figure 3. Effect of dilution rate on the production of xanthan by Xanthomonas campestris in con tinuous culture (6)

0.05

0.10

0.15 1

Dilution rate h

Exopolymer

Ο mg/ml

Figure 4. Effect of di lution rate on produc tion of an exopolysac charide by Pseudomonas sp in ammonia-limited continuous culture (Data from Williams A. G., 1975; Ph.D. Thesis Uni versity College, Cardiff, U.K.)

• mg/mg protein/h

0.25 Dilution rate h

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

2.

DEAVIN

E T AL.

Production

of Alginic

Acid

in

19

Culture

W e h a v e studied a l g i n i c a c i d synthesis b y A z o t o b a c t e r v i n e l a n d i i

in

some d e t a i l and w o u l d l i k e to pursue this argument w i t h that p a r t i c u l a r system.

A l g i n a t e as o b t a i n e d from the c o n v e n t i o n a l s o u r c e , the brown a l g a e ,

is a 1 , 4 - l i n k e d l i n e a r c o p o l y m e r of J i - D - m a n n u r o n i c a c i d and its 5 - e p i m e r « C - L - g u I u r o n i c a c i d (7)

(Figure 6).

T h e arrangement of monomers i n this

c o p o l y m e r has b e e n referred to as the b l o c k structure (8), the p o l y m e r h a v i n g b e e n shown to consist of regions o f h o m o - p o l y m e r i c b l o c k s of mannuronic a c i d and o f g u l u r o n i c a c i d together w i t h the s o - c a l l e d a l t e r n a t i n g or random s e q u e n c e s .

T h e properties of the polymer, e s p e c i a l l y w i t h respect

to its g e l l i n g in the p r e s e n c e o f c a l c i u m ions,depends both on the mannuronic a c i d to g u l u r o n i c a c i d r a t i o a n d the b l o c k structure, the higher the proportion of p o l y g u l u r o n i c a c i d blocks in the p o l y m e r the stronger and more b r i t t l e the gel formed in th produced by Azotobacter vinelandi from a l g a l sources e x c e p t that it is p a r t i a l l y a c e t y l a t e d , a p p r o x i m a t e l y o n e in ten of the C 2 α η σ / o r C 3 h y d r o x y l groups b e i n g e s t e r i f i e d w i t h a c e t a t e

(ίο, η.). T h e markets for a l g i n a t e s demand products h a v i n g a range of solution viscosities and g e l l i n g q u a l i t i e s .

A range o f a l g i n a t e types c o m p a r a b l e

a l g a l products c a n b e p r o d u c e d b y A z o t o b a c t e r v i n e l a n d i i c h o i c e o f fermentation c o n d i t i o n s .

with

by appropriate

H a u g a n d Larsen (12) showed that the

mannuronic to g u l u r o n i c a c i d r a t i o of A z o t o b a c t e r a l g i n a t e c o u l d b e i n f l u e n c e d b y the c a l c i u m i o n c o n c e n t r a t i o n o f the growth medium and they presented e v i d e n c e w h i c h suggested that mannuronic a c i d residues

were

epimerised to g u l u r o n i c a c i d residues b y a n e x t r a c e l l u l a r e n z y m e d e p e n d e n t on c a l c i u m ions for a c t i v i t y .

In a d d i t i o n

we h a v e b e e n a b l e to m a n i p u l a t e

the m o l e c u l a r w e i g h t and thus solution v i s c o s i t y of the p r o d u c t p r o d u c e d b y Azotobacter vinelandii. By a p p r o p r i a t e c h o i c e o f fermentation c o n d i t i o n s products w i t h a w i d e range of viscosities w e r e o b t a i n e d w h i c h c o m p a r e d f a v o u r a b l y w i t h c e r t a i n c o m m e r c i a l a l g a l a l g i n a t e s h a v i n g l o w , medium a n d h i g h viscosities (Figure 7 ) .

T h e results reported here a p p l y to products o b t a i n e d from

continuous cultures but products w i t h a similar range o f viscosities c a n also b e o b t a i n e d from b a t c h c u l t u r e s . T h e metabolism o f A z o t o b a c t e r v i n e l a n d i i in r e l a t i o n to p o l y s a c c h a r i d e biosynthesis is shown in F i g u r e 8.

Sucrose, the c a r b o h y d r a t e growth

substrate used , is transported into the c e l l , i n v e r t e d , a n d

glucose-6-phosphate

a n d f r u c t o s e - 6 - p h o s p h a t e formed b y their r e s p e c t i v e kinases.

Fructose-6-

phosphate enters the a l g i n a t e b i o s y n t h e t i c p a t h w a y w h i c h has b e e n shown to be v i a m a n n o s e - 6 - p h o s p h a t e / n a n n o s e - l - p h o s p h a t e

and

GDP-mannose

n u c l e o t i d e w h i c h is o x i d i s e d to G D P - m a n n u r o n i c a c i d (13).

Mannuronic

a c i d residues a r e then p o l y m e r i s e d to form p o l y m a n n u r o n a t e w h i c h is p a r t i a l l y e p i m e r i s e d e x t r a c e l l u l a r l y ( 1 2 ) , to y i e l d a l g i n a t e .

Azotobacter

is an o b l i g a t e a e r o b e , c a r b o h y d r a t e growth substrates are metabolised v i a

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

20

EXTRACELLULAR

0.05

0.10

0.Ï5

0.20

MICROBIAL

POLYSACCHARIDES

0.25

Dilutionrate(h ) Figure 5.

Exopolysaccharide production by Azotobacter vinelandii at a range of dilution rates

Monomers

^5-D-Mannuronic acid

oL -L-Guluronic acid

Block Structure

-M-M-M-M-M-M-

Figure 6. The structure of alginic acid

-G-G-G-G-G-G-M-G-M-G-M-G-

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

DEAVIN

E T A L .

Production

of Alginic

Acid

in

Culture

10,000 ρ

α

Lj

n

j

u

mxfc

Rate of shear (sec ^) Figure 7.

Apparent viscosity vs. rate of shear plots for Azotobacter algi nates and certain commercial algal alginates

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

EXTRACELLULAR

Figure 8.

MICROBIAL

POLYSACCHARIDES

Metabolism of Azotobacter vinelandii in relation to alginate synthesis

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

2.

DEAVIN

Production

E T AL.

of Alginic

Acid

in

Culture

23

the E n t n e r - D o u d o r o f f p a t h w a y , pentose phosphate c y c l e and t r i c a r b o x y l i c a c i d c y c l e (14) a n d a r e o x i d i s e d to c a r b o n d i o x i d e *

T h e products of sucrose

metabolism are e s s e n t i a l l y a l g i n a t e , biomass and carbon d i o x i d e .

With

increases in o x y g e n tension A z o t o b a c t e r e x h i b i t a n increase in respiration rate (15);

the e f f i c i e n c y o f e n e r g y c o n s e r v a t i o n f a l l i n g until at v e r y high

respiration rates as much as 9 0 % o f the sucrose u t i l i s e d c a n b e burnt off as carbon d i o x i d e .

O n e o f the problems i n d e v e l o p i n g a process for

A z o t o b a c t e r a l g i n a t e p r o d u c t i o n has therefore been to control this adverse respiration.

This was a d i f f i c u l t proposition i n b a t c h c u l t u r e w i t h

c o n t i n u a l l y c h a n g i n g biomass and o x y g e n d e m a n d , e s p e c i a l l y as o x y g e n l i m i t a t i o n has p r o v e d to be a disadvantageous production.

c o n d i t i o n for polymer

T r i a l s i n b a t c h c u l t u r e under p h o s p h a t e - d e f i c i e n t c o n d i t i o n s

i n d i c a t e d maximal o b t a i n a b l 2 5 % of the sucrose u t i l i s e d p r o d u c t i o n in continuous c u t l u r e was therefore i n v e s t i g a t e d . T h e organism was grown at a range of s p e c i f i c respiration rates o b t a i n e d b y a l t e r i n g the fermenter i m p e l l e r speed thus c h a n g i n g the rate o f o x y g e n transfer into the c u l t u r e broth (Figure 9 ) .

W e chose p h o s p h a t e - l i m i t e d

growth c o n d i t i o n s , as a phosphate d e f i c i e n t m e d i u m , as discussed e a r l i e r was known to b e c o n d u c i v e to p o l y s a c c h a r i d e synthesis in b a t c h c u l t u r e . A l t h o u g h c e l l mass, w h i c h

r e m a i n e d e s s e n t i a l l y constant,was l i m i t e d b y

a v a i l a b i l i t y of phosphate, the s p e c i f i c respiration rate was d e t e r m i n e d b y oxygen a v a i l a b i l i t y.

P o l y s a c c h a r i d e c o n c e n t r a t i o n was a l s o e s s e n t i a l l y

constant, d e c r e a s i n g o n l y at v e r y low r e s p i r a t i o n rates.

T h e rate of

a l g i n a t e synthesis was therefore l a r g e l y i n d e p e n d e n t of both the rate at w h i c h sucrose e n t e r e d the c e l l , as i n d i c a t e d b y the amount of sucrose u t i l i s e d , a n d the rate at w h i c h intermediates e n t e r e d the c a t a b o l i c pathways and were respired to c a r b o n d i o x i d e .

T h e maximum y i e l d of sodium a l g i n a t e ,

which

o c c u r r e d at the lower respiration rates, was i n the r e g i o n of 4 5 % of the sucrose u t i l i s e d as compared w i t h the y i e l d s of 2 5 % o b t a i n e d in b a t c h c u l t u r e . A t higher respiration rates the y i e l d f e l l d r a m a t i c a l l y d u e to a greater p r o p o r t i o n o f the sucrose b e i n g o x i d i s e d t o c a r b o n d i o x i d e . T h e e f f e c t of d i f f e r e n t growth limitations o n a l g i n a t e p r o d u c t i o n has also been investigated.

S t e a d y state continuous cultures w e r e o b t a i n e d w i t h

d i f f e r e n t nutrients l i m i t i n g growth but c e l l mass and also s p e c i f i c respiration rate were c o n t r o l l e d to w i t h i n narrow ranges.

Polysaccharide, determined

as isopropanol p r e c i p i t a t e d material,was p r o d u c e d under a l l limitations tested ( T a b l e 1).

M o l y b d a t e l i m i t a t i o n f o l l o w e d b y phosphate l i m i t a t i o n ,

the c o n d i t i o n r o u t i n e l y used, g a v e the most f a v o u r a b l e s p e c i f i c rates of p o l y s a c c h a r i d e synthesis.

Surprisingly,

under sucrose l i m i t a t i o n , a

c o n d i t i o n w h e r e the c e l l w o u l d be e x p e c t e d to make the most e f f i c i e n t use possible of its a v a i l a b l e carbon and e n e r g y substrate, p o l y s a c c h a r i d e was still

p r o d u c e d at similar rates to other l i m i t a t i o n s .

It is d i f f i c u l t to

c o m p a r e o x y g e n l i m i t a t i o n , o n e c o n d i t i o n tested w h e r e the s p e c i f i c rate of

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

24

EXTRACELLULAR

0

10

20

MICROBIAL

30

POLYSACCHARIDES

40

Specific respiration rate (umol O^/h/mg cell)

Figure 9.

Exopolysaccharide production by Azotobacter vinelandii at a range of respiration rates

T a b l e 1. E f f e c t of g r o w t h - l i m i t i n g

nutrient o n e x o p o l y s a c c h a r i d e

production b y Azotobacter vinelandii Growth-limiting nutrient

C e l l Mass

S p e c i f i c Rate o f

(mg/ml)

polysaccharide production (mg/mg c e l l / h )

1.1

0.34

1.9

0.28

Fe-H-

1.4

0.25

C(sucrose)

1.3

0.25

N

1.5

0.22

1.2

0.20

1.9

0.16

1.2

0.06

M004

2

C a "

o D

2

=

0.15 + 0.01

h

_ 1

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

2.

DEAViN E T A L .

Production

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Acid

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25

Culture

p o l y s a c c h a r i d e p r o d u c t i o n was v e r y much l o w e r , w i t h other c o n d i t i o n s s i n c e under these c o n d i t i o n s the c e l l mass was p r o b a b l y less a c t i v e due to i n t r a c e l l u l a r a c c u m u l a t i o n of p o l y - j i - h y d r o x y b u t y r a t e (16).

With

the e x c e p t i o n of O 2 - I i m i t a t i o n the s p e c i f i c rate o f p o l y s a c c h a r i d e p r o d u c t i o n v a r i e d b y just a l i t t l e over t w o f o l d w h i c h c o n s i d e r i n g the large #

changes i n the p h y s i o l o g y o f the c e l l w h i c h a r e l i k e l y under the various l i m i t a t i o n s i s not v e r y g r e a t . Some c h a n g e was found however i n the /

p h y s i c a l properties o f the p o l y s a c c h a r i d e p r o d u c e d under the various limitations.

T h e r e f o r e a l t h o u g h the s p e c i f i c rate of a l g i n a t e p r o d u c t i o n does

not v a r y g r e a t l y w i t h changes i n fermentation c o n d i t i o n s the y i e l d o f a l g i n a t e i n terms o f the amount o f sucrose u t i l i s e d is m a i n l y d e t e r m i n e d b y o x y g e n a v a i l a b i l i t y a n d thu c u l t u r e studies h a v e g i v e n a l g i n a t e biosynthesis to choose conditions where improved y i e l d s of a l g i n a t e can be obtained. In summary, t h e rate o f a l g i n a t e synthesis per unit c e l l mass remains r e l a t i v e l y constant o v e r a range o f c o n d i t i o n s where the p h y s i o l o g i c a l

state

of the c e l l w o u l d b e e x p e c t e d to v a r y w i d e l y , that is o v e r a range o f growth rates, o v e r a range o f respiration rates a n d w i t h a v a r i e t y o f growth l i m i t i n g nutrients.

How this constant rate is o b t a i n e d i n terms o f control

remains u n c l e a r .

mechanisms

A s y e t w e a r e u n a b l e to distinguish whether it is a

r e l a t i v e l y u n c o n t r o l l e d process or whether f i n e controls a r e necessary to o b t a i n this constant r a t e .

From these findings a n d our observations o n other

e x o p o l y s a c c h a r i d e p r o d u c i n g organisms, n a m e l y Xanthomonas

campestris

a n d a Pseudomonas s p . the a b i l i t y to p r o d u c e e x o p o l y s a c c h a r i d e at similar rates under a v a r i e t y o f c o n d i t i o n s c o u l d b e much more general than has hitherto b e e n r e c o g n i s e d .

Literature Cited (1) (2) (3) (4) (5) (6) (7)

Herbert, C., Ellsworth, R. and Telling, R.C. J. Gen. Microbiol. (1965), 14, 601-622. Moraine, R.A. and Rogovin, P. Biotechnol. Bioeng. (1973), 14 225-237 Lawson, C.J. and Symes, K.C. Unpublished data. Williams, A.C. Ph.D. Thesis, University College, Cardiff, U.K. (1974) Neijssel, O.M. and Tempest, D.W. Arch. Microbiol. (1976), 107, 215-221 Silman, R.W. and Rogovin, P. Biotechnol. Bioeng. (1972), 14 23-31 Drummond, D.W., Hurst, E.L. and Percival, E. (1961). J. Chem. Soc., London, p. 1208-1216.

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

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(8)

Larsen, B.,Sandsrød,O . , Haug, A. and Painter, T. Acta Chem. Scand. (1969), 23, 2375-2388. (9) Smidsrød, O. Disc. Faraday Soc. (1974),57, 263-274. (10) Gorin, P.A.J. and Spencer, J.F.t. Can. J. Chem. (1966) 44, 993-998 (11) Larsen, B. and Haug, A.Carbohyd.Res.(1971), 17, 287-296. (12) Haug, A. and Larsen B. Carbohyd. Res. (1971 ),17, 297-308. (13) Pindar, D.F. and Bucke, C. Biochem. J. (1975), 152, 617-622. (14) Still, G.C. and Wang, C.H. Arch. Biochem. Biophys. (1964) 105, 126-132. (15) Downs, A.J. and Jones, C.W., FEBS Lett.(1975),60,42-46. (16) Dawes,Ε.A.and Senior 10, 135-266

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

3 Xanthan G u m from A c i d W h e y MARVIN CHARLES and MOHAMMED K. RADJAI Department of Chemical Engineering, Lehigh University, Bethlehem, PA 18015

Xanthan gum (fro 1459A) has been produced from media containing deproteinized acid-set or culture-set cottage cheese wheys, the lactose contents of which were hydrolyzed to glucose and galactose by means of immobilized lactase. Both glucose and galactose were used almost completely to give gum yields, productivities and final concentrations which were generally as good as, and in some cases better than, those obtained with comparable conventional media.With the exception of an anomalous pH history (the pH increased rather than decreased) when culture-set whey permeate was used, the fermentations followed courses typical of those previously reported. Details of media preparation, fermentation conditions, and experimental results w i l l follow a brief discussion of cottage cheese whey and whey permeate. C o t t a g e Cheese Whey and Whey Permeate A c i d whey i s t h e h i g h BOD waste r e s u l t i n g from t h e manufacture o f c o t t a g e cheese. I t s composition {!) (see T a b l e I) v a r i e s somewhat w i t h t h e c u r d - s e t t i n g p r o c e s s employed (and w i t h m i l k c o m p o s i t i o n , e t c . ) b u t i n g e n e r a l i t c o n t a i n s around 4% t o 5% l a c t o s e , 0.8% t o 1.0% p r o t e i n ( l a c t a l b u m i n ) , and l e s s e r quant i t i e s o f a c i d s , m i n e r a l s , v i t a m i n s , e t c . Most o f t h e a c i d whey produced each y e a r i s r u n t o waste r e s u l t i n g i n c o n s i d e r a b l e c o s t s t o d a i r i e s and communities. Furthermore, such d i s p o s a l r e s u l t s i n y e a r l y l o s s e s o f o v e r 100 m i l l i o n l b s . o f v a l u a b l e and m a r k e t a b l e whey p r o t e i n ( l a c t a l b u m i n ) , which has e x c e l l e n t n u t r i t i o n a l and f u n c t i o n a l p r o p e r t i e s , and o v e r 500 m i l l i o n l b s . of lactose along with l e s s e r but s i g n i f i c a n t 27

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

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q u a n t i t i e s o f o r g a n i c a c i d s and v i t a m i n s . Therefore t h e r e i s c o n s i d e r a b l e economic i n c e n t i v e f o r t h e development o f p r o c e s s e s f o r d i r e c t u t i l i z a t i o n o f a c i d whey o r f o r r e c o v e r y and subsequent use o f i n d i v i d u a l a c i d whey components b u t t h e l a t t e r approach appears t o have g r e a t e r p o t e n t i a l i n t h e f o r s e e a b l e f u t u r e . Table

I.

A c i d Whey C o m p o s i t i o n

L a c t o s e (wt %) P r o t e i n (wt %) Ash (wt %) L a c t i c A c i d (wt %) Glucono-6-Lactone(wt% C a l c i u m (G/L) Phosphorous (G/L) T o t a l S o l i d s (wt %) PH

Culture Set 4.3-4.4 0.8-1.0 0.7-0.8

1.2-1.3 0.7-0.8 6.9-7.0 4.3-4.7

(Typical) Acid Set 4.6-4.9 0.9 0.8-0.9

—

1.3-1.4 1.9-2.1 7.0-7.2 4.1-4.5

Recovery o f l a c t a l b u m i n by t h e proven t e c h n o l o g y o f u l t r a f i l t r a t i o n o f f e r s c o n s i d e r a b l e economic promi s e t h r o u g h o u t most o f t h e c o u n t r y and a l r e a d y has been operated commercially. However, an i m p o r t a n t f a c t o r i n f l u e n c i n g t h e economics o f t h e r e c o v e r y i s t h e u l t i mate use o f whey permeate which i s t h e b y - p r o d u c t o f u l t r a f i l t r a t i o n and which c o n t a i n s a l a r g e q u a n t i t y o f l a c t o s e , some low m o l e c u l a r weight p r o t e i n , o r g a n i c a c i d s , m i n e r a l s , v i t a m i n s , and some o t h e r minor comp o n e n t s . We r e q u i r e , t h e n , e c o n o m i c a l uses f o r whey permeate (2) . Many s u g g e s t i o n s have been made f o r d i r e c t u t i l i z a t i o n o f permeate i n c l u d i n g c o n v e r s i o n t o y e a s t a n d / o r a l c o h o l (_3) · F e r m e n t a t i o n t e c h n o l o g i e s f o r b o t h a r e w e l l known and i t seems r e a s o n a b l e t o e x p e c t t h a t t h e r e may be some c a s e s i n which such p r o c e s s e s w i l l be e c o n o m i c a l l y f e a s i b l e a l t h o u g h i t must be r e c o g n i z e d t h a t t h e r e l a t i v e l y low economic v a l u e s o f t h e p r o d u c t s might be a d e t e r r e n t t o i n v e s t m e n t . However, i n t h e absence o f r e c e n t well-documented economic s t u d i e s i t i s d i f f i c u l t t o make a s a t i s f a c t o r y a n a l y s i s p a r t i c u l a r l y i n l i g h t o f t h e p o t e n t i a l , b u t somewhat u n c e r t a i n , l a r g e - s c a l e use o f e t h y l a l c o h o l as a f u e l . A n o t h e r approach i n v o l v e s t h e h y d r o l y s i s o f whey permeate l a c t o s e t o g l u c o s e and g a l a c t o s e by means o f i m m o b i l i z e d l a c t a s e (£,!5,£, 1) . W i d e l y d i s c u s s e d food r e l a t e d a p p l i c a t i o n s o f t h e "sweet permeate" so p r o duced a r e based on t h e d e s i r e t o r e c y c l e whey permeate so as t o e l i m i n a t e d i s p o s a l c o s t s , t o d e c r e a s e sweetener c o s t s , and t o c i r c u m v e n t n u t r i t i o n a l and

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

3.

CHARLES

A N D

RADjAi

Xanthan Gum from Acid Whey

29

f u n c t i o n a l problems a s s o c i a t e d w i t h l a c t o s e . However, d e s p i t e t h e f a c t t h a t t h e h y d r o l y s i s c a n be performed f o r w e l l under ΙΟΦ/lb o f l a c t o s e (5) , t h e "sweet p e r meate" may s t i l l meet w i t h s t i f f c o m p e t i t i o n from a v a i l a b l e c o r n and h i g h f r u c t o s e s y r u p s s i n c e g a l a c t o s e i s n o t as sweet as g l u c o s e and hence on a pound f o r pound ( s o l i d s ) b a s i s t h e h y d r o l y z a t e i s n o t a s sweet as t h e a l r e a d y a v a i l a b l e s y r u p s . Furthermore, i t appears t h a t d e m i n e r a l i z a t i o n w i l l be r e q u i r e d t o make t h e h y d r o l y z a t e a c c e p t a b l e as a food i n g r e d i e n t and t h i s w i l l add c o n s i d e r a b l y t o i t s c o s t (15) . These f a c t s , c o u p l e d w i t h t h e d e c l i n e i n sugar p r i c e s have c a s t some doubt on t h e v e r y p r o m i s i n g economic progno s i s which e x i s t e d f o t h f "sweet permeate" food i n g r e d i e n t j u s An a l t e r n a t i v e use o f t h e h y d r o l y z a t e i s as a f e r m e n t a t i o n medium. There a r e many organisms which w i l l m e t a b o l i z e b o t h g l u c o s e and g a l a c t o s e (but n o t l a c t o s e ) t o p r o d u c t s c o n s i d e r a b l y more v a l u a b l e than y e a s t o r a l c o h o l and whose n i t r o g e n r e q u i r e m e n t s a r e s a t i s f i e d p a r t i a l l y o r c o m p l e t e l y by t h e low m o l e c u l a r weight permeate p r o t e i n s . T h i s i s p a r t i c u l a r l y t r u e i n c a s e s where p r o d u c t i o n o f l a r g e q u a n t i t i e s o f c e l l mass i s n o t r e q u i r e d o r even p a r t i c u l a r l y d e s i r a b l e (e.g., i n p r o d u c t i o n o f x a n t h a n ) . F u r t h e r m o r e , demin e r a l i z a t i o n o f the hydrolyzate i s g e n e r a l l y not r e quired f o r this application. Thus, i n s o f a r as use as a f e r m e n t a t i o n medium i s c o n c e r n e d , h y d r o l y z e d p e r meate has t h e f o l l o w i n g advantages: • carbohydrate cost competitive with glucose • adequate n i t r o g e n and o t h e r growth f a c t o r s f o r many a p p l i c a t i o n s » u t i l i z e s a h i g h BOD waste stream •enhances economics o f whey p r o t e i n r e c o v e r y . I t s h o u l d a l s o be n o t e d t h a t even i f c o n d e n s a t i o n i s required to f a c i l i t a t e t r a n s p o r t a t i o n , the cost o f h y d r o l y z a t e would s t i l l be c o m p e t i t i v e w i t h commercial dextrose. The m i c r o b i a l p r o d u c t i o n o f xanthan gum i s a p a r t i c u l a r example o f an a l r e a d y s u c c e s s f u l commercial f e r m e n t a t i o n which uses a c o n v e n t i o n a l g l u c o s e - c o n t a i n i n g medium b u t which c a n be conducted as w e l l o r b e t t e r w i t h a h y d r o l y z e d whey permeate medium. The

Fermentation

Process

Medium F o r m u l a t i o n . The medium c a n be produced from e i t h e r c u l t u r e - s e t o r a c i d - s e t c o t t a g e cheese whey by means o f t h e p r o c e s s i l l u s t r a t e d i n F i g u r e 1: (a) Whey i s f i l t e r e d t h r o u g h a h o l l o w - f i b e r

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

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(c)

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u l t r a f i l t e r h a v i n g a m o l e c u l a r weight c u t o f f o f 50,000 (HF 26.5-45 - XM50 c a r t r i d g e , Romicon, I n c . , Woburn, Ma s s . ) . The permeate, which has a pH o f 4.1-4.6, i s hydrolyzed i n a p i l o t - p l a n t f l u i d i z e d - b e d r e a c t o r c o n t a i n i n g A.niger l a c t a s e (Lactase, L.P., W a l l e r s t e i n , C h i c a g o , IL) i m m o b i l i z e d on alumina p a r t i c l e s (£,7). The h y d r o l y z e d permeate Ts then s t e r i l i z e d and supplemented w i t h s t e r i l e K 2 H P O 4 and MgS04«7H20 t o y i e l d a medium whose composit i o n i s given i n Table I I . The pH o f t h e medium i s a d j u s t e d t o 7.0.

Table I I .

Hydrolyze ( F u l l S t r e n g t h - C u l t u r e Set)

G l u c o s e (wt %) Galactose Lactose K Mg 2

HPO4 S 0 . 7 H 4

2

0

P r o t e i n (Lowry) Whey A c i d pH

2.05 2.05 0.30 0.50 0.01

0.20 0.70 7.0

(a) Medium a l s o c o n t a i n s whey a s h , a c i d s , vitamins, e t c . W h i l e e i t h e r a c i d - s e t o r c u l t u r e - s e t whey may be used, i t i s i m p o r t a n t t o note t h a t t h e two a r e n o t e q u i v a l e n t as w i l l be i l l u s t r a t e d below. In some c a s e s we have used t h e media d e s c r i b e d as i s w h i l e i n o t h e r s t h e y have been d i l u t e d t o a p p r o x i m a t e l y h a l f - s t r e n g t h , F u r t h e r m o r e , we o c c a s i o n a l l y have added s m a l l q u a n t i t i e s o f supplemental n i t r o g e n i n t h e form o f e n z y m i c a l l y - h y d r o l y z e d l a c t a l b u m i n (Edamin, S h e f f i e l d C h e m i c a l , Union, N J ) . T h i s was p r o v e n t o be p a r t i c u l a r l y v a l u a b l e when a c i d - s e t whey was u s e d . Sterilization. H y d r o l y z e d whey permeate i s a complex medium c o n t a i n i n g sugars and low m o l e c u l a r weight p r o t e i n a l o n g w i t h a c i d s and v a r i o u s m i n e r a l s and hence some c a u t i o n i s n e c e s s a r y d u r i n g steam s t e r i l i z a t i o n , p a r t i c u l a r l y when an a u t o c l a v e i s used as i t was i n o u r c a s e . We found t h a t i f t h e permeate was s t e r i l i z e d a t i t s n a t u r a l pH (4.1-4.6) t h e r e was obs e r v a b l e browning b u t t h e r e was almost no l o s s o f n u t r i e n t s and i n h i b i t o r y p r o d u c t s were n o t formed t o any a p p r e c i a b l e e x t e n t . Indeed, medium steam

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

3.

C H A R L E S A N D RADJAI

Xanthan

Gum

from Acid

Whey

31

s t e r i l i z e d a t t h e n a t u r a l permeate pH behaved a s w e l l as f i l t e r - s t e r i l i z e d medium. On t h e o t h e r hand, steam s t e r i l i z a t i o n a t pH 6.0 o r g r e a t e r r e s u l t e d i n s e v e r e browning, c o n s i d e r a b l e p r e c i p i t a t i o n , l o s s o f n u t r i e n t s , apparent formation o f r e l a t i v e l y high l e v e l s o f i n h i b i t o r y compounds and a g e n e r a l l y i n f e r i o r medium. Fermentation C o n d i t i o n s . Bench-scale fermentat i o n s were conducted i n 7 l i t e r a e r a t e d , n o n - b a f f l e d f e r m e n t o r s equipped w i t h t h r e e p i t c h e d - b l a d e t u r b i n e i m p e l l e r s h a v i n g tank diameter t o i m p e l l e r d i a m e t e r r a t i o s o f 1.8 t o 1.0. We found t h a t t h e use o f m u l t i p l e l a r g e i m p e l l e r s and t h e i n t e n t i o n a l removal o f baffles resulted i bette mixing transfer and p r o d u c t i v i t y whe viscous, p a r t i c u l a r l y g r e a t e r t h a n 1% (8_) . The f e r m e n t o r s were a l s o equipped w i t h a u t o m a t i c foam c o n t r o l l e r s , d i s s o l v e d oxygen m o n i t o r s , and pH c o n t r o l systems which added e i t h e r 4 N KOH o r gaseous N H 3 . The seed c u l t u r e was d e v e l o p e d as suggested by Moraine and h i s coworkers (9,10,11) and a 5% (V/V) seed was used t o i n o c u l a t e t h e main f e r m e n t o r s i n a l l c a s e s . Temperature was always m a i n t a i n e d a t 28°C and pH a t 7.0 e x c e p t when t h e pH remained above 7 as was t y p i c a l l y t h e c a s e when c u l t u r e - s e t whey was used. Analytical G l u c o s e , g a l a c t o s e , and xanthaa c o n c e n t r a t i o n s were measured a t r e g u l a r i n t e r v a l s . G l u c o s e was d e t e r m i n e d by means o f a g l u c o s e - o x i d a s e impregnated membrane and g a l a c t o s e by means o f a g a l a c t o s e - o x i d a s e impregnated membrane. Both were used i n c o n j u n c t i o n w i t h a YSI Model 23A g l u c o s e a n a l y z e r (YSI I n s t r u m e n t s , Y e l l o w S p r i n g s , O h i o ) . The l a c t o s e c o n t e n t o f unhydrol y z e d whey was u s u a l l y d e t e r m i n e d by f i r s t c o m p l e t e l y h y d r o l y z i n g i t w i t h e x c e s s A . n i g e r l a c t a s e ( L a c t a s e LP, W a l l e r s t e i n , C h i c a g o , IL) and t h e n measuring t h e r e s u l t i n g g l u c o s e o r g a l a c t o s e . In some c a s e s t h e g a l a c t o s e o x i d a s e membrane, which responds t o l a c t o s e t o an e x t e n t o f 10-15% o f i t s r e s p o n s e t o g a l a c t o s e , was used t o determine whey permeate l a c t o s e d i r e c t l y . The l a c t o s e c o n c e n t r a t i o n i n whey permeate used f o r f e r m e n t a t i o n s was c a l c u l a t e d from t h e h y d r o l y z a t e g l u c o s e c o n c e n t r a t i o n (which i s e q u a l t o t h e g a l a c t o s e c o n c e n t r a t i o n p r i o r t o i n o c u l a t i o n ) and t h e known i n i t i a l permeate l a c t o s e c o n c e n t r a t i o n . The l a c t o s e c o n c e n t r a t i o n remained e s s e n t i a l l y c o n s t a n t t h r o u g h o u t a l l t h e f e r m e n t a t i o n s performed as i t was n o t

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

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m e t a b o l i z e d by X . c a m p e s t r i s under t h e c o n d i t i o n s employed. Xanthan was d e t e r m i n e d by f i r s t f i l t e r i n g f e r m e n t a t i o n samples t o remove a l l suspended s o l i d s , p r e c i p i t a t i n g the xanthan i n t h e f i l t r a t e by a d d i t i o n o f KC1(2%) and methanol (50-60%) and f i n a l l y d e t e r m i n i n g t h e d r y weight o f t h e p r e c i p i t a t e d gum. F e r m e n t a t i o n Modes Both b a t c h and r e p e a t e d - b a t c h f e r m e n t a t i o n s were performed. In r e p e a t e d - b a t c h o p e r a t i o n a g i v e n f e r m e n t a t i o n c y c l e was t e r m i n a t e d when t h e g a l a c t o s e conc e n t r a t i o n dropped t o a p p r o x i m a t e l y 0.1% o r when t h e xanthan p r o d u c t i o n t h a t time, approximatel t e n t s were r e p l a c e d w i t h f r e s h medium and a new c y c l e was i n i t i a t e d . R e s u l t s and D i s c u s s i o n G l u c o s e / G a l a c t o s e Medium. F e r m e n t a t i o n s were c o n d u c t e d u s i n g media based on 50/50 m i x t u r e s o f pure g l u c o s e and g a l a c t o s e t o p r o v i d e b a s e - l i n e d a t a f r e e o f a m b i g u i t i e s t h a t might a r i s e as a r e s u l t o f t h e complex n a t u r e o f whey-based media. The h i s t o r y o f a t y p i c a l f e r m e n t a t i o n i s g i v e n i n F i g u r e 2 and t h e comp o s i t i o n o f t h e medium used i n T a b l e I I I . Table I I I .

G l u c o s e - G a l a c t o s e Medium

G l u c o s e (wt %) Galactose Edamin K2 HP04 Mg S 0 . 7 H 0 pH 4

2

1.3 1.3 0.06 0.50 0.01 7.0

The most i n t e r e s t i n g p o i n t i l l u s t r a t e d by t h e s e r e s u l t s i s t h e s i m u l t a n e o u s use o f b o t h s u g a r s . A l though g a l a c t o s e was used l e s s r a p i d l y t h a n g l u c o s e t h e r e was c l e a r l y no d i a u x i e . Furthermore, both sugars were u t i l i z e d f o r gum p r o d u c t i o n . Otherwise t h e c o u r s e o f the f e r m e n t a t i o n was t y p i c a l o f t h o s e r e p o r t e d by Moraine and h i s coworkers (£, 10/11.) · The f i n a l gum c o n c e n t r a t i o n o f a p p r o x i m a t e l y 2% which r e p r e s e n t e d a 77% y i e l d was a c h i e v e d i n about 50 h o u r s . C u l t u r e Set Whey; B a t c h F e r m e n t a t i o n . The h i s t o r y o f a t y p i c a l b a t c h f e r m e n t a t i o n based on c u l t u r e - s e t

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

CHARLES

AND

Xanthan

RADJAI

Gum from Acid

Whey

Mgso

4

IME

K HPO 2

4

Reactor

UltraFilter Raw Whey "

KOH

Permeate

Concentrate S?Sterilize

Fermentor Figure 1.

Medium preparation 8.0 Ck

4J

U

20

30

Time (Hrs) Figure 2. Batch fermentation; glucose-galactose medium

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

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MICROBIAL

POLYSACCHARIDES

whey permeate medium i s i l l u s t r a t e d i n F i g u r e 3 and t h e c o m p o s i t i o n o f t h e medium used i s g i v e n i n T a b l e I . The most s i g n i f i c a n t p o i n t t o be noted here i s t h a t the pH b e h a v i o r was v e r y d i f f e r e n t from t h a t observed by o t h e r s u s i n g c o n v e n t i o n a l g l u c o s e media o r by o u r s e l v e s when we used t h e g l u c o s e / g a l a c t o s e medium. We w i l l return to this later. The o t h e r p o i n t worth n o t i n g i s t h a t t h e f i n a l gum c o n c e n t r a t i o n o f 3.5% ( i n a p p r o x i m a t e l y 90 hours) r e p r e s e n t s an 85* y i e l d from t h e a s s i m i l a b l e sugars which was c o n s i d e r a b l y g r e a t e r t h a n would have been expected on t h e b a s i s o f p r e v i o u s r e p o r t s . A g a i n , we w i l l r e t u r n t o t h i s l a t e r . A c i d - S e t Whey h Fermentation Result f batch fermentation usin medium supplemented w i t h Edamin and h a v i n g t h e compo s i t i o n g i v e n i n T a b l e IV a r e p r e s e n t e d i n F i g u r e 4. T a b l e IV.

Hydrolyzed-Permeate/Edamin M e d i u m ^ (Half Strength-Acid Set)

G l u c o s e (wt %) 1.3 Galactose 1.3 Lactose 0.2 Whey P r o t e i n (Lowry) 0.1 Edamin 0.06 K HP0 0.25 Mg S 0 . 7 H 0 0.005 pH 7.0 (a) Medium a l s o c o n t a i n s whey a s h , a c i d s , vitamins, e t c . 2

4

4

2

I n g e n e r a l , t h i s h i s t o r y i s t h e same a s t h a t f o r t h e f e r m e n t a t i o n i n which t h e g l u c o s e / g a l a c t o s e medium was used a l t h o u g h i t d i d p r o c e e d somewhat more r a p i d l y . In p a r t i c u l a r , t h e pH b e h a v i o r was t y p i c a l and t h e y i e l d was w i t h i n t h e range e x p e c t e d . I t should a l s o be noted t h a t media c o n t a i n i n g a c i d s e t whey b u t no Edamin gave somewhat lower y i e l d s and l o n g e r fermentations. The r e a s o n s f o r t h e enhanced gum p r o d u c t i o n and anomalous pH b e h a v i o r o b s e r v e d when c u l t u r e - s e t whey permeate was used a r e n o t c l e a r . A t t h i s time we c a n o n l y s p e c u l a t e t h a t d i f f e r e n c e s i n whey permeate comp o s i t i o n s must be r e s p o n s i b l e the primary d i f f e r e n c e s b e i n g i n t h e c o n c e n t r a t i o n s o f low m o l e c u l a r weight whey p r o t e i n , and i n t h e c o n c e n t r a t i o n s and composit i o n s o f t h e whey a c i d f r a c t i o n s . However, we c a n n o t r u l e o u t o t h e r f a c t o r s such as d i f f e r e n c e s i n v i t a m i n content. f

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

CHARLES

AND

0

Xanthan

RADJAI

10

Gum from Acid

©

Glucose

• Δ 0

Galactose Xanthan Viscosity

20

30

40

50

60

70

80

Whey

90 100

Time (Hrs) Figure S.

Batch fermentation; full-strength culture-set whey medium

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

36

EXTRACELLULAR

0

Glucose

•

Galactose

Δ

Xanthan

Ο

0

10

MICROBIAL

POLYSACCHARIDES

Viscosity

20

30

40

50

Time (Hrs) Figure 4.

Batch fermentation; half-strength acid-set whey medium

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

3.

CHARLES

AND

RADJAI

Xanthan

Gum from Acid

Whey

Time (Hrs)

Figure 5.

Repeated batch fermentation; full-strength culture-set whey medium

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

37

38

EXTRACELLULAR

MICROBIAL

POLYSACCHARIDES

Repeated-Batch F e r m e n t a t i o n . Results of a threec y c l e repeated batch fermentation with f u l l - s t r e n g t h c u l t u r e - s e t whey medium (no Edamin) a r e i l l u s t r a t e d i n F i g u r e 5. Other t h a n t h e anomalous pH b e h a v i o r and g r e a t e r - t h a n - e x p e c t e d y i e l d s t h e most n o t a b l e f e a t u r e o f t h e s e r e s u l t s i s t h a t t h e r e was l i t t l e change i n f e r m e n t a t i o n h i s t o r y from c y c l e t o c y c l e . However, i t s h o u l d be o b s e r v e d t h a t t h e r e i s a p e r c e p t i b l e i n c r e a s e i n l a g time from one c y c l e t o t h e n e x t . A t t h i s time we c a n n o t say w i t h c e r t a i n t y t h a t t h i s was a c t u a l l y a t r e n d n o r , i f i t was, c a n we p r e d i c t t h e number o f c y c l e s which c o u l d be performed b e f o r e t h e l a g would become p r o h i b i t i v e l long However s h o u l d note t h a t becaus fermentor t h e c u l t u r next c y c l e always came from t h e v e r y bottom o f t h e v e s s e l where m i x i n g and a e r a t i o n were p a r t i c u l a r l y poor d u r i n g t h e l a s t hours o f each c y c l e . T h i s may have been t h e cause o f t h e i n c r e a s e d l a g t i m e s . Conclusion H y d r o l y z e d whey permeate has been shown t o be a s u i t a b l e and c o m p e t i t i v e medium f o r t h e p r o d u c t i o n o f xanthan gum by X . c a m p e s t r i s . I t supports e x c e l l e n t y i e l d s and h i g h f i n a l c o n c e n t r a t i o n s i n b o t h b a t c h and r e p e a t e d b a t c h o p e r a t i o n p a r t i c u l a r l y when modif i e d n o n - b a f f l e d a g i t a t i o n systems employing m u l t i p l e l a r g e p i t c h e d - b l a d e t u r b i n e i m p e l l e r s a r e used.

The a u t h o r s w i s h t o e x p r e s s t h e i r g r a t i t u d e t o t h e P e n n s y l v a n i a S c i e n c e and E n g i n e e r i n g F o u n d a t i o n f o r s u p p o r t i n g t h i s work under PSEF Agreement #273 and t o Romicon, I n c . f o r t h e i r generous g i f t o f u l t r a f i l t r a t i o n c a r t r i d g e s used i n t h i s work.

Literature Cited 1. 2. 3.

Personal communication, Lehigh Valley Dairy, Allentown, PA. Melicouris, N . , paper presented at Enzyme Technology Transfer and Utilization Conference, Lehigh University, Bethlehem, PA, May 27, 1976. Goulet, J., paper presented at the First International Congress on Food and Engineering, Boston, Mass., August 10, 1976.

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

3.

CHARLES

A N D RADjAI

Xanthan

Gum from Acid Whey

4.

Si

Coughlin, R. W., Charles, Μ., in "Enzyme Engin eering" ed. Oye, Ε. Κ., and Wingard, L . Β., Plenum Press, N.Y., 1974. 5. Pitcher, W. H., in "Immobilized Enzymes for Indus trial Reactors" ed. R. A. Messing, Academic Press, New York (1975). 6. Charles, Μ., Coughlin, R. W., paper presented at NSF/RANN Grantees Conference, University of V i r ginia, Charlottesville, VA, May 19-21, 1976. 7. Charles, Μ., Coughlin, R. W., Allen, B. R., Paruchuri, Ε. Κ., Hasselberger, F. X . , in "Immobilized Biochemicals and Affinity Chromatography", ed. Dunlay, R. Β., Plenum Press, N . Y . , 1974. 8. Charles, Μ., Zmuda J., paper presented at AIChE Meeting, Nov. 28-Dec 9. Moraine, R. Α . , Rogovin, , Bioeng., , 511 (1966). 10. Moraine, R. Α . , Rogovin, P . , Biotech. Bioeng., 13, 381 (1971). 11. Moraine, R. Α . , Rogovin, P . , Biotech. Bioeng., 15, 225 (1973).

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

4 Microbial Exopolysaccharide Synthesis I. W. SUTHERLAND Department of Microbiology, University of Edinburgh, West Mains Road, Edinburgh, EH9 3JG, Scotland

The fate of a carbohydrate (or other) substrate supplied to an exopolysaccharide-producin microbial species chosen from bacterial species, this review will be concerned essentially with the synthesis of exopolysaccharides by bacteria. In some bacteria, given the correct substrate, exopolysaccharide may be formed without penetration of the cell membrane by the substrate. This is seen in dextran and levan-forming cells supplied with sucrose or several of its analogues. Examples are to be found in Leuconostoc mesenterioides, Streptococcus or Bacillus species. Although this process has been studied by various workers, (1,2) the polysaccharides formed are more limited in their applications and current interest is centred rather on species which form their polymer intracellularly then excrete it into the medium. The aim is therefore to consider a series of processes by which substrates enter the microbial cells, are modified by a series of enzymic processes and finally are excreted in polymeric form from the microbial surface. Much of the information about these reactions has been gained from strains producing polymers which have little or no commercial value, but it is nevertheless possible to extrapolate many of the results and thereby obtain a reasonable hypothesis for the mode of synthesis of a polymer of given structure and to propose mechanisms for the regulation of its biosynthesis. Substrate Uptake The s u b s t r a t e may enter the c e l l by one of three mechanisms - f a c i l i t a t e d d i f f u s i o n , a c t i v e t r a n s p o r t or group t r a n s l o c a t i o n . The l a t t e r two processes, both of which a r e endergonic, are of p a r t i c u l a r i n t e r e s t i n the present context. In a c t i v e t r a n s p o r t , the substrate enters the c e l l u n a l t e r e d , but the group t r a n s l o c a t i o n process i n v o l v e s the phosphorylation o f the s u b s t r a t e , the o v e r a l l process being represented by: X

+

PEP

• X-P

+

pyruvate

40

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

4.

SUTHERLAND

Microbial

Exopolysaccharide

Synthesis

41

The i n i t i a l f a t e of the substrate i s summarised i n F i g . l . In E s c h e r i c h i a c o l i , the r a t e at which the b a c t e r i a grow on v a r i o u s substrates i s dependent on substrate uptake, i r r e s p e c t i v e of whether a c t i v e t r a n s p o r t or group t r a n s l o c a t i o n systems are involved ( 3 ) . Thus substrate uptake i s one of the f i r s t l i m i t a t i o n s on exopolysaccharide production. As y e t , no attempts to increase c e l l growth and hence exopolysaccharide production by d u p l i c a t i o n of the genes concerned with a c t i v e t r a n s p o r t or with group t r a n s l o c a t i o n appears to have been made. In many b a c t e r i a , t h i s might not even be necessary, as s e v e r a l uptake mechanisms may e x i s t f o r each substrate i . e . Although a s p e c i f i c substrate may be transported by d i f f e r e n t mechanisms i n d i f f e r e n t microorganisms b a c t e r i a such as E* c o l i possess various mechanisms f o r uptake of a s i n g l e substrate such as g a l a c t o s e D i f f e r e n c e s can c e r t a i n l y be expected between Gra between pseudomonads an The group t r a n s l o c a t i o n mechanisms i n v o l v i n g phosphorylation from PEP have been studied by Roseman and h i s colleagues (4) but i t i s not c l e a r whether the u t i l i z a t i o n of r e l a t i v e l y l a r g e amounts of PEP f o r substrate uptake lead to a r e d u c t i o n i n the amount of PEP a v a i l a b l e f o r other purposes. I f t h i s does r e s u l t under c o n d i t i o n s i n which growth i s l i m i t e d by substrate uptake and where high growth r a t e s are used, the r e s u l t might be a r e d u c t i o n i n the degree of p y r u v y l a t i o n observed i n the polymer excreted. Intermediary Metabolism and D i r e c t i o n to Polymer Synthesis F o l l o w i n g the entry of the substrate i n t o the c e l l and i t s phosphorylation by e i t h e r the group t r a n s l o c a t i o n mechanism or by a hexokinase u t i l i z i n g ATP, the substrate can be committed to e i t h e r anabolic processes or to m i c r o b i a l catabolism ( F i g . 2). I f i t s u f f e r s the l a t t e r f a t e , i t i s i n e f f e c t wasted as f a r as polymer production i s concerned, although i f i t enters the TCA c y c l e i t may be converted to pyruvate or to acetate and thus incorporated at a l a t e r stage i n t o polymer. The c o n t r o l of c a t a b o l i c processes w i l l not be considered here. The a n a b o l i c f a t e of the substrate can s t i l l take one of s e v e r a l l i n e s at t h i s stage. I f the m i c r o b i a l species under c o n s i d e r a t i o n i s a Gram negative species, forming exopolysaccharide, l i p o p o l y s a c c h a r i d e and glycogen, the carbohydrate may be converted to any one of these. In the p r o l i f e r a t i n g bacterium, glycogen i s r a r e l y synthesized, but i t s production i s a l s o d i f f e r e n t i a t e d from w a l l polymer or e x t r a c e l l u l a r polymer synthesis through the lack of involvement of i s o p r e n o i d l i p i d s . The c o n t r o l of glycogen synt h e s i s i s exerted through a l l o s t e r i c r e g u l a t i o n of ADP-glucose synthesis (5), the f i r s t enzymic step i n the pathway, which i s unique to glycogen synthesis ( F i g . 3). I t may thus be worth c o n s i d e r i n g the i s o l a t i o n of ADP-glucose pyrophosphorylase mutants i f the b a c t e r i a l s t r a i n i n which we are i n t e r e s t e d produces large

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

42

EXTRACELLULAR

SUBSTRATE

MICROBIAL

SUBSTRATE

POLYSACCHARIDES

SUBSTRATE

EXTRACELLULAR ENZYMES f POLYMER

(DEXTRANS, L E V A N S , ETC.)

f

>

HISTIDINYL+ PEP +

PERMEASE

PROTEIN MEMBRANE

ENZYME M SUBSTRATE

SUBSTRAT

j KINASE + ATP SUBSTRATE -

+

PHOSPHATE + ADP Figure 1.

HEXOSE-

Initial pathways for extracellular substrates

•HEXOSE

6 Ρ—-HEXOSE

1 P- -CATABOLISM ENERGY

ANABOLISM POLYMERS Figure 2.

GLUCOSE

Fate of hexose substrate

G L C - 6 P — - G L C - 1 P UDP-GLUCOSE

PYROPHOSPHORYLASE

UDP-GLUCOSE

ADP-GLUCOSE PYROPHOSPHORYLASE

ADP-GLC

(GLC1^4GLC) GLYCOGEN EXOPOLYSACCHARIDE Figure 3.

LPS Anabolic fate of glucose

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

4.

SUTHERLAND

Microbial

Exopolysacchande

Synthesis

43

amounts of glycogen and thus converts s u b s t r a t e to an unwanted product. T h i s would e l i m i n a t e the " d r a i n " of glucose-l-phosphate i n t o glycogen synthesis and away from the d e s i r e d product. Such mutants would be p a r t i c u l a r l y v a l u a b l e i f a two-stage p r o d u c t i o n process was envisaged i n which the second stage contained c e l l s i n an e s s e n t i a l l y n o n - p r o l i f e r a t i n g environment, i . e . c o n d i t i o n s under which l a r g e q u a n t i t i e s of glycogen are normally s y n t h e s i z e d . ( S i m i l a r arguments would apply i f the micro-organism produce p o l y hydroxybutyric a c i d or t r e h a l o s e r a t h e r than glycogen.) The next precursor through which c o n t r o l can be exerted i s the sugar n u c l e o t i d e such as UDP-glucose. UDP-glucose pyrophosphorylase i s a key enzyme producing i n many micro-organisms a precursor f o r both w a l synthesis. The l e v e l appears to be almost u n a l t e r e d i n mutants d e f e c t i v e i n these polymers and t h i s i s r e f l e c t e d at l e a s t i n the E n t e r o b a c t e r i a c e a e , i n the l e v e l of UDP-glucose found i n n u c l e o t i d e pools of s e v e r a l strains (6). The s t r i c t c o n t r o l exerted by such enzymes as UDPglucose pyrophosphorylase or TDP-glucose pyrophosphorylase (7) enables some micro-organisms to channel intermediates to one p o l y mer or another. Thus, TDP-glucose i s a precursor of TDP-rhamnose: f o r i n c o r p o r a t i o n i n t o one or more polymers. I n species poss e s s i n g both enzymes mutual cross i n h i b i t i o n was observed, UDPglucose i n h i b i t i n g TDP-glucose pyrophosphorylase and TDP-glucose i n h i b i t i n g UDP-glucose pyrophosphorylase (7). T h i s could perhaps be p r e d i c t e d , as l o s s of s y n t h e s i s of p o l y s a c c h a r i d e would lead to the accumulation of both g l u c o s e - c o n t a i n i n g sugar n u c l e o t i d e s . T h i s double c o n t r o l i s apparently r e s t r i c t e d to micro-organisms i n which polymers c o n t a i n i n g both sugars are found and i s absent from micro-organisms l a c k i n g rhamnose-containing p o l y s a c c h a r i d e s . S i m i l a r c o n t r o l mechanisms are found i n the formation of fucose as GDP-fucose from GDP-mannose. T h i s was s t u d i e d i n b a c t e r i a l species c o n t a i n i n g ( i ) D-mannose i n t h e i r p o l y s a c charides; ( i i ) c o n t a i n i n g L-fucose; and ( i i i ) c o n t a i n i n g both D-mannose and L-fucose ( 8 ) . In the f i r s t type, c o n t r o l of the r a t e of GDP-mannose s y n t h e s i s occurred through GDP-mannose pyrophosphorylase. In those b a c t e r i a i n which GDP-mannose i s s o l e l y a precursor i n fucose s y n t h e s i s , GDP-fucose c o n t r o l l e d both GDPmannose pyrophosphorylase and GDP-mannose hydrolyase through feedback i n h i b i t i o n . When both mannose and fucose are present i n polysaccharides produced by a s i n g l e bacterium, each sugar nucleot i d e c o n t r o l l e d i t s own s y n t h e s i s ( F i g . 4 ) . Xanthomonas campestris i s of p a r t i c u l a r i n t e r e s t because GDP-mannose and UDPglucose most probably both serve as precursors f o r l i p o p o l y s a c charide and exopolysaccharide. Further c o n t r o l o f the n u c l e o t i d e pool can occur through UDPsugar hydrolases (9,10), although, as these enzymes i n E. c o l i are

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

44

EXTRACELLULAR

MICROBIAL

POLYSACCHARIDES

p e r i p l a s m i c , they may not n e c e s s a r i l y have access to a l l the sugar n u c l e o t i d e formed by a c e l l but may be s p a t i a l l y separated from i t i n normal c e l l s . As s e v e r a l of the enzymes i n v o l v e d i n sugar n u c l e o t i d e synthesis are membrane-bound, i t i s by no means c l e a r whether t h e i r products occur f r e e l y w i t h i n the cytoplasm or whether they are produced i n c l o s e proximity to the enzymes which r e q u i r e them f o r polymer s y n t h e s i s . There i s a l s o the p o s s i b i l i t y of genetic r e g u l a t i o n of precursors s p e c i f i c to a p a r t i c u l a r polymer. The example of t h i s which has probably r e c e i v e d most study, through the work of Markovitz and h i s colleagues (11,12,13), i s c o l a n i c a c i d synthesis i n c e r t a i n b a c t e r i a of the Enterobacteriaceae. Knowledge of the s t r u c t u r e of c o l a n i c a c i c h a r i d e s , D-glucose and and to w a l l polymers and two others, L-fucose and D-glucuronic a c i d , unique to the polymer. C o n t r o l of the exopolysaccharide synthesis involved r e g u l a t o r genes; mutations i n these genes l e d to derepression and increased polysaccharide s y n t h e s i s . As a r e s u l t of the derepression, increased production of the three enzymes l e a d i n g to GDP-fucose synthesis and under the c o n t r o l of one r e g u l a t o r gene, was detected; increased formation of UDPglucose dehydrogenase ( r e s p o n s i b l e f o r conversion of UDP-glucose to UDP-glucuronic acid) occurred from mutation i n another r e g u l a t o r gene. As y e t , the concept of such r e g u l a t o r genes as those found i n c o l a n i c a c i d formation, dominant on episomes but r e c e s s i v e when located on the b a c t e r i a l chromosome, i s confined to a few s t r a i n s of E. c o l i , Salmonella e t c . One should not discount the p o s s i b i l i t y that polysaccharide production i n other genera and species i s under s i m i l a r genetic c o n t r o l , e s p e c i a l l y as so l i t t l e i s known about the genetic systems of most exopolysacchar ide-producing micro-organisms. Formation of Exopolysaccharide The c o n s t r u c t i o n of the r e p e a t i n g u n i t s of the polymer i s dependent on t r a n s f e r of the appropriate monosaccharides from sugar n u c l e o t i d e s to a c a r r i e r l i p i d i s o p r e n o i d a l c o h o l phosphate. The sequence of r e a c t i o n s has been w e l l c h a r a c t e r i z e d through i s o l a t i o n of the products at each t r a n s f e r step (16) and through i s o l a t i o n and i d e n t i f i c a t i o n of mutants (17) i n two Enterobacter aerogenes systems. The s e r i e s of r e a c t i o n s f o r the s t r a i n studied by Troy et ail. (16) was: UDP-Gal

+ P-lipid « = ±

+

UMP

Gal-P-P-lipid

+ GDP-Man

• Man-Gal-P-P-lipid

+

(GDP)

Man-Gal-P-P-lipid

+ UDP-GlcA

• GlcA-Man-Gal-P-P-lipid +

(UDP)

y Gal-Man-Gal-P-P-lipid

(UDP)

GlcA-Man-Gal-P-P-lipd + UDP-Gal

Gal-P-P-lipid

GlcA

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

+

SUTHERLAND

Microbial

Exopolysaccharide

Synthesis

f" — s Man - I - Ρ — - — - G D P - M a n ii)

Man- l-P J-GDP-Man

iii)

Man- I - P-^GDP-Man

- POLYMER

(-Man-)

n

GDP - Fuc - * P O L Y M E R ( - F u c - ) ^

-i

GDP - F u c — P O L Y M E R S (-Man-)

n

1 = GDP-mannos 2=

GDP-mannos

3= G D P - f u c o s e s y n t h e t a s e Figure 4. Control of mannose and fucose synthesis (after Kornfeld Gloser, 1966)

and

Ί

P Y R U V = ^ Gal 4 β GIcA 1 '3 Gal

3 \ β Glc 1

I

»· F u d

Figure 5

t

*4Fuc 1

Ac

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

n

46

EXTRACELLULAR

MICROBIAL

POLYSACCHARIDES

In the other s t r a i n studied (17), the f i r s t r e a c t i o n a l s o involved t r a n s f e r of a hexose-l-phosphate. The methods employed i n these studies l i m i t e d the s i z e of fragment which could be i d e n t i f i e d as being attached to the l i p i d . The l a r g e s t o l i g o s a c c h a r i d e charac t e r i z e d was an octasaccharide equivalent to two r e p e a t i n g u n i t s (16). The exact mechanisms involved i n f u r t h e r chain e l o n g a t i o n and e x t r u s i o n of exopolysaccharides i s s t i l l unknown. Recently, two of the enzymes involved i n Ε. aerogenes have been shown to be extremely l i p o p h i l i c p r o t e i n s e x t r a c t a b l e from membrane prepara t i o n s with a c i d butanol. In t h i s they resemble the i s o p r e n o i d a l c o h o l phosphokinase p u r i f i e d e a r l i e r from Staphylococcus aureus (18) and a s i m i l a r but not i d e n t i c a l p r o t e i n prepared from E. aerogenes (19,20). The s i t e of s y n t h e s i requirement f o r c a r r i e r l i p i d and a l s o f o r c e r t a i n of the sugar n u c l e o t i d e s , has been i d e n t i f i e d as the cytoplasmic membrane (21). P r e l i m i n a r y experiments i n our labo .ratory have shown that i t i s a l s o the s i t e of exopolysaccharide synthesis (Table 1). Attempts to p u r i f y the t r a n s f e r a s e enzymes by detergent s o l u b i l i z a t i o n were u n s u c c e s s f u l ; membrane p r o t e i n s were s o l u b i l i z e d but the procedure u s u a l l y l e d to p a r t i a l or complete i n a c t i v a t i o n . Although studies of t h i s k i n d have only been a p p l i e d to a l i m i t e d number of micro-organisms, the general mechanisms appear to be the same. In the synthesis of the phosphorylated mannan °f Hansenula capsulata, both mannose and phosphate were derived from GDP-mannose (22). Although i n t h i s p a r t i c u l a r study there was no attempt to demonstrate the involvement of l i p i d i n t e r mediates, they f u n c t i o n i n the formation of s i m i l a r polymers i n m i c r o b i a l w a l l s (23). As the enzyme preparations used i n these studies were crude membranes, nothing i s known about t h e i r r e g u l a t i o n , although i n a s e r i e s of non-polysaccharide-forming Ε. aerogenes mutants, the amount of t r a n s f e r a s e a c t i v i t y appeared to be lower than that found i n w i l d type b a c t e r i a (17). Isoprenoid L i p i d s i n Exopolysaccharide Synthesis The requirement f o r i s o p r e n o i d l i p i d s f o r exopolysaccharide synthesis i s a l s o common to other repeating u n i t - c o n t a i n i n g glycan polymers l o c a t e d e x t e r n a l to the c e l l membrane i . e . the same c a r r i e r l i p i d s are used f o r synthesis of peptidoglycan, t e i c h o i c a c i d s , l i p o p o l y saccharide and exopolysaccharides. Considerable i n d i r e c t evidence suggests that the a v a i l a b i l i t y of i s o p r e n o i d l i p i d phosphate i s one of the most c r i t i c a l f a c t o r s a f f e c t i n g exopoly saccharide synthesis (24). Any mutation a f f e c t i n g i s o p r e n o i d l i p i d synthesis w i l l thus a f f e c t exopolysaccharide production. Various authors have i n d i c a t e d that b a c t e r i a c o n t a i n 6.5-20 mg i s o p r e n o i d l i p i d % dry weight ( c a l c u l a t e d from r e s u l t s i n 25,26). I t has a l s o been suggested that i t s a v a i l a b i l i t y could be c o n t r o l led through phosphorylation of the f r e e a l c o h o l and dephosphory-

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

r~~

GO

U

Table 1.

Location of Sugar Transferase A c t i v i t i e s

72

Ο

68

3

Cytoplasmic

Outer membrane

membrane

81

lOO*

75

100*

Gal Transfer (%)

Spheroplast membrane

Crude membrane

G l c - l - P Transfer (%)

techniques.

* A c t i v i t i e s were of the order of 0.154 nmol/mg p r o t e i n / h and 0.282 nmol r e s p e c t i v e l y .

ci

η

£J£. aeroqenes type 8, G l c - l - P and Gal I + I I t r a n s f e r a s e s assayed by standard

^

is

CO

I

2. 8CO

Ci

ο "Ρ

Ci 3 ft

48

EXTRACELLULAR

MICROBIAL

POLYSACCHARIDES

l a t i o n of the a l c o h o l phosphate and pyrophosphate (27). Unfortu n a t e l y , Gram negative b a c t e r i a do not take up mevalonic a c i d and i t i s not p o s s i b l e to l a b e l the l i p i d precursors and thus o b t a i n more accurate e s t i m a t i o n of the amount present i n c e l l s than can be found from d i r e c t e x t r a c t i o n . However, one p o s s i b l e way of i n c r e a s i n g the i s o p r e n o i d l i p i d content appeared to be through s e l e c t i o n f o r b a c i t r a c i n r e s i s t a n c e , s i n c e t h i s a n t i b i o t i c binds very s t r o n g l y to i s o p r e n o i d l i p i d s and e f f e c t i v e l y removes them from b i o s y n t h e t i c processess. Mutants with c o n s i d e r a b l y elevated b a c i t r a c i n r e s i s t a n c e have been i s o l a t e d i n our l a b o r a t o r y and some undoubtedly y i e l d more exopolysaccharide and show increased t r a n s f e r of monsaccharides to l i p i d . (Other mutants were l i t t l e d i f f e r e n t from w i l d type i n a l l respects tested or had l o s t the a b i l i t y to synthesize exopolysaccharide. I t i s a l s o p o s s i b l e that some mutants d e f e c t i v e i n p e p t i d o glycan synthesis might r e q u i r e l e s s i s o p r e n o i d l i p i d than w i l d type c e l l s , thus r e l e a s i n g more f o r exopolysaccharide s y n t h e s i s . A mutant of t h i s type has r e c e n t l y been i s o l a t e d from E. c o l i Β and, u n l i k e the parent b a c t e r i a , produces exopolysaccharide (R.W. North, unpublished r e s u l t s ) . S i m i l a r observations have a l s o been reported during attempts to prepare mutants f o r genetic engineering. The reverse s i t u a t i o n , reduced i s o p r e n o i d l i p i d content, i s a l s o d i f f i c u l t to study and can only be checked i n d i r e c t l y . Mutants with l e s s l i p i d than w i l d type b a c t e r i a have not been c h a r a c t e r i z e d , but a group of CR (crenated) mutants i s o l a t e d from E. aerogenes have c h a r a c t e r i s t i c s which i n d i c a t e that they may be c o n d i t i o n a l mutants of t h i s type (28). These b a c t e r i a have rough c o l o n i a l appearance at lowered i n c u b a t i o n temperature and t h i s has been a s c r i b e d to a reduced content of l i p o p o l y s a c c h a r i d e . Exopolysaccharide i s not synthesized u n t i l growth has ceased. The enzymes f o r p o l y s a c c h a r i d e synthesis are present i n the b a c t e r i a grown a t low temperature and on t r a n s f e r to washed c e l l suspensions ( n o n - p r o l i f e r a t i n g c o n d i t i o n s ) exopolysaccharide i s immediately formed i n the presence or absence of chloramphenical. Thus no new enzymes have to be formed but at low temperature the synthesis of peptidoglycan - e s s e n t i a l f o r c e l l v i a b i l i t y appears to take precedence over exopolysaccharide production and, to a l e s s e r extent l i p o p o l y s a c c h a r i d e s y n t h e s i s . At 37°C, the mutants are i d e n t i c a l i n a l l respects t e s t e d to w i l d type b a c t e r i a . The mutants are not l i k e c l a s s i c a l membrane mutants, d e f i c i e n t i n membrane p h o s p h o l i p i d and s u s c e p t i b l e to various detergents. S i m i l a r c h a r a c t e r i s t i c s were observed i n a polysaccharide-forming pseudomonad (29). The e x t r a c e l l u l a r polymer was only produced l a t e i n the l o g phase of growth and i n the s t a t i o n a r y phase, having s e v e r a l of the a t t r i b u t e s of a secondary m e t a b o l i t e . Could t h i s too be due to i n s u f f i c i e n t i s o p r e n o i d l i p i d i n the growing and peptidoglycan-forming b a c t e r i a ?

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

4.

SUTHERLAND

Microbial

Exopolysaccharide

Synthesis

49

In the l i t e r a t u r e , frequent r e p o r t s of exopolysaccharide production being favoured by growth a t low temperature a r e to be found; a l t e r n a t i v e l y the polymer i s s a i d to be a product of the c e l l s a f t e r growth has ceased. No s a t i s f a c t o r y explanation f o r these observations has been provided, y e t b a c t e r i a from the l o g or e a r l y s t a t i o n a r y phases of growth appear to produce exopolysaccharide i n washed suspension a t s i m i l a r r a t e s . T h i s could be due to l i m i t a t i o n of exopolysaccharide synthesis during a c t i v e growth through the a v a i l a b i l i t y of i s o p r e n o i d l i p i d ; i t would be needed f o r the formation of w a l l polymers u n t i l l a t e i n the l o g phase of growth. L i m i t a t i o n of c a r r i e r l i p i d a l s o occurs i n c e r t a i n Salmonella mutants d e f e c t i v e i n l i p o p o l y s a c c h a r i d e formation. Mutants forming the l i p i d - l i n k e d O-antigen but unable to t r a n s f e r i t to the appropriate accepto p a r t of the normal i s o p r e n o i other processess, e f f e c t i v e l y reducing the t o t a l present i n the bacteria. Mutants of t h i s type could not produce exopolysaccharide although others d e f e c t i v e i n l i p o p o l y s a c c h a r i d e synthesis but not accumulating l i p i d - l i n k e d glycans, had t h i s c a p a c i t y (31). Several d i f f e r e n t types of mutations can thus a f f e c t i s o p r e noid l i p i d a v a i l a b i l i t y and consequently exopolysaccharide production. These are summarized i n F i g . 6. The i n d i r e c t evidence suggests a d i s t i n c t s e r i e s of p r i o r i t i e s f o r i s o p r e n o i d l i p i d utilization. The e s s e n t i a l w a l l polymer peptidoglycan has p r i o r i t y over l i p o p o l y s a c c h a r i d e which i n turn has p r i o r i t y over exopolysaccharide synthesis ( F i g s . 7 and 8 ) . T h i s could to some extent be achieved through s p a t i a l s e p a r a t i o n of the polysaccharide s y n t h e s i z i n g systems w i t h i n the m i c r o b i a l membrane but obviously requires further elucidation. The f i n a l stages - m o d i f i c a t i o n and e x t r u s i o n As already discussed, the o l i g o s a c c h a r i d e r e p e a t i n g u n i t s accumulate on the c a r r i e r l i p i d and t h i s type of mechanism probably a p p l i e s to a l l exopolysaccharides other than dextrans, levans and r e l a t e d polymers (24). The mechanism could accommodate b a c t e r i a l a l g i n a t e synthesis i f i t i s regarded i n i t i a l l y as a homopolymer of Dmannuronic a c i d and i s probably a l s o v a l i d f o r the glucans secreted by Agrobacterium species. However, many exopolysaccharides c o n t a i n a c y l and k e t a l s u b s t i t u e n t s . Are these added while the repeating u n i t s are attached to l i p i d or at some l a t e r stage? (Fig.9). P r e l i m i n a r y evidence suggests that a c y l a t i o n occurs while the o l i g o s a c c h a r i d e i s s t i l l attached to the l i p i d , but f u r t h e r s t u d i e s are needed. This might i n d i c a t e the lower degree of p y r u v y l a t i o n o c c u r r i n g i n polysaccharide produced a t higher growth r a t e s (and higher r e s u l t a n t l i p i d turnover rates) reported i n some s p e c i e s . The carbon source probably has no d i r e c t e f f e c t (Table 2). Considerable v a r i a t i o n s i n a c y l a t i o n are found w i t h i n a s i n g l e polysaccharide. Thus, a c e t y l groups may occur on each r e p e a t i n g u n i t or on every second repeating u n i t i n one E. aero gene s

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

50

EXTRACELLULAR

MICROBIAL

POLYSACCHARIDES

Wild-type bacteria (lipopolysaccharides, capsules, slime)

/ i

CR mutants* (decreased lipopolysaccharides, no slime or capsule until growth ceases)

SL mutants (lipopolysaccharides, slime)

\ Ο mutants (lipopolysaccharides)

« • Bacitracin-resistance

CRO mutants (decreased lipopolysaccharides)

SR mutants (1 repeat unit of lipopolysaccharid

R mutants* (core lipopolysaccharides, side chains unattached)

R mutants (inner core only)

* Mutations affecting isoprenoid lipidsfdirectly or indirectly) Biochemical Society Transactions

Figure 6.

How mutations affect the production of exopolysaccharides (31)

\

GROWING

CELLS

E N D OF L O G P H A S E -

Figure 7.

EXOPOLYSACCHARIDE

OR L O W I N C U B A T I O N

TEMPERATURE

Carrier lipid utilization

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

Microbial

SUTHERLAND

Exopolysaccharide

IPP-

C 5 5 - ISOPRENYL

IPA -

C 5 5 - ISOPRENOID ALCOHOL

PYROPHOSPHATE

ISOPENTENYL

1 1

IP-

PYROPHOSPHATE +

51

Synthesis

C - I S O P R E N Y L PHOSPHATE 5 5

FARNESYL

PYROPHOSPHATE

POLYMER -

MUCOPEPTIDE LPS orTEICHOIC ACID EXOPOLYSACCHARIDE

INTRACELLULAR and MEMBRANE - BOUND PRECURSORS

IPA

Figure 8.

Regulation of carrier lipids

L I P I D - P - P - GIc-GIc I Man I GIcA I Man • A c e t y l CoA

[GIC- Glc] I Man I GIcA I Man

n

OR

• A c e t y l CoA

+ PEP

I

• PEP

r

1

L I P I D - P - P - G I c - GIc [Glc-GlcJ I I Man-O-Ac Man-O-Ac I I GIcA GIcA I I Man = Pyr Man = Pyr Figure 9. Possible exopolysaccharide acylation mechanisms n

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977. 1.92 1.74

5.48

3.61 3.67

14.5

72.8

Galactose

17.1

79.4

78.8

Sodium pyruvate

Sodium succinate

18.5

18.0

74.0

Raffinose

3.40

6.10 6.22

3.46 3.52

1.30

1.55

0.60

6.96

3.90

2.65 2.87

6.28 5. 81

3.75

16.9

73.6

Sucrose

Maltose

4.03

17.4

79.7

Lactose

1.89

3.78

5.20

5.36

3.16

16.7

75.7

17.9

72.1

Ribose

1.14 5. 70

3.46

16.5

70. Ο

Arabinose

8.75

3.36

0.43

2.20

6.46

3.84

18.2

74.4

Rhamnose

15.8

67.3

Xylose

3.39

6.64

Fructose

3.37

18.5 15.8

74.0

69.4

Mannose

3.23

5. 55 6.32

3.52

15.2

Exopolys acc har ide mg/ml

75. Ο

Carbon Source

Glucose

of

Pyruvic A c i d %

r

Acetate %

R e s u l t s

of a Pseudomonas Exopolysaccharide Derived from Growth on Various Substrates ^ Williams, 1974)

Deoxyhexose %

The Composition

Hexose %

Table 2.

Ω Ω

>

F *d Ο F *

>

W

ο

§

M F F

> Ω

αϊ to

4.

SUTHERLAND

Microbial

Exopolysaccharide

Synthesis

53

s t r a i n (32). I t has a l s o been demonstrated that a c e t y l a t i o n can be l o s t from a s t r a i n without l o s s of exopolysaccharides y n t h e s i z i n g capacity (33) . In c o n t r a s t , loss of any enzyme c o n t r i b u t i n g to the polysaccharide s t r u c t u r e would lead to a non-mucoid v a r i a n t . S i m i l a r l y , p y r u v y l a t i o n a l s o appears i n e s s e n t i a l f o r polysaccharide synthesis as, under c e r t a i n growth c o n d i t i o n s pyruvate groups can be l o s t but polysaccharide of apparently normal carbohydrate composition produced. The exopolysaccharides studied so f a r , have mainly comprised repeating u n i t s with a s i n g l e attached monosaccharide s i d e - c h a i n . I t i s p o s s i b l e that c o n s t r u c t i o n of the longer s i d e chains- found i n xanthan gum or c o l a n i c a c i d might r e q u i r e some other mechanism such as c o n s t r u c t i o n o separate c a r r i e r l i p i d found i n l y s o g e n i c conversion, 34.) The mode of f i n a l r e l e a s e from the i s o p r e n o i d l i p i d has not yet been demonstrated. I t i s u n l i k e l y that the process occurs through non-enzymic r e l e a s e of the i n c r e a s i n g l y h y d r o p h i l i c elongating polysaccharide chain. T h i s would probably leave the c a r r i e r l i p i d u n a v a i l a b l e f o r f u r t h e r polysaccharide s y n t h e s i s . In capsuleproducing s t r a i n s , a l i g a s e r e a c t i o n may remove the polymer chain and attach i t to the c e l l s u r f a c e . It is unlikely that h y d r o l y s i s of the polysaccharide chain occurs at t h i s stage unless a h i g h l y s p e c i f i c enzyme cleaves the t e r m i n a l , phosphate l i n k e d monosaccharide: L i p i d - Ρ - Ρ - Glucose - Galactose

etc.

Enzymes reducing the degree of polymerization have been i d e n t i f i e d i n alginate-producing b a c t e r i a (35) but the f u n c t i o n of the enzyme i s probably unconnected with polymer r e l e a s e of t h i s type. Mutants unable to attach to the c e l l surface (SI mutants) have been widely found, presumably through l o s s of the capsule a t t a c h ment s i t e s on the c e l l s u r f a c e ; other micro-organisms always produce exopolysaccharide as e x t r a c e l l u l a r s l i m e . The chain length of the polymer may a l s o depend on the growth rate i n a manner analogous to l i p o p o l y s a c c h a r i d e side-chains (36), but t h i s needs f u r t h e r study. Higher growth r a t e might lead to more r a p i d turnover of the c a r r i e r l i p i d and r e l e a s e of polymer of lower molecular weight. This i s obviously important to the commercial producer. I t may a l s o be advantageous to use rough mutants ( i . e . s t r a i n s with surface defects) which autoagglutinate of f l o c c u l a t e and lead to e a s i e r polymer recovery. Thus exopoly saccharide production should be examined along with the synthesis of other polysaccharides and not i n i s o l a t i o n .

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

EXTRACELLULAR

54

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

Hexokinase Phosphoglucomutase Phosphoglucose Isomerase Phosphomannose Isomerase Phosphomannomutase UDP-Glc Pyrophosphorylase GDP-Man Pyrophosphorylase UDP - Gal Epimerase UDP-Glc Dehydrogenase Glc Transferase I Glc Transferase 1 Man Transferase I Man Transferase Π GIcA Transferase Polymerase (s) Ketalase Acetylase Figure 10.

MICROBIAL

POLYSACCHARIDES

- [Glc - Glc] | Man - Ο - Ac QJ ^ J_ _ ipc Man « ryr ^ ^ ^* / \ \ • ' / _ \ \ " GDP-Man UDP - Glc A « j UDP- Glc ^ UDP-Gal F7 T* Man-I-P Glc-l-P N

C

%

Î5

Man-6-P

T*

Glc-6-P

Fruct J3

Biosynthesis of Xanthomonas polysaccharides

REACTION

CONTROL

Substrate

Substrate entry

Membrane CYTOPLASM

Hexose-phosphate

Hexose-phosphate level

X D P - hexose

XDP-hexose pyrophosphorylase X D P - hexose hydrolase

i

Lipid - Ρ - Ρ - hexose Isoprenoid lipid availability Lipid-P - Ρ - oligosaccharide

Ψ Polysaccharide Figure 11.

?

The control of ρ dysaccharide synthesis

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

4.

SUTHERLAND

Microbial

Exopolysaccharide

Synthesis

55

Summary The production of m i c r o b i a l exopolysaccharides i n v o l v e s a r e l a t i v e l y l a r g e number of enzymes, some of which are i n v o l v e d i n the formation of other polysaccharides while others are unique to exopolysaccharide s y n t h e s i s . By e x t r a p o l a t i o n from r e s u l t s obtained with other s p e c i e s , a b i o s y n t h e t i c pathway f o r X. campestris polysaccharide can be constructed ( F i g . 10). Loss of most of these enzymes leads to l o s s o f polysaccharide product i o n , but v a r i a t i o n s i n a c y l a t i o n or k e t a l a t i o n occur and may be of importance to the i n d u s t r i a l m i c r o b i o l o g i s t . C o n t r o l of p o l y s a c c h a r i d e synthesis probably occurs at a number of l e v e l s ( F i g . 11), and some mutations with a l t e r e d p o l y s a c c h a r i d e r e g u l a t i o n may have advantageou

Literature Cited 1. Gibbons, R.J. and Nygaard, M. Arch. oral Biol., 13, 12491249 (1968). 2. Smith, E.E.

FEBS Letters, 12, 33-37 (1970).

3. Herbert, D. andKornberg,H.L. Biochem. J., 156, 477-480 (1976). 4. Roseman, S. In'MetabolicPathways'Ed. Hokin, L.E., 6, 41-89 (1972). Academic Press, London and New York. 5. Preiss, J . In'CurrentTopics in Cellular Regulation' 1, pp. 125-160 (1969). 6. Grant, W.D., Sutherland, I.W. and Wilkinson, J.F. J . Bact., 103, 89-96 (1970). 7. Bernstein, R.L. and Robbins, P.W. J . Biol. Chem., 240, 391-397 (1965). 8. Kornfeld, R.H. and Ginsburg, V. Biochim. Biophys. Acta, 117, 79-87 (1966). 9.

Ward, J.B. and Glaser, L. Biochem. Biophys. Res. Commun., 31, 671-6 (1968).

10. Ward, J.B. and Glaser, L. Arch. Biochem., 134, 612-622 (1969). 11.

Lieberman, M.M. and Markovitz, A. J . Bact., 101, 965-972 (1970).

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

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12.

EXTRACELLULAR

MICROBIAL

POLYSACCHARIDES

Lieberman, M.M., Shaparis, A. and Markovitz, A. J. Bact., 101, 959-964 (1970).

13. Markovitz, A. In 'Surface Carbohydrates of Prokaryotes', Ed. Sutherland, I.W., Academic Press, London and New York (In press). 14.

Lawson, C.J., McCleary, C.W., Nakada, H.I., Rees, D.A., Sutherland, I.W. and Wilkinson, J.F. Biochem. J., 115, 947-958 (1969).

15.

Sutherland, I.W. Biochem. J., 115, 935-945 (1969).

16.

Troy, F.A., Frerman F.A 246, 118-133

17.

Sutherland, I.W. and Norval, M. Biochem. J., 120, 567-576 (1970).

18.

Sandermann, H. and Strominger, J.L. J . Biol. Chem., 247, 5123-5131 (1972).

19.

Poxton, I.R., Lomax, J.A. and Sutherland, I.W. J . Gen. Microbiol., 84, 231-233 (1974).

20.

Lomax, J.Α., Poxton, I.R. and Sutherland, I.W. FEBS Letters 34, 232-234 (1973).

21.

Osborn, M.J., Gander, J.E. and Parisi, E. J . Biol. Chem.,

d Heath E.C

J Biol Chem.

247, 3973-3986 (1972). 22.

Mayer, R.M.

23.

Lennarz, W.J. and Scher, M.G. Biochim. Biophys Acta, 265, 417-441 (1972). Sutherland, I.W. In"SurfaceCarbohydrates of Prokaryotes", pp. - , Academic Press, London and New York (In press).

24.

Bio chim. Biophys. Acta, 252, 39-47 (1971).

25.

Umbreit, J.N., Stone, K.J. and Strominger, J.L. J . Bacteriol. 112, 1302-1305 (1972).

26.

Dankert, M., Wright, Α., Kelley, W.S. and Robbins, P.W. Arch. Biochem., 116, 425-435 (1966).

27.

Willoughby, E . , Higashi, Y. and Strominger, J.L. J . Biol. Chem.,247, 5113-5115 (1972).

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

4.

28.

SUTHERLAND

Microbial

Exopolysaccharide

Synthesis

57

Norval, M. and Sutherland, I.W. J . Gen. Microbiol., 57, 369-377 (1969)

29.

Williams, A. Ph.D. Thesis, University College, Cardiff. (1974)

30.

Kent, J.L. and Osborn, M.J. Biochemistry, 7, 4396-4408 (1968). 31. Sutherland, I.W. Biochem. Soc. Trans., 3, 840-843 (1975). 32. Sutherland, I.W. In "Surface Carbohydrates of Prokaryotes", pp. - , Academic Press, London and New York, (In press). 33.

Garegg, P.J., Lindberg Scand., 25, 1185-1194 (1971).

34.

Wright, A. J . Bacteriol., 105, 927-936 (1971).

35.

Madgwick, J., Haug, A. and Larsen, B. Acta Chem. Scand., 27, 711-712 (1973). Collins, F.M. Aust. J . Exp. Biol. Med., 42, 255- 2 (1964).

36.

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

5 Polysaccharide Formation by a Methylomonas ΚΑΙ T. ΤΑΜ and R. K. FINN School of Chemical Engineering, Cornell University, Ithaca, NY 14853

Extracellular microbial polysaccharides show great diversity as well as novelty in thei applications of some o fiers, or thickeners in foods; as additives for recovery of pe troleum by water flooding; as plasma extenders or as selective adsorbents in laboratory research, are well documented (2,3,4). A new polysaccharide-producing bacterium called Methylomonas mucosa NRRL B-5696, was isolated from s o i l as an obligate methylotroph and the batch production of polymer and some of its prop erties have been described (5,6). Kinetics for growth of the cells and for polymer production in shake flasks and chemostats are reported here. Materials and Methods The bacteria were maintained on agar plates with a 3% (v/v) methanol basal medium which contained 3.0 g ΚΗ ΡO , 3.7 g Na HPO , 2.5 g NaNO , 0.4 g MgSO ·7 H O, 0.07 g Fe (NH SO ) , 0.025 g Ca (NO ) ·4 H O, 0.001 g ZnSO ·H O, in one liter of d i s t i l l e d water. Methanol concentration was determined by a gas chromatograph with a flame ionization detector using ethanol as the internal standard. Cell dry weight was calibrated against a modified Lowry's protein assay (7), and the latter was used for routine measurements. Polysaccharide concentration was expressed as glu cose equivalent by the phenol-sulfuric acid method of Dubios et. al. (8) with D-glucose as standard. Effluent gas composition was analyzed by a Fisher-Hamilton gas partitioner, model 29, using helium as a carrier gas. Dissolved oxygen measurements were made with membrane probes constructed as described by Johnson and Borkowski (9, 10). Polymer was recovered by acetone precipita tion (11). Viscosity was measured in a Brookfield SynchroLectric Viscometer, model LVT with U. L. adaptor. Fermenter broths diluted in the range 1:5 to 1:10, were f i r s t degassed in vacuum, and then viscosity measurements for each dilution were made at various shearing rates. In some cases, viscosity of the 2

3

3 2

2

4

2

4

4

4

2

4 2

2

58

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

4

5.

ΤΑΜ

Polysaccharide

A N D FINN

Formation

by a Methylomonas

59

f i n a l b r o t h was d e t e r m i n e d a t a s h e a r i n g r a t e o f 30 RPM w i t h a No. 3 s p i n d l e u s i n g 150 m l o f b r o t h c o n t a i n e d i n a 200 m l b e a k e r . M e t h a n o l i s a t o x i c s u b s t r a t e f o r b a c t e r i a ; e v e n f o r metha n o l u t i l i z i n g o r g a n i s m s a c o n c e n t r a t i o n b e l o w l.O/ may i n h i b i t t h e g r o w t h o f many s t r a i n s (12,13). T h e r e f o r e t h e e f f e c t o f meth a n o l c o n c e n t r a t i o n o n t h e g r o w t h o f M. mucosa was s t u d i e d i n shake f l a s k s . To do t h i s , 250 m l p o r t i o n s o f l o w p h o s p h a t e m e d i um ( b a s a l medium b u t w i t h o n l y h a l f t h e amount o f p h o s p h a t e ) i n 1 - l i t e r i n d e n t e d f l a s k s were i n o c u l a t e d w i t h s e e d f r o m a chemos t a t o p e r a t i n g a t a d i l u t i o n r a t e o f 0.25 h r " a n d a t a s t e a d y s t a t e e f f l u e n t m e t h a n o l c o n c e n t r a t i o n o f 1.0 v/v$>. M e t h a n o l c o n c e n t r a t i o n s i n t h e r a n g e 0.1k t o 2.0/o ( v / v ) were i n v e s t i g a t e d . S p e c i f i c g r o w t h r a t e s a t 30°C a n d 350 RPM r o t a t i o n o f t h e s h a k e r i n c u b a t o r were d e t e r m i n e d i n t h e t i m e p e r i o d when t h e maximum change i n t h e m e t h a n o l i n i t i a l value i n the f l a s k t i o n o f m e t h a n o l c o n c e n t r a t i o n was t h e n p l o t t e d . 0

1

R e s u l t s and

Discussion

K i n e t i c s o f Growth. The e x p o n e n t i a l g r o w t h d a t a f r o m shake f l a s k s i n d i c a t e t h a t m e t h a n o l c o n c e n t r a t i o n s above 1$ v / v a r e i n h i b i t o r y ( F i g u r e 1). F u r t h e r m o r e , a L i n e w e a v e r - B u r k p l o t shows t h a t a t c o n c e n t r a t i o n s l e s s t h a n 1$ t h e d a t a f i t a Monod m o d e l f o r s u b s t r a t e - l i m i t e d g r o w t h . The e x t r a p o l a t e d m a x i m a l s p e c i f i c g r o w t h r a t e , \x ., f r o m F i g u r e 2 i s 0.725 h r " , ( e q u i v a l e n t t o a g e n e r a t i o n t i m e o f 0.956 h r ) . T h i s i s a b o u t 3 t i m e s h i g h e r t h a n t h e a v e r a g e v a l u e f o r most o f t h e m e t h a n o l u t i l i z i n g b a c t e r i a r e p o r t e d i n t h e l i t e r a t u r e (11), a n d i s a b o u t t w i c e t h a t o f P s e u domonad C ( ώ ) , t h e f a s t e s t g r o w i n g m e t h a n o l b a c t e r i a r e p o r t e d . S u c h a f a s t g r o w t h r a t e makes M. mucosa a t t r a c t i v e a s a n o t h e r bacterium f o r s i n g l e - c e l l p r o t e i n production. The h i g h s p e c i f i c g r o w t h r a t e o b s e r v e d i n s h a k e f l a s k s was l a t e r c o n f i r m e d b y a c e l l w a s h o u t e x p e r i m e n t i n a c h e m o s t a t , where t h e m a x i m a l s p e c i f i c g r o w t h r a t e was m e a s u r e d a s 0.719 h r " . The o t h e r k i n e t i c c o n s t a n t , Ks i n t h e Monod m o d e l , was f o u n d t o b e 0.20 M m e t h a n o l . T h i s v a l u e i s two o r d e r s o f m a g n i t u d e l a r g e r t h a n t h e v a l u e o f 0.00375M (120 m g / l ) r e p o r t e d f o r H a n s e n u l a p o l y m o r p h a - a t h e r m o p h i l i c m e t h a n o l - u t i i i z i n g y e a s t whose g r o w t h k i n e t i c s a l s o f i t t h e Monod m o d e l (15). Recent s t u d i e s on the growth o f Candida b o i d i n i i , another m e t h a n o l - u t i l i z i n g yeast, show a K v a l u e a s h i g h a s 0.02M ( l 6 ) . No o t h e r l i t e r a t u r e v a l u e s o f K s f o r m e t h a n o l - a s s i m i l a t i n g b a c t e r i a a r e a v a i l a b l e f o r compar ison. The v a l u e o f K o b t a i n e d i s a l s o much h i g h e r t h a n v a l u e s o b t a i n e d f o r m i c r o b i a l g r o w t h o n o t h e r c a r b o n s o u r c e s , w h i c h gen e r a l l y r a n g e f r o m 1 t o 50 m g / l (17). S i n c e M. mucosa i s sub c u l t u r e d i n yjo m e t h a n o l - s a l t s medium w h i c h i s i n h i b i t o r y f o r most o t h e r m e t h a n o l - a s s i m i l a t i n g b a c t e r i a , t h e b a c t e r i u m must h a v e d e v e l o p e d a t r a n s p o r t mechanism t h a t r e g u l a t e s a slow p e r m e a t i o n of substrate i n t o the c e l l i n order t o reduce the i n h i b i t o r y 1

m

1

s

s

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

60

EXTRACELLULAR

MICROBIAL

POLYSACCHARIDES

Figure 1. Specific growth rate at different initial sub strate concentrations

Figure 2. Lineweaver-Burk plot for the specific growth rate data

-L

( % Μ · ! Η α η · Ι )r l

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

5.

ΤΑΜ

A N D FINN

Polysaccharide

Formation

by a Methylomonas

61

e f f e c t o f t h e methanol. A l s o t h e p o l y s a c c h a r i d e s l i m e i s an a d d i t i o n a l b a r r i e r f o r t h e d i f f u s i o n o f m e t h a n o l i n t o t h e c e l l . The h i g h K i m p l i e s t h a t a low o v e r a l l a f f i n i t y f o r methanol should be e x p e c t e d . The good agreement o f t h e e x t r a p o l a t e d μ w i t h t h e w a s h o u t datum adds c o n f i d e n c e t o t h e a c c u r a c y o f t h e k i n e t i c c o n stants. The i m p l i c a t i o n o f s u c h a h i g h v a l u e f o r K g i s t h a t a s t a b l e r e a c t o r c a n be o p e r a t e d a t a d o u b l i n g t i m e as s h o r t as 1.8 h r f o r M. mucosa i n a c a r b o n - l i m i t e d c h e m o s t a t . F i g u r e 3 shows t h a t d a t a f o r t h e s u b s t r a t e - i n h i b i t o r y r e g i o n f i t t h e model, s

ηι

μ

where

μ

=

1 + S/K.

(

1

)

= 6.05 h r "

Κ. = 1 8 Λ m M o l a r ι The f a c t t h a t d a t a f i t t h e t w o - p a r a m e t e r m o d e l s does n o t t e l l u s t h e e x a c t mechanism o f i n h i b i t i o n o r growth s t i m u l a t i o n a t t h e molecular l e v e l . However, n o f u r t h e r e x p e r i m e n t s were done t o e l u c i d a t e t h e mechanism o r s i t e o f i n h i b i t i o n b e c a u s e t h e p r i m e o b j e c t i v e o f d e t e r m i n i n g t h e safe o p e r a t i o n range f o r a carbonl i m i t e d c h e m o s t a t was o b t a i n e d i n t h i s s e t o f e x p e r i m e n t s . Respiration Kinetics. From t h e d e p l e t i o n r a t e o f d i s s o l v e d o x y g e n a n d a n a v e r a g e c e l l mass o f 0.152 mg i n t h e Y e l l o w S p r i n g s D i s s o l v e d Oxygen m o n i t o r i n g chambers, t h e s p e c i f i c r e s p i r a t i o n r a t e s were c a l c u l a t e d f o r d i f f e r e n t i n i t i a l m e t h a n o l c o n c e n t r a tions. I n t h e absence o f s u b s t r a t e i n h i b i t i o n , M i c h a e l i s - M e n t e n k i n e t i c s f i t t h e r e s p i r a t i o n r a t e d a t a as i n d i c a t e d b y t h e s t r a i g h t l i n e i nt h e Lineweaver-Burk p l o t (Figure 4). The c e l l s demonstrate a h i g h a f f i n i t y f o r m e t h a n o l as s u g g e s t e d b y t h e l o w v a l u e o f Κ , 8.1 pmolar methanol. The maximum r e s p i r a t i o n r a t e ( e x t r a p o l a t e d ) i s 33 mMole 0 / ( g c e l l , h r ) , w h i c h i s s l i g h t l y h i g h e r t h a n t h e a v e r a g e v a l u e o f 2 6 . 6 mMole 0 / ( g , h r ) o b t a i n e d from an oxygen balance i n t h e chemostat o p e r a t i n g w i t h a s t e a d y s t a t e e f f l u e n t m e t h a n o l c o n c e n t r a t i o n b e t w e e n 0.6$ a n d 1.5$ (ν/ )· The l o w e r c h e m o s t a t v a l u e o f V m i g h t be due t o s u b s t r a t e i n h i b i tion. Compared w i t h l i t e r a t u r e v a l u e s ( T a b l e I ) , M. mucosa h a s a K i n t h e same o r d e r o f m a g n i t u d e a s H y p h o m i c r o b i u m . The m a x i m a l s p e c i f i c r e s p i r a t i o n r a t e i s about t w i c e t h e h i g h e s t r a t e l i s t e d i n t h e Table. A h i g h e r r e s p i r a t i o n r a t e i s e x p e c t e d f o r M. mucosa because o f i t s v e r y h i g h s p e c i f i c growth r a t e . Another piece o f evidence t h a t agrees w i t h t h e e x t r a p o l a t e d s p e c i f i c r e s p i r a t i o n r a t e comes f r o m s e p a r a t e b a t c h e x p e r i m e n t s . A t t h e p o i n t when t h e d i s s o l v e d o x y g e n r e a c h e s z e r o , t h e o x y g e n demand c a l c u l a t e d f r o m t h e s p e c i f i c r e s p i r a t i o n r a t e s h o u l d j u s t equal t h e o x y g e n 2

2

ν

m

r

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

EXTRACELLULAR

MICROBIAL

POLYSACCHARIDES

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

5.

ΤΑΜ

A N D FINN

Polysaccharide

Formation

by a Methylomonas

63

supplied. The o x y g e n s u p p l y , b a s e d o n g a s c h r o m a t o g r a p h i c a n a l y s i s o f t h e i n f l u e n t a n d e f f l u e n t g a s , was k6k0 mg 0 / h r , a n d t h e p r e d i c t e d o x y g e n demand b a s e d o n t h e above r e s p i r o m e t e r d a t a ( 33 mMole 0 p e r g c e l l p e r h r ) was 4780 mg Cg/hr. The endogeneous r e s p i r a t i o n r a t e i n m e t h a n o l - f r e e medium i s 1.21 ± 0.05 mMole 0 / ( g c e l l , h r ) w h i c h a g r e e s w i t h t h e a v e r a g e endogeneous r a t e o f 1.3 ± 0.3 mMole 0 / ( g c e l l , h r ) o b t a i n e d i n t h e Y e l l o w S p r i n g s D i s s o l v e d - O x y g e n M o n i t o r Chamber, u s i n g c e l l s grown i n d i f f e r e n t shake f l a s k s . 2

2

2

2

Table I Michaelis-Menten K i n e t i c Constants f o r the R e s p i r a t i o n o f B a c t e r i a Growing i n Methanol Reference Harrison

(l8)

Harrison

(l8)

Pseudomonas e x t o r q u e n s

20Λ

methane u t i l i z i n g

50.0

10.5 4.15

Pseudomonad Wilkinson

(19)

Hyphomi c r o b i u m

Wilkinson

(19)

mixed c u l t u r e

T h i s work Kim

& R y u (20)

8.53 29400

M e t h y l o m o n a s mucosa

8.1

0.0215 0.024 33.0 18.0

Methylomonas sp.

C a r b o n - l i m i t e d Chemostat. The s t r a i g h t l i n e i n t h e L i n e weaver-Burk p l o t f o r t h e s p e c i f i c growth r a t e i n t h e chemostat, w i t h m e t h a n o l as t h e l i m i t i n g s u b s t r a t e , s u g g e s t s t h a t Monod-type growth k i n e t i c s f i t t h e data ( F i g u r e 5).

s = 1.43 h r

where Κ

.1

=0.583 Molar

However, t h e s e c o n s t a n t s do n o t a g r e e w i t h t h e s h a k e f l a s k d a t a , where ^ = 0.725 h r " , a n d K = 0.20 M o l a r . Such a d i s c r e p a n c y i s t o o l a r g e t o be e x p l a i n e d b y t h e s h o r t - c i r c u i t o f t h e f l o w o r o t h e r e x p e r i m e n t a l e r r o r s . The o n l y l o g i c a l e x p l a n a t i o n i s t h a t b y c o n t i n u o u s l y c u l t i v a t i n g M. mucosa i n t h e c h e m o s t a t f o r o v e r a week, some v a r i a n t was s e l e c t e d t h a t h a d a l o w e r a f f i n i t y f o r methanol and a f a s t e r growing r a t e . When t h e s p e c i f i c m e t h a n o l u t i l i z a t i o n r a t e (Q^) i s p l o t t e d a g a i n s t d i l u t i o n r a t e (D), a s t r a i g h t l i n e i s o b t a i n e d ( F i g u r e 6) 1

s

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

Figure 6.

Specific substrate utilization rate correlation

0.1

0.2

Dilution

0J

Rat* ,

1

Hr*

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

5.

Polysaccharide

Τ Α Μ A N D FINN

Formation

by a

65

Methylomonas

This r e s u l t confirms t h e v a l i d i t y o f the e m p i r i c a l equation b y P i r t (17) a n d N a g a i e t a l . (21 ) :

used

(3) be

F o r s t e a d y - s t a t e i n a chemostat w i t h no r e c y c l e , e q u a t i o n comes

x/s where

m = maintenance c o e f f i c i e n t f o r methanol = 0.26 g methanol/( Υ ^ = 0.3*1-5 g c e l l / χ

3

The e x t r a p o l a t e d m i s t h e same o r d e r o f m a g n i t u d e as t h a t r e p o r t e d f o r A. v i n e l a n d i i (22 ), b u t a n o r d e r o f m a g n i t u d e h i g h e r t h a n t h a t f o r other microorganisms (Table II). U n l i k e most o f t h e m i c r o o r g a i s m s l i s t e d , ( w i t h t h e e x c e p t i o n o f A. v i n e l a n d i i w h i c h f o r m s p o l y - b e t a - h y d r o x y b u t y r i c a c i d ) M. mucosa p r o d u c e s e x t r a c e l l u l a r polysaccharides i n a d d i t i o n t o c e l l t i s s u e s and carbon d i o x i d e . The f o r m a t i o n o f e x t r a s t o r a g e p r o d u c t o r p o l y m e r r e q u i r e s more carbon uptake, and t h e r e f o r e a h i g h e r value o f t h e c e l l mainte n a n c e c o e f f i c i e n t s h o u l d b e e x p e c t e d f o r A. v i n e l a n d i i a n d M. mucosa a s i n d i c a t e d i n T a b l e 2. The e x p e r i m e n t a l y i e l d c o e f f i c i e n t Y / = 0.3^5 g c e l l / g CH3OH i s q u i t e r e a s o n a b l e , b e c a u s e M a t e l e s e t a l . r e p o r t e d Y / = 0.31 f o r t h e i r polymer-producing Pseudomonad C i n shake f l a s k s (24) a n d Y / = 0 . 5 4 i n a c h e m o s t a t t h a t f a v o r e d c e l l p r o d u c t i o n (iJT). The c e l l y i e l d s o f o t h e r m e t h a n o l - u t i l i z i n g b a c t e r i a , w i t h no polymer p r o d u c t i o n , range f r o m 0 . 2 t o 0 . 4 (11). H a g g s t r o m (2£) e s t i m a t e d t h a t f o r h i s m e t h a n o l - u t i l i z i n g b a c t e r i a , t h e e f f i c i e n c y o f transforming the carbon from methanol i n t o t h e c a r b o n i n c e l l s w o u l d be 4 l $ ( C M c - m a s s / m e t h a n o l ) · we assume t h e c o m p o s i t i o n o f M. mucosa i s C 5 H 8 O 3 N a n d c a l c u l a t e the e f f i c i e n c y o f carbon t r a n s f o r m a t i o n t o biomass from the exper i m e n t a l y i e l d Y / = Ο.345, t h e e f f i c i e n c y i s 42.7$, about t h e same a s t h e number a s o b t a i n e d b y H a g g s t r o m . The e m p i r i c a l f o r mula C 5 H 8 O 3 N i s used i n s t e a d o f the formula o f C H80 N based on Hamer a n d J o h n s o n s d a t a b e c a u s e t h e l a t t e r p r e d i c t s 13-7$ i n t h e c e l l , w h e r e a s t h e n i t r o g e n c o n t e n t o f M. mucosa i s 11.0 + 0 . 5 $ a v a l u e i n c l o s e r agreement w i t h t h e f o r m u l a C 5 H 8 O 3 N (Table 3). The a v e r a g e y i e l d c o e f f i c i e n t s Y n / = 0.175 a n d Y"c0 /s = 0.483 a r e c o n s t a n t w i t h i n t h e r a n g e o r d i l u t i o n r a t e s s t u d i e d (Figure 7). I f t h e hypothesis i s c o r r e c t , t h a t t h e carbon i n t h e methanol o n l y t u r n s i n t o c e l l s , polymer and carbon d i o x i d e then a c a r b o n b a l a n c e b a s e d o n t h e sum o f t h e t h r e e y i e l d c o e f f i c i e n t s x

S

x

S

x

S

c

x

I

S

4

2

1

S

2

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

f

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

methane

methanol

Methane m i x b a c t e r i a (23)

Methylomonas mucosa

oxygen

glycerol

glucose

(22)

(22)

Saccharomyces c e r e v i s i a e (22)

Azotobacter vinelandii

Aerobacter aerogenes

Organism

Limiting Factor

methanol

methane

glucose

glucose

glycerol

Substrate

0.26

0.12

0.02

0.15

0.08

m g sub — cell, hr

Growth Y i e l d and Maintenance C o e f f i c i e n t s and O t h e r M i c r o o r g a n i s m s

Table I I

0.345

0.7

O.5O

0.26

0.56

x/s g cell g sub.

f o r M^ mucosa

ο 2

0.039

0.06

0.02

0.18

0.10

g c e l l , hr

g o

0.425

Ο.38

1.10

0.4l

2

cell

g o 0.94

g

M

o

>

η

>

CO

o

S W >

53

>

r r

ο M

>

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

Haggstrom

Harrison et a l .

2.4

7.1

6.15

11.4

11.0

io.9

io.8

45.Ο

47.5

46.2

methanol

average :

methanol

36.9

32.12

ash

48.0

4.0

methanol

30.1

7.0

11.0

5

8

3

(28)

(27)

(23)

(26)

assuming a f o r m u l a of C H 0 N

(25)

Hamer e t a l .

Sheehan e t a l .

47-9

Ρ

methane

1.62

29.44

7.14

11.7

50.1

Vary & Johnson

Reference

methane

-

36.72

7.1

Other Elements

9.48

0

Η

Ν

46.7

C

methane

Substrate

E l e m e n t a l A n a l y s e s o f Methane a n d M e t h a n o l U t i l i z i n g Bacteria

Table I I I

68

EXTRACELLULAR

MICROBIAL

POLYSACCHARIDES

s h o u l d add up t o u n i t y . I n o r d e r t o do t h e c a r b o n b a l a n c e , t h e f o l l o w i n g two a s s u m p t i o n s were made: l ) t h e r e i s U6.2# c a r b o n i n t h e c e l l s as i n d i c a t e d b y t h e e m p i r i c a l f o r m u l a C5H8O3ÏÏ and 2 ) t h e r e i s 4θ$> c a r b o n i n t h e p o l y m e r . Since the polymer i s a h e t e r o p o l y s a c c h a r i d e , t h e g e n e r a l f o r m u l a f o r c a r b o h y d r a t e CH 0 i s a good a p p r o x i m a t i o n . The o v e r a l l c a r b o n b a l a n c e comes o u t t o b e (Figure 7): 2

ν fys Ô 3 7 5 ο

Λ

ν 0^62 . V s Ô375

ο

+

v Y

0.2725 C 0 / s 0.375

n =

2

s °* Q

R

9 6 5

S t r i p p i n g o f t h e v o l a t i l e m e t h a n o l o r a t r a c e amount o f b y p r o d uct f o r m a t i o n d u r i n g f e r m e n t a t i o n , s u c h as t h e y e l l o w p i g m e n t , w i l l a c c o u n t f o r t h e 3· 5$ d i s c r e p a n c y i n t h e c a r b o n b a l a n c e . Thus t h e d a t a a r e i n t e r n a l l c o n s i s t e n t To a c c o u n t f o r a l m e t r i c e q u a t i o n can be (ll) 22

CH3OH+I5.5 0 + 2 N 0 ~ 2 H 2

^ 2 C H 0 N + 4 CH 0+33 H 0

+

3

5

8

2

3

2

T h i s e q u a t i o n p r e d i c t s Y / = 0.171, Y / = Ο . 3 6 9 , Y c o / s = 0 . 5 0 . These numbers a g r e e w i t h t h e Y / = 0.175, Y / = 0.3^5, Y C 0 / s = 0.483 o b t a i n e d f r o m t h e e x p e r i m e n t . The e x p e r i m e n t a l p o l y m e r y i e l d o f 17· 5$ i - "too l°w £ ° a n y p r a c t i c a l polymer p r o d u c t i o n process. However, p r e v i o u s s h a k e f l a s k experiments performed w i t h n i t r o g e n l i m i t a t i o n suggested t h a t t h e p o l y m e r y i e l d c o u l d be i m p r o v e d a t t h e e x p e n s e o f c e l l yield (ll). The f e a s i b i l i t y o f a c o n t i n u o u s p o l y m e r p r o d u c t i o n scheme w i t h n i t r o g e n a s t h e l i m i t i n g s u b s t r a t e w i l l be i n v e s t i gated i n the f o l l o w i n g section. p

S

x

p

S

S

2

x

S

2

s

r

N i t r o g e n - l m i t e d C h e m o s t a t . To a c h i e v e n i t r o g e n - l i m i t e d g r o w t h , 1 g/L N a N 0 was u s e d i n t h e f e e d and t h e f l o w r a t e o f medium was a d j u s t e d s o t h a t t h e c e l l d e n s i t y was 1.63 ± 0 . 0 3 g c e l l / L f o r a l l t h e d i l u t i o n r a t e s ( 0 . l 4 t o Ο . 3 2 h r " i ) . The g r o w t h o f t h e c e l l s was n o t o x y g e n l i m i t e d s i n c e t h e d i s s o l v e d oxygen,D. 0., was a l w a y s more t h a n t h a t e q u i v a l e n t t o 30$ a i r s a t u ration. The s p e c i f i c m e t h a n o l u t i l i z a t i o n r a t e , Qj^, r e m a i n e d c o n s t a n t i n s t e a d o f i n c r e a s i n g l i n e a r l y w i t h d i l u t i o n r a t e as was t h e c a s e f o r c a r b o n - l i m i t e d g r o w t h ( F i g u r e 6 ) . The a v e r a g e i s 0.97 + 0.015 g m e t h a n o l / ( g c e l l , h r ) . S i n c e t h e c e l l c o n c e n t r a t i o n a n d t h e Çfa were e s s e n t i a l l y c o n s t a n t f o r a l l t h e d i l u t i o n r a t e s i n v e s t i g a t e d , an i n c r e a s e i n r e s i d e n c e time i m p l i e d t h a t more m e t h a n o l w o u l d be c o n v e r t e d i n t o p o l y m e r . Thus t h e s p e c i f i c p o l y m e r p r o d u c t i o n r a t e , Qp, s h o u l d i n c r e a s e l i n e a r l y w i t h r e s i d e n c e t i m e , and t h i s i s shown i n F i g u r e 8 where p o l y m e r f o r m a t i o n i s e x p r e s s e d b o t h as g l u c o s e e q u i v a l e n t , Qg, and a l s o as d r y w e i g h t , Q ,. The d a t a f o r Q a r e s c a t t e r e d b e c a u s e o f e r r o r s i n d r i e d weight determinations. However, t h e s l o p e s o f Qg and Q s h o u l d b e t h e same, and a t z e r o r e s i d e n c e t i m e , n o 3

p

p

p

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

5.

Τ Α Μ AND

Polysaccharide Formation

FINN

.3 Dilution

Figure 7.

.4 rat» ,

Hr."

RISIDENCI

Figure 8.

by a Methylomonas

TIM!

69

Yield coefficients for the car bon-limited chemostat

(hr)

9

Polymer production in nitrogen-limited chemostat

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

70

EXTRACELLULAR

MICROBIAL

POLYSACCHARIDES

p o l y m e r s h o u l d be p r o d u c e d . A s t r a i g h t l i n e w i t h s l o p e p a r a l l e l t o Qg a n d w i t h z e r o i n t e r c e p t i s drawn t h r o u g h t h e d a t a f o r t h e s p e c i f i c polymer p r o d u c t i o n r a t e . F o ra given c e l l population (X g / l ) , t h e t o t a l amount o f p o l y m e r f o r m e d u n d e r n i t r o g e n - l i m i t ed c o n d i t i o n s i s g i v e n b y : t

2

Op d0 = 0.035X ( t | - t f ) ( g p o l y m e r / 1 )

X

Jtx The y i e l d s o f c a r b o n d i o x i d e b a s e d o n m e t h a n o l consumed d i d n o t v a r y w i t h d i l u t i o n r a t e , D. The a v e r a g e v a l u e o f Y c o / s °.47 agrees w e l l w i t h t h e averag v a l u f 0.48 f o t h c a r t o n - l i m i t i n g c a s e . However Yp/ as d i l u t i o n r a t e was c h a n g e ( F i g u r 9) , extrapolate Υχ/s i s z e r o a n d Yp/s i s Ο . 5 6 , w h i c h s u g g e s t s t h a t t h e maximum y i e l d f o r t h e p o l y m e r i s a b o u t 56$ o f t h e m e t h a n o l consumed. This a p p a r e n t h i g h p r o j e c t e d y i e l d m i g h t n o t be a t t a i n a b l e i n p r a c t i c e because o f d i s s o l v e d oxygen l i m i t a t i o n d u r i n g t h e polymer forma t i o n phase. E v e n i f t h e s y s t e m were o p e r a t e d a t h a l f t h e maximum y i e l d , s a y a t Y / = 0 . 2 8 , t h e p e r f o r m a n c e w o u l d s t i l l be b e t t e r t h a n f o r t h e c a r o o n - l i m i t e d c a s e where Y / s 0-175The y i e l d data s t r o n g l y suggest use o f a n i t r o g e n - l i m i t i n g process f o r p o l y mer p r o d u c t i o n . A c h e c k f o r c o n s i s t e n c y o f t h e d a t a was made b y t a k i n g a c a r b o n b a l a n c e w i t h t h e same a s s u m p t i o n s a s b e f o r e , i . e . 4 6 . 2 $ c a r b o n i n c e l l s a n d 40$ c a r b o n i n p o l y m e r . =

2

p

S

=

p

Y

p / s

(i.o 8) 5

+

x

x / s

(i.2 2) 5

+

Y

C

0

2

/

S

(O.T26)

=c

The a v e r a g e v a l u e f o r C t u r n e d o u t t o be 0.990 i n s t e a d o f 0. 965 as i n t h e c a r b o n - l i m i t i n g c a s e . I n o t h e r w o r d s , t h e r e was o n l y 1$ e r r o r i n t h e c a r b o n b a l a n c e . When Yp/s i s z e r o , t h e e x t r a p o l a t e d maximum c e l l y i e l d Y / s i s 0-555Assuming t h a t t h e carbon d i o x i d e y i e l d remains c o n s t a n t a t 0.47 i n t h e a b s e n c e o f p o l y m e r f o r m a t i o n , a c a r b o n b a l a n c e g i v e s a v a l u e o f C a s 1.02, i . e . 2$ e r r o r i n t h e c a r b o n b a l a n c e when o n l y c e l l s a n d C 0 a r e formed. The c o i n c i d e n c e o f t h e m a x i mum v a l u e s f o r t h e y i e l d c o e f f i c i e n t s Υχ/s = 0.56 a n d Yp/s = 0.555 s u g g e s t s t h a t t h e e n e r g y d e r i v e d f r o m c a t a b o l i c p r o c e s s e s i s u s e d w i t h a p p r o x i m a t e l y t h e same maximum e f f i c i e n c y f o r t h e b i o s y n t h e s i s o f e i t h e r c e l l s o r polymer. I n f a c t , these y i e l d d a t a agree c l o s e l y w i t h t h e p r e d i c t i o n s based on " t h e o r e t i c a l " m o l a r g r o w t h y i e l d s f r o m A T P (29). From gas c h r o m a t o g r a p h i c a n a l y s i s , t h e e f f l u e n t a i r h a d a n a v e r a g e c o m p o s i t i o n o f Ο . 6 7 ± 0.02$ c a r b o n d i o x i d e a n d 19-4 ± 0.15$ oxygen. By an oxygen and carbon d i o x i d e b a l a n c e , t h e r e s p i r a t o r y q u o t i e n t (R.Q.) was f o u n d t o be 0 . 4 l 8 m o l e C 0 / m o l e 0 . The a v e r a g e s p e c i f i c o x y g e n c o n s u m p t i o n r a t e was 2 6 . 6 m m o l e x

2

2

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

2

5.

ΤΑΜ

Polysaccharide

A N D FINN

Formation

by a Methylomonas

71

°2/(g c e l l , h r ) , w h i c h comes c l o s e t o t h e e x t r a p o l a t e d maximum v a l u e o f 33 m m o l e 0 / ( g c e l l , h r ) f r o m t h e p r e v i o u s r e s p i r a t i o n study. An i n t e r e s t i n g f l o c c u l a t i o n phenomenon was o b s e r v e d a t t h e h i g h d i l u t i o n r a t e s (F>1.2 l / h r ) . C e l l s tended t o f l o c c u l a t e and s e t t l e much f a s t e r u p o n s t a n d i n g i n a t e s t t u b e a t room t e m p e r a ture. However, a f t e r a s h i f t t o l o w d i l u t i o n r a t e where more p o l y m e r was p r o d u c e d , t h e f l o c c u l a t i n g phenomenon d i s a p p e a r e d . There are two p o s s i b l e e x p l a n a t i o n s : e i t h e r a mutant i s formed o r t h e f l o c c u l a t i o n i s due t o a c o n c e n t r a t i o n e f f e c t o f t h e p o l y m e r . Only a t a p a r t i c u l a r c o n c e n t r a t i o n o f the a n i o n i c polymer t h a t t h e i n t e r a c t i o n b e t w e e n t h e f i x e d amount o f c e l l a n d t h e c o l l o i d a l p h o s p h a t e c a t i o n c o m p l e x i n t h e b a s a l medium w o u l d b r i n g t h e system t o the i s o e l e c t r i p o i n i agglomeratio precipitation. A g a r p l a t e s i n o c u l a t e d w i t h t h e p r e c i p i t a t i n g c e l l s gave t h e same t y p e o f c o l o n y as t h e n o r m a l c e l l s . A l s o the r a p i d r e v e r s i b i l i t y o f t h e c o a g u l a t i n g phenomenon a c h i e v e d b y c h a n g i n g t h e d i l u t i o n r a t e ( i . e . t h e amount o f p o l y m e r f o r m e d ) s u g g e s t s t h a t the second reason provides a b e t t e r e x p l a n a t i o n . 2

Hon-growth A s s o c i a t e d C o e f f i c i e n t . I t i s apparent from the n i t r o g e n - l i m i t e d growth data t h a t polymer p r o d u c t i o n i s nongrowth a s s o c i a t e d . I n order t o t e s t the e x t r a p o l a t e d polymer y i e l d d a t a (Yp/s °·56) f o r a non-growth s i t u a t i o n ( Y / = θ ) and to f i n d c o e f f i c i e n t f o r non-growth a s s o c i a t e d polymer p r o d u c t i o n i n t h e L u e d e k i n g (30 ) e q u a t i o n , d P / d t = a d X / d t + bX, a shake f l a s k e x p e r i m e n t was. done u s i n g w a s h e d c e l l s . D i f f e r e n t amounts o f w a s h e d c e l l s s u s p e n d e d i n p h o s p h a t e b u f f e r were u s e d t o i n o c u l a t e n i t r o g e n - f r e e b r o t h i n i n d e n t e d f l a s k s c o n t a i n i n g 1.29$ m e t h a n o l . The p o l y m e r p r o d u c t i o n r a t e s were l i n e a r f o r t h e f i r s t s i x t o e i g h t h o u r s b u t d e c r e a s e d when t h e t i m e o f i n c u b a t i o n i n c r e a s e d beyond f o u r generation times. The i n i t i a l p o l y m e r p r o d u c t i o n r a t e was p l o t t e d a g a i n s t t h e d r i e d c e l l w e i g h t . A s t r a i g h t l i n e was o b t a i n e d as shown i n F i g u r e 10. The n o n - g r o w t h a s s o c i a t e d c o e f f i c i e n t , b, o b t a i n e d from t h e s l o p e i s Ο.39 g polymer ( g c e l l , h r ) . The a v e r a g e p o l y m e r y i e l d f o r t h e f i v e f l a s k s was 0.59 ± 0.15 w h i c h a g r e e s w e l l w i t h t h e e x t r a p o l a t e d v a l u e o f 0.56 f r o m F i g u r e 9. The r e l a t i v e l y l a r g e e r r o r i n t h e Yp/s c a l c u l a t i o n i s due t o t h e s m a l l q u a n t i t i e s o f m e t h a n o l consumed i n t h e f i r s t s i x h o u r s ; a d i f f e r e n c e o f 0.01$ m e t h a n o l c o n t e n t w o u l d g i v e 1 0 $ e r r o r i n =

x

S

V*· N i t r o g e n - l i m i t i n g Batch. Based on the p r e v i o u s o b s e r v a t i o n s , a p o l y m e r p r o d u c t i o n scheme w i t h p e r i o d i c n i t r o g e n s t a r v a t i o n was investigated. A b a s a l medium c o n t a i n i n g 1.5 g/L N a N 0 was u s e d and t w o p u l s e s o f a d d i t i o n a l c a r b o n a n d n i t r o g e n (65 m l m e t h a n o l and 2.15 g ammonium s u l f a t e ) were a d d e d a t 9 l / h o u r s a n d a t 23 hours a f t e r the i n i t i a t i o n o f the batch run. For these e x p e r i ments t h e Magnaferm f e r m e n t o r h a d a n a e r a t i o n r a t e o f 5 l i t e r s o f 3

2

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

72

EXTRACELLULAR

MICROBIAL

POLYSACCHARIDES

Cproducts Cmethanol

Figure

9.

Yield

coefficients in nitrogen-limited chemostat

.25,

Figure

10. Polymer formation washed cell suspension

rate in

Dried Cell

(g/i)

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

5.

ΤΑΜ

AND

Polysaccharide

FINN

Formation

by

a Methylomonas

73

a i r p e r m i n u t e , and a s t i r r e r s p e e d o f 800 REM. For t h e e x p o n e n t i a l growth phase, the s p e c i f i c c e l l growth r a t e was 0.278 h r " (ta. = 2.5 h r ) w h i c h s h i f t e d t o 0.102 h r " u p o n the a d d i t i o n o f t h e f i r s t p u l s e o f carbon and n i t r o g e n . The l o w c e l l p r o d u c t i o n r a t e was l a r g e l y due t o d i s s o l v e d o x y g e n l i m i t a t i o n , as shown i n F i g u r e 11. The c o n c e n t r a t i o n o f d i s s o l v e d o x y gen r e m a i n e d z e r o a f t e r t h e c o n s u m p t i o n o f lfo m e t h a n o l . The f i r s t - o r d e r r a t e c o n s t a n t f o r g l u c o s e p r o d u c t i o n i s 0.24 hr" . A l a g o f a b o u t two h o u r s was o b s e r v e d b e f o r e p r o d u c t i o n o f polymer resumed a f t e r t h e p u l s e a d d i t i o n o f c a r b o n and n i t r o g e n . S i n c e ammonium s u l f a t e was u s e d as a n i t r o g e n s o u r c e , no n i t r i t e should accumulate t o i n h i b i t polymer p r o d u c t i o n . Perhaps i t t a k e s t i m e f o r M. mucosa t o a d j u s t t o t h e c o n c e n t r a t i o n s h o c k p r o d u c e d by a step increase of methanol inhibitin level Th impor t a n t t h i n g t o note her does n o t d e c r e a s e a p p r e c i a b l y non-growt t h e l 4 t h a n d 23rd h o u r s . A f t e r t h e d i s s o l v e d o x y g e n c o n t e n t r e a c h e d z e r o , t h e mass t r a n s f e r c o e f f i c i e n t Kjja, as c a l c u l a t e d f r o m o x y g e n b a l a n c e , was about c o n s t a n t . The a v e r a g e K a was 165 h r " i . With the aeration a n d s t i r r i n g p a r a m e t e r s k e p t c o n s t a n t , t h e e f f e c t o f t h e foam b r e a k e r on t h e o x y g e n mass t r a n s f e r c o e f f i c i e n t c o u l d be s e e n b y a sudden d e c r e a s e i n K^a f r o m 165 t o 120 h r " when t h e s u r f a c e o f t h e b r o t h f a i l e d t o r e a c h t h e foam b r e a k e r ( a t t h e 34th h o u r ) . The t e r m i n a t i o n o f p o l y m e r p r o d u c t i o n c o i n c i d e d w i t h t h e d r o p i n K^a v a l u e . T h i s o b s e r v a t i o n s u g g e s t s t h a t mass t r a n s f e r o f d i s s o l v e d o x y g e n may l i m i t p o l y m e r p r o d u c t i o n o r e l s e t h e r e i s a c c u mulation of t o x i c by-products i n the batch. A n o t h e r i n t e r e s t i n g p o i n t was t h e c o n t i n u e d m e t h a n o l consump t i o n a t a l i n e a r r a t e o f Ο.332 g m e t h a n o l / ( 1 , h r ) a f t e r b o t h g r o w t h and polymer s y n t h e s i s had stopped. T h i s decrease i n methanol c o u l d p e r h a p s be a c c o u n t e d f o r b y t h e c e l l m a i n t e n a n c e r e q u i r e m e n t and t h e s t r i p p i n g l o s s . The f i n a l y i e l d d a t a a t t h e e n d o f t h e 56 h o u r s w e r e : Y / = 0.118, Y / = 0.4θ8, a n d 1.897fo s o l i d p r o d u c e d f o r 4.55$> m e t h a n o l consumed. The maximum y i e l d c o e f f i c i e n t s f o r p o l y m e r p r o d u c t i o n i n t h e b a t c h s h o u l d be c a l c u l a t e d a t t h e 38th h o u r when p o l y m e r p r o d u c t i o n was t e r m i n a t e d . Thus, d i s r e g a r d i n g t h e methanol l o s s i n c e l l maintenance and s t r i p p i n g d u r i n g t h e l a s t 18 h o u r s o f i n c u b a t i o n , t h e y i e l d c o e f f i c i e n t s s h o u l d be Υχ/s =.0.122 a n d Y / = 0.452 w h i l e t h e t o t a l s o l i d y i e l d s h o u l d be 0.574. The p o l y m e r y i e l d a p p r o a c h e d t h e e x t r a p o l a t e d maximum o f Ο.56 ( F i g u r e 8 ) . 1

1

1

L

1

x

S

p

p

S

S

S e m i - c o n t i n u o u s F e r m e n t a t i o n . An a t t e m p t t o c u l t u r e t h e b a c t e r i a c o n t i n u o u s l y a t l o w d i l u t i o n r a t e was n o t v e r y s u c c e s s f u l . The y i e l d o f p o l y m e r d e c r e a s e d a f t e r one week o f c o n t i n u o u s f e r mentation. C o n t a m i n a t i o n a t l o w s t e a d y - s t a t e m e t h a n o l l e v e l and/ o r p o s s i b l y c u l t u r e d e g e n e r a t i o n became t h e m a j o r o b s t a c l e s t o s u c c e s s f u l o p e r a t i o n at low d i l u t i o n rate. Operation o f a carbonl i m i t e d chemostat a t a h i g h e r d i l u t i o n r a t e had p r e v i o u s l y r e -

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

EXTRACELLULAR

MICROBIAL

POLYSACCHARIDES

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

RPM

dt

dS

jo

hr

CH3OH

dP g g l u c o s e d t 1, h r

dX g c e l l d t 1, h r

Time, h r

.37 t o

500

.065

.033

.16

0 t o 32

Semicontinuous

First

0.4 t o

.175

500

500

350

.175

hO

.018

.014

30 t o

.038

.057

.Oik

3 t o 47

.054

hi

Semi - c o n t i n u o u s

Second C y c l e Batch

.175

550

.045

.031

.044

42 t o 64

Fermentor

.049

.109

0

33 t o

Shake flask

Cycle

Rate Constants f o r the Semi-continuous Operation i n the l4 L

Table IV

76

EXTRACELLULAR

MICROBIAL

POLYSACCHARIDES

s u i t e d i n s e l e c t i n g a f a s t g r o w i n g v a r i a n t t h a t was a p o o r p o l y m e r former. I n o r d e r t o a v o i d p u t t i n g s e l e c t i v e p r e s s u r e on M. mucosa and t o i m p r o v e t h e p r o c e s s e c o n o m i c s , a s e m i - c o n t i n u o u s o p e r a t i o n scheme was t h e r e f o r e c o n s i d e r e d . The s e t - u p c o n s i s t e d o f t w o t a n k s i n s e r i e s ; t h e f i r s t f e r m e n t o r was t h e Magnaferm w h i c h s e r v e d as a c o n t i n u o u s c e l l p r o p a g a t o r o p e r a t i n g a t r e l a t i v e l y h i g h d i l u t i o n r a t e (0.42 l / h r ) . I n t h i s t a n k t h e s t e a d y s t a t e m e t h a n o l c o n c e n t r a t i o n was k e p t above lfo s o a s t o p r e v e n t g r o w t h o f c o n t a m i n a n t s . B a s a l medium w i t h 3$ m e t h a n o l a n d 2.5 g / l N a N 0 was f e d t o t h e Magnaferm c o n t i n u o u s l y . The e f f l u e n t f r o m t h e f i r s t t a n k was d i r e c t e d i n t o t h e l 4 - l f e r m e n t o r where n i t r o g e n - l i m i t e d g r o w t h began. The n i t r o g e n l i m i t a t i o n not o n l y f a v o r e d polymer formation but a l s o h e l p e d t o prevent the growth o f contaminants A f t e r a f i x e d volume h a d a c c u m u l a t e d i n the second tank, e f f l u e n d i v e r t e d i n t o another fermento t h e s e c o n d t a n k was a l l o w e d t o r u n a s a b a t c h p r o c e s s . A pulse o f 1.7 g N a N 0 a n d 84 m l m e t h a n o l was a d d e d t o t h e l 4 - l i t e r f e r m e n t o r when c o n t i n u o u s f e e d s t o p p e d ( F i g u r e 12). A t t h e 33**d h o u r o f o p e r a t i o n , t h e s e c o n d t a n k was e m p t i e d a n d 250 m l o f t h e b r o t h was p u t i n a n i n d e n t e d f l a s k a n d i n c u b a t e d a t 350 REM i n a c o n s t a n t t e m p e r a t u r e (30°C) s h a k e r . Then t h e s e c o n d c y c l e o f t h e c o n t i n u o u s f e e d t o t h e s e c o n d t a n k b e g a n a n d no a d d i t i o n a l n i t r a t e was a d d e d i n t h i s c y c l e . The r e s u l t s o f t h e t w o c y c l e s o f s e m i - c o n t i n u o u s o p e r a t i o n f o r t h e s e c o n d t a n k a r e shown i n F i g u r e s 12 a n d 13 a n d t h e r a t e c o n s t a n t s a r e summarized i n TableIV. I n t h e f i r s t c y c l e , 2.776 g / l N a N 0 was consumed i n 33 h o u r s a n d 4.22 g c e l l / l was p r o d u c e d . I f a l l t h e a v a i l a b l e n i t r o g e n h a d ended up i n t h e c e l l s , t h e p e r c e n t a g e o f n i t r o g e n i n t h e c e l l s w o u l d be 10.82$> w h i c h a g r e e s w i t h t h e v a l u e o f 10.8ofo p r e d i c t e d b y t h e e m p i r i c a l f o r m u l a C H s 0 N . The m e a s u r e d c e l l a n d p o l y m e r p r o d u c t i o n r a t e s were a l l l i n e a r (Table 4). I f t h e b r o t h were a l l o w e d t o i n c u b a t e l o n g e r t h a n 30 h o u r s i n t h e a e r a t e d t a n k , t h e r a t e o f p o l y m e r p r o d u c t i o n s h o u l d s l o w down t o a b o u t o n e - t h i r d o f t h e i n i t i a l v a l u e as s u g g e s t e d b y d a t a i n t h e s e c o n d c y c l e ( F i g u r e 12). However, t h e r a t e o f p o l y m e r p r o d u c t i o n a c t u a l l y i n c r e a s e d f r o m Ο.Ο65 t o 0.109 g g l u c o s e / l , h r i n t h e shake f l a s k ( a t 350 RFM). T h i s i n d i c a t e d t h a t f o r t h e v i s c o u s b r o t h , a shake f l a s k h a d b e t t e r a e r a t i o n , a n d t h a t a K L a v a l u e o f 98 h r " i n t h e a e r a t e d t a n k was n o t h i g h enough t o meet t h e o x y g e n demand. A n o t h e r p i e c e o f e v i d e n c e f o r o x y g e n l i m i t a t i o n was f o u n d i n t h e s e c o n d c y c l e o f t h e o p e r a t i o n i n the second tank. A 10fo i n c r e a s e i n t h e a g i t a t i o n r a t e o f t h e i m p e l l e r , f r o m 500 t o 550 RPM, r a i s e d t h e p o l y m e r f o r m a t i o n r a t e f r o m 0.018 t o 0.031 g g l u c o s e / ( L , h r ) , a n d t h e c e l l p r o d u c t i o n r a t e f r o m 0.014 t o 0.044 g c e l l / ( L , h r ) . However, t h e K a showed l i t t l e o b s e r v a b l e change a n d r e m a i n e d c o n s t a n t a t 120 h r - i . The r e s p i r a t o r y q u o t i e n t (R.Q.) i s a v e r y s e n s i t i v e p a r a m e t e r t h a t t e l l s t h e age d i s t r i b u t i o n o f t h e p o p u l a t i o n b e c a u s e t h e d e mand f o r o x y g e n a n d t h e e v o l u t i o n o f c a r b o n d i o x i d e a r e n o t c o n 3

3

3

5

1

L

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

3

5.

Polysaccharide

ΤΑΜ A N D FINN

Formation

by a Methylomonas

HOUR Figure 12.

I

Ο

.

ι

10

First cycle semi-continuous fermentation

J—Λ

20

,

ι

30

,

i _ U

40

1

50

.

•

60

•

I

70

HOUR

Figure 13.

Second cycle semi-continuous fermentation

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

77

78

EXTRACELLULAR

MICROBIAL

POLYSACCHARIDES

s t a n t d u r i n g t h e d i f f e r e n t phases o f the b a c t e r i a l growth c y c l e . For example, i n t h e f i r s t c y c l e o f t h e s e m i - c o n t i n u o u s o p e r a t i o n , a p r e s s u r e l e a k was d e v e l o p e d i n t h e f i r s t t a n k r e s u l t i n g i n no o v e r f l o w i n t o t h e second t a n k and b r o t h a c c u m u l a t i o n i n t h e c e l l p r o p a g a t o r . A b o u t a n h o u r l a t e r (10-1/2 h o u r i n F i g u r e 1 2 ) t h e p r e s s u r e was r e a d j u s t e d a n d a b o u t 0 . 6 l i t e r o f c e l l s f r o m t h e e x p o n e n t i a l g r o w t h p h a s e was f o r c e d i n t o t h e s e c o n d t a n k . The R. Q. i m m e d i a t e l y jumped f r o m 0.2 t o 0-31 as i n d i c a t e d b y t h e d a s h e d l i n e i n F i g u r e 12. The maximum i n t h e R. Q. c u r v e a l s o i n d i c a t e d t h a t a r e l a t i v e l y l a r g e p o r t i o n o f e x p o n e n t i a l l y growing c e l l s was i n t h e p o p u l a t i o n d u r i n g t h e c o n t i n u o u s f e e d i n g s t a g e i n the second c y c l e ( F i g u r e 12). T h e o r e t i c a l l y , t h e maximum r e s p i r a t o r y q u o t i e n t (R. Q. = 0 . 6 6 ) o c c u r s when c a r b o n i n m e t h a n o is completely oxidised t 2

CH3OH

+

3 0

> 4H 0 + 2 C 0

2

2

3^7 K c a l / m o l e CH 0H

2

3

However, when p a r t o f t h e c a r b o n i s d i v e r t e d t o c e l l a n d p o l y m e r s y n t h e s i s , l e s s c a r b o n d i o x i d e s h o u l d be f o r m e d a n d t h e R. Q. s h o u l d be l e s s t h a n 0.66. S i n c e p o l y m e r i z a t i o n r e q u i r e s l e s s energy than c e l l s y n t h e s i s , the r e s p i r a t o r y quotient s h o u l d de c r e a s e m o n o t o n i c a l l y as more p o l y m e r a n d f e w e r c e l l s a r e formed. The e x p e r i m e n t a l R. Q. d a t a f a l l b e t w e e n 0 . 4 t o 0.1 a n d t h e d e c r e a s e i n t h e r e s p i r a t o r y q u o t i e n t w i t h t h e i n c r e a s e i n t h e amount o f p o l y m e r f o r m e d does i n f a c t a g r e e w i t h t h e p r e d i c t e d g e n e r a l trend. The f i n a l y i e l d d a t a f o r t h e s e m i - c o n t i n u o u s e x p e r i m e n t a r e s u m m a r i z e d i n T a b l e V. Table V F i n a l Y i e l d Data f o r the Semi-continuous

Yield Constant Y

t o t a l solid/s

Y

,

T

a

n

k

Experiment

2

F i r s t Cycle

Second C y c l e

·3

·3*

2

(-W

.121». ( . ι 8 )

.128 (.160)

.196 (.229)

.212 (.264)

5

Χ/ S

Y^

g

Numbers i n t h e b r a c k e t s show t h e y i e l d c o n s t a n t s , c o r r e c t e d f o r l o s s due t o m e t h a n o l s t r i p p i n g a t a r a t e o f 0.12 g M e t h a n o l / ( 1 , h r ) f o r 60 h o u r s . The y i e l d d a t a a r e l o w e r t h a n t h e b e s t o b s e r v e d v a l u e s o f Y / = 0.122, Y p / = ΟΛ52 a n d Y t o t a l s o l i d / s = Ο . 5 6 i n t h e n i t r o g e n - l i m i t e d b a t c h ( F i g u r e 11). The p o o r e r y i e l d i s due t o t h e x

S

S

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

5.

ΤΑΜ

A N D FINN

Polysaccharide

Formation

by a Methylomonas

79

i n f e r i o r o x y g e n t r a n s f e r c a p a c i t y o f t h e s e c o n d t a n k and, more i m p o r t a n t l y , t o t h e n i t r o g e n dosage scheme. I n t h e n i t r o g e n - l i m i t e d b a t c h c u l t u r e , a d d i t i o n a l n i t r o g e n s o u r c e was a d d e d i n a way t h a t avoided p r o l o n g e d p e r i o d s o f n i t r o g e n exhaustion, and conse q u e n t l y t h e r e was a r e l a t i v e l y l a r g e p o p u l a t i o n o f y o u n g c e l l s i n the broth. The f a c t t h a t t h e R. Q. was b e t w e e n 0.4 a n d 0.33 i n t h e f i r s t 50 h o u r s o f t h e b a t c h o p e r a t i o n a s compared t o t h e a v e r a g e R. Q. o f l e s s t h a n 0.2 i n t h e s e m i - c o n t i n u o u s o p e r a t i o n , s u p p o r t s t h e above argument. I n c o n c l u s i o n , a s e m i - c o n t i n u o u s o p e r a t i o n seems f e a s i b l e b e cause i t i s r e p r o d u c i b l e and because i t m i n i m i z e s problems o f contamination o r c u l t u r e degeneration. The p r e s e n t o p e r a t i o n a l p r o c e d u r e i s n o t t h e o p t i m a l one. Improvements i n a e r a t i o n b y i n s t a l l i n g a foam b r e a k e w i l l help t o bring the n i t r o g e n dosage c a n be large portio o f y o u n g c e l l s i n t h e s e c o n d f e r m e n t o r (R. Q. b e t w e e n 0.3 t o 0 . 4 ) .

Literature Cited 1. Bikales, Ν. M. (ed.) in "Water Soluble Polymers", pp 227-42, Plenum Publishing Corp., New York, N.Y., 1973. 2. McNeely, W. H. in "Microbial Technology", H. Peppler (ed.), 381-402, Reinhold Publishing Corp., New York, N.Y., 1967. 3. MacWilliams, D.C., Rogers, J. Η., and West, T. J. in "Ency clopedia of Polymer Science and Technology", Vol. II, pp 105-126, Wiley-Interscience, New York, N.Y., 1973. 4. Moraine, R. A. and Rogovin, P., Biotechnol. Bioeng. (1971), 13, 381-91. 5. Tannahill, Alex L. and Finn, R. Κ., U.S. Patent 3,878,045, April 15, 1975. 6. Finn, R. Κ., Tannahill, Alex L . , and Laptewicz, J. E. Jr., U.S. Patent 3,923,782, Dec. 2, 1975. 7. Herbert, D., Phipp, P. J., and Strange, R. E. in "Methods in Microbiology", Norris, J. R. and Ribbons, D. W. (eds.), Vol. 5B, pp 249-51, Academic Press, London, 1971. 8. Dubios, M. et al., Anal. Chem. (1956), 28, 350-56. 9. Johnson, M. J., Borkowski, J., and Engblom, C., Biotechnol. Bioeng. (1964), 6, 457-68. 10. ibid. 9, 635-39. 11. Tam, K. T., Ph.D. Thesis, Cornell University, Ithaca, Ν. Υ., 1975. 12. Van Dijken, J. P. and Harder, W., J. Gen. Microbiol. (1974), 84, 409-11. 13. Whittenburg, R., Phillips, K. C., and Wilkinson, J. F., J. Gen. Microbiol. (1970), 6 1, 205-18. 14. Battat, E . , Goldberg, I . , and Mateles, R. I., Appl. Microbiol. (1974), 28, 906-11. 15. Levine, P. W. and Cooney, C. L . , Appl. Microbiol. (1973), 26, 982-90.

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16. Pilat, P. and Prokop, Α., Biotechnol. Bioeng. (1975), 17 1717-28. 17. Pirt, S. J., "Principles of Microbe and Cell Cultivation", 10-12, Blackwell Scientific Publications, Oxford, England, 1975. 18. Harrison, D. E. F., J. Appl. Bacteriol. (1973), 36, 301-8. 19. Wilkinson, T. G. and Harrison, D. E.F., J. Appl. Bacteriol. (1973), 309-13. 20. Kim, J. H. and Ryu, D. Y . , J. Fermentation Technol. (1976), 54, 427-36. 21. Nagai, S., Mori, T., and Aiba, Α., J. Appl. Chem. Biotechnol. (1973), 23, 540-62. 22. Nagai, S. and Aiba, S., J. Gen. Microbiol. (1972), 73, 531. 23. Sheehan, Β. T. and Johnson M J., Appl Microbiol (1972), 21, 511-15. 24. Mateles, R. I. and Appl (1972) 135-40. 25. Haggstrom, L . , Biotechnol. Bioeng. (1969), 11, 1043-54. 26. Vary, P. S. and Johnson, Μ. Η., Appl. Microbiol. (1967), 15, 1473. 27. Wilkinson, T. G., Topiwala, Η. Η., and Hamer, G., Biotechnol. Bioeng. (1974), 16, 41-59. 28. Harrison, D. E. F., Topiwala, Η. Η., and Hamer, G., pp 4915, "Fermentation Technology Today: Proc. IVth Int'l Ferm. Symp.", G. Terui (ed.), Soc. Ferm. Technol., Osaka, Japan, 1972. 29. Abbott, B. J. and Gledhill, W. Ε., Adv. Appl. Microbiol. (1971), 14, 249-60. 30. Luedeking, R. and Piret, E. L . , J. Biochem. Microbiol. Technol. Eng. (1959), 1, 393.

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6 Molecular Origin of Xanthan Solution Properties E. R. MORRIS Unilever Research, Colworth/Welwyn Laboratory, Sharnbrook, Bedford. MK44 1LQ., Great Britain

The technological importance of Xanthan gum rests principally on the following unusua properties in aqueous solution 1) Remarkable emulsion stabilising and particle suspending ability. 2) Extremely large shear dependence of viscosity, leading to pronounced thixotropy. 3) L i t t l e variation in viscosity with temperature under normal conditions of industrial utilisation. 4) High salt tolerance. The aim of this paper is to provide a unified explanation of the origin of these properties, at a molecular level. Solution Viscosity. Normally polyelectrolytes adopt a highly expanded conformation under conditions of low ionic strength, but collapse to a more compact coil on addition of salt, due to charge screening. Since polymer solution rheology i s c r i t i c a l l y dependent on molecular shape, these variations i n coil dimensions are normally reflected in large changes i n solution viscosity (4). Since the xanthan molecule is a polyanion, its maintenance of viscosity with increasing ionic strength is therefore particularly surprising, and indicates a considerable departure from normal random coil behaviour. The temperature dependence of i t s solution viscosity i s also complex. In the presence of moderate amounts of salt xanthan viscosity shows virtually no variation with temperature, i n contrast to the normal marked decrease i n polymer solution viscosity on heating. Under low ionic strength conditions, such as exist when the polymer i s dissolved i n distilled water, the temperature dependence of xanthan rheology i s even more unusual, showing an anomolous increase i n solution viscosity on heating, over a specific fairly narrow temperature range (l), suggesting a sharp change i n molecular conformation over this range. 81

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Optical Activity Changes i n polysaccharide conformation are f r e q u e n t l y accompanied by l a r g e changes i n o p t i c a l a c t i v i t y ( 5 - 1 3 ) , and i n p a r t i c u l a r s i n g l e wavelength o p t i c a l r o t a t i o n provides a s e n s i t i v e and convenient index of chain conformation. We have t h e r e f o r e used t h i s approach t o i n v e s t i g a t e f u r t h e r the o r i g i n of t h i s p e c u l i a r temperature p r o f i l e (14,15). As shown i n F i g u r e 1 the anomolous v i s c o s i t y behaviour c o i n c i d e s e x a c t l y with a l a r g e sigmoidal i n c r e a s e i n o p t i c a l r o t a t i o n , such as has been shown to accompany o r d e r - d i s o r d e r t r a n s i t i o n s i n other polysaccharide systems (6-10). Indeed a simple q u a n t i t a t i v e r e l a t i o n s h i p has been developed (5) t o p r e d i c t changes i n o p t i c a l r o t a t i o n a r i s i n g from changes i sugar r e s i d u e s i n the polyme I n t e r p r e t a t i o n of xanthan o p t i c a l r o t a t i o n i s , however, complicated by the presence of a c e t a t e , pyruvate, and uronate groups, a l l of which absorb l i g h t a t l o n g e r wavelengths than the polymer backbone and might t h e r e f o r e dominate o p t i c a l r o t a t i o n measurements i n the v i s i b l e r e g i o n . To explore t h i s p o s s i b i l i t y we have used c i r c u l a r dichroism t o monitor d i r e c t l y the temperature dependence of the o p t i c a l a c t i v i t y of these chromophores. As shown i n F i g u r e 2, there i s a l a r g e negative s h i f t i n c.d. on h e a t i n g . The observed o p t i c a l r o t a t i o n s h i f t t o l e s s negative values a t h i g h temperatures i s opposite i n sense, and must t h e r e f o r e a r i s e from changes i n the f a r - u l t r a v i o l e t where the e l e c t r o n i c t r a n s i t i o n s of the polymer backbone are known t o occur ( l 6 , 1 7 ) . N.m.r. R e l a x a t i o n C h i r o p t i c a l and r h e o l o g i c a l evidence t h e r e f o r e i n d i c a t e s that the xanthan molecule e x i s t s i n s o l u t i o n a t moderate temperatures i n an ordered conformation which, under s u i t a b l e c o n d i t i o n s , can be melted out. To f u r t h e r t e s t t h i s c o n c l u s i o n we have used time-domain pulsed n.m.r. t o probe molecular m o b i l i t y . N.m.r. r e l a x a t i o n by energy t r a n s f e r between adjacent n u c l e i provides a s e n s i t i v e index of polymer f l e x i b i l i t y , being extremely r a p i d f o r r i g i d molecules, but much slower f o r f l e x i b l e c o i l s , where t h e r mal motions i n t e r f e r e w i t h the exchange. At e l e v a t e d temperat u r e s , s a l t - f r e e xanthan s o l u t i o n s show o n l y the m i l l i s e c o n d r e l a x a t i o n processes normal f o r d i s o r d e r e d p o l y s a c c h a r i d e s . At ambient temperatures, however, a much more r a p i d r e l a x a t i o n i s observed i n the microsecond range t y p i c a l of r i g i d , ordered structures (18). High r e s o l u t i o n n.m.r. l i n e w i d t h i s i n v e r s e l y r e l a t e d t o the rate of decay of magnetisation, and so f r e e l y moving molecules show sharp n.m.r. spectra, while f o r r i g i d polymers the l i n e w i d t h i s so great that the high r e s o l u t i o n spectrum i s so f l a t t e n e d

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83

,4-0

t

3Ό VISCOSITY (CPSxlO" ) 3

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20

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80

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with temperature for Xanthomonas polysaccharide ("KeltroY')

arc

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2

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Keltrol. Effect of tem perature on CD.

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that no peaks are v i s i b l e . Thus normal h i g h - r e s o l u t i o n n.m.r. can be used to monitor o r d e r — d i s o r d e r t r a n s i t i o n s (9-10). At temperatures above the d i s c o n t i n u i t y i n o p t i c a l r o t a t i o n and s o l u t i o n v i s c o s i t y , xanthan s o l u t i o n s show n.m.r. spectra t y p i c a l of a normal d i s o r d e r e d polysaccharide c o i l . On c o o l i n g through the t r a n s i t i o n r e g i o n , however, the spectrum g r a d u a l l y c o l l a p s e s , u n t i l f i n a l l y no d i s c e r n a b l e h i g h r e s o l u t i o n spectrum can be detected. This decay i s conveniently monitored q u a n t i t a t i v e l y by measuring the area of the acetate and pyruvate resonances, which occur as w e l l r e s o l v e d s i n g l e t s at 2.1 and 1.5 ppm r e s p e c t i v e l y (Figure 3 ) . As shown i n F i g u r e 4, the n.m.r. r e l a x a t i o n behaviour f o l l o w s the same sigmoidal temperature course as o p t i c a l a c t i v i t y and s o l u t i o n v i s c o s i t y (see F i g u r e l ) . The Ordered State R h e o l o g i c a l evidence ( l ) i n d i c a t e s that xanthan conformation i s c r i t i c a l l y dependent on the presence or absence of s a l t . To i n v e s t i g a t e t h i s we have followed the o r d e r - d i s o r d e r t r a n s i t i o n at v a r i o u s i o n i c strengths, using o p t i c a l r o t a t i o n as a convenient index of conformational change. As shown i n F i g u r e 5, the t r a n s i t i o n s h i f t s to higher temperature with i n c r e a s i n g s a l t l e v e l , u n t i l f o r i o n i c strengths above about 0.15 M, the ordered c o n f o r mation p e r s i s t s up to 100 C. S i m i l a r s t a b i l i s a t i o n of ordered s t r u c t u r e s by a d d i t i o n of s a l t i s observed i n other charged polysaccharides ( 6 - 8 ) , and i s presumably due to the r e d u c t i o n of e l e c t r o s t a t i c r e p u l s i o n s between neighbouring charged groups i n the compact, ordered s t a t e . At constant i o n i c strength the temperature course of the t r a n s i t i o n appears to be independent of polymer c o n c e n t r a t i o n (Figure 6 ) . This i n d i c a t e s that e i t h e r the o r d e r - d i s o r d e r process i s unimolecular, or that i t i s extremely co-operative, as i n the case of DNA (19). The breadth of the xanthan t r a n s i t i o n argues against the l a t t e r explanation, and suggests i n t r a m o l e c u l a r order. The covalent s t r u c t u r e of xanthan has only r e c e n t l y been d e t e r mined ( 2 0 , 2 1 ) , and c o n s i s t s of a c e l l u l o s e backbone s u b s t i t u t e d on a l t e r n a t e residues with charged t r i s a c c h a r i d e sidechains, as shown i n F i g u r e 7· We suggest that i n the ordered conformation the sidechains are a l i g n e d with the main chain to give a r i g i d s t r u c t u r e s t a b i l i s e d by i n t r a m o l e c u l a r non—covalent bonding. D e f i n i t i v e d e s c r i p t i o n of the ordered n a t i v e conformation, however, must await X-ray evidence. Such work i s at present i n progress i n Purdue u n i v e r s i t y , and i s d e s c r i b e d i n the f o l l o w i n g paper. Molecular I n t e r p r e t a t i o n of S o l u t i o n P r o p e r t i e s Whatever the d e t a i l of the ordered s t a t e , i t s existence i n s o l u t i o n o f f e r s a s a t i s f a c t o r y u n i f y i n g explanation of the unusual and v a l u a b l e r h e o l o g i c a l p r o p e r t i e s of xanthan. In most

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

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Solution

Properties

Figure 3.

Figure

4.

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

85

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In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

6.

MORRIS

Molecular Origin of Xanthan Solution Properties

-> 4 ) - p - B - G l c p - ( l - > 4 ) - p - D - G l c p - 0 - >

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t e c h n o l o g i c a l l y important a p p l i c a t i o n s , s u f f i c i e n t s a l t i s present t o maintain the ordered conformation a t a l l temperatures. The r e l a t i v e i n s e n s i t i v i t y of s o l u t i o n v i s c o s i t y t o a d d i t i o n of f u r t h e r s a l t i s then a d i r e c t consequence of molecular r i g i d i t y . The emulsion s t a b i l i s i n g , p a r t i c l e suspending and t h i x o t r o p i c behaviour a l l point t o the existence of a p p r e c i a b l e i n t e r molecular s t r u c t u r e i n xanthan s o l u t i o n s . Such an i n t e r p r e t a t i o n i s e n t i r e l y c o n s i s t e n t with the known tendency o f r o d - l i k e molecules i n s o l u t i o n t o a l i g n (22). Indeed, b i r e f r i n g e n c e s t u d i e s (2) give d i r e c t evidence of c o n s i d e r a b l e molecular o r i e n t a t i o n i n xanthan s o l u t i o n s . We t h e r e f o r e suggest that weak non-covalent a s s o c i a t i o n s between a l i g n e d molecules b u i l d up a tenuous g e l - l i k e network capable of supporting s o l i d p a r t i c l e s , l i q u i d d r o p l e t s , or a i r bubbles P r o g r e s s i v e breakdown of t h i s network with i n c r e a s i n of the remarkable t h i x o t r o p p r o p e r t y of the p o l y s a c c h a r i d e .

Abstract Xanthan exists in solution at moderate temperatures in a native, ordered conformation. At low salt levels this order may be melted out, as monitored by n.m.r. relaxation, optical rotation, circular dichroism, and intrinsic viscosity. We suggest that in the ordered conformation the charged trisaccharide side -chains fold back around the cellulose backbone, to give a rigid, rod—like structure. Increasing salt concentration stabilises this conformation by minimising electrostatic repulsions between the sidechains. At the salt levels encountered in most industrial situations, the ordered form is stable to above 100°C, hence the relative insensitivity of xanthan solution viscosity to tempera ture or further increase in ionic strength. Stacking of the rigid molecules in solution builds up a tenuous intermolecular network, giving rise to the other commercially attractive properties, such as suspending ability, emulsion stabilisation, and thixotropy. Literature Cited 1. 2. 3. 4. 5. 6.

Jeanes, Α., Pittsley, J.E. & Senti, F.R. J. Applied Polymer Sci. (1961). 5, 519-526. Jeanes, A. In"Proceedingsof the ACS Conference on Water Soluble Polymers" (Bikales, N.M., ed.), pp. 227-242, Plenum Press, New York, (1973). Glicksman, H."PolysaccharideGums in Food Technology", Academic Press, New York, (1970). Smidsrød, O. & Haug, A. Biopolymers. (1971). 10, 1213. Rees, D.A. J . Chem. Soc. (B). (1970). 877-884. Rees, D.A. & Scott, W.E. J . Chem. Soc. (B). (l971). 469-479.

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7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22.

Molecular

Origin

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Solution

Properties

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Rees, D . A . , Scott, W.E. & Williamson, F . B . Nature. (1970). 227, 390-393. Rees, D . A . , Steel, I.W. & Williamson, F . B . J . Polymer S c i . (C). (1969). 28, 261-276. Bryce, T . A . , McKinnon, Α . Α . , Morris, E . R . , Rees, D.A. & Thom, D. Faraday Discuss. Chem. Soc. (1974). 57, 221. Dea, I.C.M., McKinnon, A.A. & Rees, D.A. J . Mol. B i o l . (1972). 68, 153-172. Grant, G . T . , Morris, E . R . , Rees, D . A . , Smith, P . J . C . & Thom, D. Febs. Lett. (1973). 32, 195-197. Morris, E . R . , Rees, D.A. & Thom, D. J . Chem. Soc. Chem. Commun. (1973). p. 245. Morris, E.R. & Sanderson, G.R. In "New Techniques i n Biophysics and Cell Biology" Joh Wiley London (1972) Morris, E . R . , Rees Darke, A. J . Mol. B i o l . Submitted. (1976) Rees, D.A. Biochem. J. (1972). 126, 257-273. Balcerski, J . S . , Pysh, E . S . , Chen, G.C. & Yang, J . T . J . Am. Chem. Soc. (1975). 97, 6274-6275. Pysh, E . S . Ann. Rev. Biophys. Bioeng. (1976). 5, 63-75. Darke, Α . , Finer, E . G . , Moorhouse, R. & Rees, D.A. J . Mol. Biol. (1975). 99, 477-486. Zimm, B.H. J . Chem. Phys. (1960). 33, 1349-1356. Jansson, P . E . , Kenne, L . & Lindberg, B. Carbohyd. Res. (1975). 45, 275-282. Melton, L . D . , Mindt, L., Rees, D.A. & Sanderson, G.R. Carbohyd. Res. (1976). 46, 245-257. Flory, P . J . Proc. Roy. Soc. ser. A. (1956). 234, 50-73.

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

7 Xanthan Gum—Molecular Conformation and Interactions R. MOORHOUSE, M. D. WALKINSHAW, and S. ARNOTT Department of Biological Sciences, Purdue University, West Lafayette, IN 47907

Xanthan Gum, the extracellular the microorganism Xanthomonas campestris has found widespread industrial use (1,2,3) because o f its unique r h e o l o g i c a l properties. The p o l y s a c c h a r i d e forms homogeneous aqueous d i s p e r s i o n s and s o l u t i o n s e x h i b i t i n g high viscosity, as w e l l as having characteristics of both p s e u d o p l a s t i c and plastic polymer systems ( 4 , 5 ) . Of particular s i g n i f i c a n c e is the a t y p i c a l insensitivity of s o l u t i o n viscosity to s a l t e f f e c t s and to heat, e s p e c i a l l y at h i g h ionic s t r e n g t h . Molecular weight measurements (6) i n d i c a t e p o l y d i s p e r s e systems of h i g h molecular weight (>2x10 ). The primary s t r u c t u r e of xanthan has r e c e n t l y been r e i n v e s t i g a t e d (7,8) and found to c o n s i s t of pentasaccharide r e p e a t i n g u n i t s (I). 6

Pyruvate is attached on average to about o n e - h a l f of the t e r m i n a l mannose r e s i d u e s ; 0 - a c e t y l groups correspond to one residue f o r each pentassaccharide r e p e a t i n g u n i t . When p r e v i o u s l y detected in bacterial p o l y s a c c h a r i d e s , pyruvate has u s u a l l y been observed on every r e p e a t i n g u n i t ( 9 , 1 0 ) . However, the c l o s e l y r e l a t e d 90

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Interactions

91

polysaccharides from other Xanthomonas species (11,12) also show differing pyruvate contents (13). We have prepared f i b e r s of both xanthan and the r e l a t e d p o l y s a c c h a r i d e from Xanthomonas p h a s e o l i (14). Using e s t a b l i s h e d techniques f o r f i b e r d i f f r a c t i o n and computer aided model b u i l d i n g (15,16,17,18,19) we have been able to examine the p o s s i b l e molecular conformations of xanthan. The almost i n d e n t i c a l X-ray d i f f r a c t i o n patterns,from a l a r g e number of p o l y s a c c h a r i d e samples from both X. campestris and X. p h a s e o l i , i n d i c a t e s an o v e r a l l s i m i l a r i t y of molecular conformation and primary sequence. R e s u l t s and D i s c u s s i o n It i s usually possibl t specimen f lon helical polymers i n which the molecule parallel. Often f u r t h e degree of a t h r e e - d i m e n s i o n a l l y ordered s i n g l e c r y s t a l . The xanthan X-ray d i f f r a c t i o n p a t t e r n (Figure 1) showing both c o n t i n uous i n t e n s i t y d i s t r i b u t i o n and Bragg maxima, i s c h a r a c t e r i s t i c of an ordered a r r a y of h e l i c e s which have t h e i r axes p a r a l l e l but are not f u r t h e r ordered (20). The presence of continuous d i f f r a c t i o n along the l a y e r l i n e s i n d i c a t e s that the i n d i v i d u a l molecules have random t r a n s l a t i o n s along and r o t a t i o n s about t h e i r axes and are not packed i n t o a w e l l developed c r y s t a l l a t t i c e . However, d e s t r u c t i v e i n t e r f e r e n c e has occurred near the center of the equator, l e a v i n g one broad Bragg r e f l e c t i o n of spacing 1.9 nm, the a r r a y of molecules t h e r e f o r e has some order when viewed down a molecular screw a x i s at s u f f i c i e n t l y low r e s o l u t i o n . The l a y e r l i n e spacing i s c o n s i s t e n t with a h e l i x of p i t c h 4.70 nm; the m e r i d i o n a l r e f l e c t i o n s (0,0,£) o c c u r r i n g only when Z=5n, suggests a 5-fold helix. T h i s gives a r i s e per backbone d i s a c c h a r i d e of 0.94 nm (Figure 2). The s t e r i c e f f e c t of the branching mannose r e s i d u e together with the consequent removal of the c e l l u l o s e 0(3)A—0(5) hydrogen bond across a l t e r n a t e 8-1,4 l i n k a g e s (u9ing the n o t a t i o n i n Figure 2) means that the backbone can no longer have the 2^ screw symmetry of c e l l u l o s e . Instead of the usual extended β-1,4 ribbon (21), a more sinuous h e l i x of the type shown i n F i g u r e 3 i s obtained. A p r i o r i we could have no preference f o r any of the four p o s s i b l e 5 - f o l d h e l i c a l models. The 5/1 and 5/4 conformations are r i g h t and left-handed r e s p e c t i v e l y and have a s i n g l e t u r n per h e l i x p i t c h while the two other (5/2 and 5/3) models a l s o d i f f e r by being r i g h t and left-handed and have two turns per h e l i x p i t c h . I n i t i a l l y molecular models f o r each of these f o u r s i n g l e h e l i c a l p o s s i b i l i t i e s , were examined assuming standard bondlengths, bond-angles and sugar r i n g conformation angles (15). The models were f u r t h e r c o n s t r a i n e d to e x h i b i t symmetry and p e r i o d i c i t y c o n s i s t e n t with the d i f f r a c t i o n p a t t e r n . On the b a s i s of a minimum s t e r i c compression comparison, the 5/1 (Figure 3) and 5/2 (Figure 4) right-handed h e l i c e s were most favored,

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

92

EXTRACELLULAR

MICROBIAL

POLYSACCHARIDES

Figure 1. Diffraction pattern typ ical for both Xanthomonas cam pestris and Xanthomonas phaseoli polysaccharides showing five-fold helical symmetry. The sharp Bragg reflection on the equator has a spacing of 1.9 nm.

Figure 2. The pentasaccharide repeat ing unit of xanthan showing atom label ing and aisaccharide backbone height. The unlettered residue and residue A are Ό-glucose, Β and Ε are O-mannose, and C is O-glucuronate.

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

7.

MOORHOUSE

Figure 3.

E TA L .

Xanthan

Gum Conformation

and

Interactions

The isolated 5/1 xanthan helix viewed perpendicular to the helix axis

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

93

Figure 4.

The isolated 5/2 xanthan helix viewed perpendicular to the helix axis

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

7.

MOORHOUSE E T A L .

Xanthan

Gum

Conformation

and

Interactions

95

having g l y c o s i d i c conformation angles w i t h i n the normal o l i g o saccharide ranges (Table 1) and no overshort non-bonded separat i o n s . With the left-handed h e l i c e s (5/3 and 5/4), minimization did not r e l i e v e a l l of the unacceptably short i n t e r a t o m i c cont r a c t s even a f t e r o p t i m i z a t i o n . In i s o l a t i o n there i s no d r i v i n g f o r c e to h o l d the s i d e chains c l o s e to the backbone and the i s o l a t e d chain models suggest a diameter of 3.8 nm as opposed to a value of 1.9 nm obtained from l a t e r a l p e r i o d i c i t i e s i n the d i f f r a c t i o n p a t t e r n . Studies on other branched polysaccharides favor the s i d e chains l y i n g roughly p a r a l l e l to the backbone (18), and we have t h e r e f o r e undertaken a second study i n which both packing and conformational v a r i a t i o n s were considered f o r each of the models. The most symmetrical and commonly observed c l o s e packing of polymeric molecules, havin hexagonal packing i n whic 6 nearest neighbors but not n e c e s s a r i l y f u r t h e r r e l a t e d . We t h e r e f o r e placed one xanthan h e l i c a l chain i n a hexagonal u n i t c e l l of s i d e a. = 2.19nm, c_ = 4.70nm, that i s c o n s i s t a n t with the e q u a t o r i a l Bragg r e f l e c t i o n indexed as (100). Minimizing s t e r i c r e p u l s i o n i n t h i s environment causes the side chain to f o l d down against the backbone. Stereochemically both the 5/4 and 5/3 h e l i c e s are u n l i k e l y as an unacceptable number of i n t r a m o l e c u l a r overshort contacts p e r s i s t a f t e r refinement. This r e i n f o r c e s our previous c o n c l u s i o n of right-handedness f o r the i s o l a t e d chains. Although the 5/1 and 5/2 h e l i c e s are s t e r i c a l l y acceptable, the 5/1 e x h i b i t s the more f a v o r a b l e comparison with o l i g o s a c c h a r i d e conformation angles. I t i s of i n t e r e s t to note that the backbone conformation angles shown i n Table I have v a r i e d l i t t l e during the process of wrapping the s i d e chains around the backbone. Further, the 5/1 packed' h e l i x (Figure 5) shows a number of p o t e n t i a l intramolec u l a r hydrogen bonds (Table I I and Figure 6). Relaxing the a t t r a c t i v e i n t e r a c t i o n (hydrogen bond) terms i n the refinement did not a l t e r the molecular conformation. Only the a d d i t i o n a l i n f l u e n c e of small p e r t u r b a t i o n s to the conformation angles about the branching mannose l i n k a g e caused the s t a b i l i s i n g i n f l u e n c e of the hydrogen bonds to be l o s t . The 'packed 5/2 h e l i x presents a much t i g h t e r s t r u c t u r e than the 5/1 model while e x h i b i t i n g some overshort i n t r a m o l e c u l a r contacts and few p o t e n t i a l hydrogen bonds and was considered u n l i k e l y on the b a s i s of t h i s a n a l y s i s . Our reasoning so f a r has been based on the premise that the e q u a t o r i a l Bragg r e f l e c t i o n on the d i f f r a c t i o n p a t t e r n (Figure 1), a r i s e s from the packing of s i n g l e molecular e n t i t i e s , the p a t t e r n does not t e l l us what form these take. In our examination of i n t e r - c h a i n i n t e r a c t i o n s , we have thus considered those i n t e r a c t i o n s that can a r i s e from some side-by-side arrangement of the 5/1 h e l i c e s and a l s o the case of c o a x i a l m u l t i p l e h e l i c e s . 1

1

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

96

EXTRACELLULAR MICROBIAL POLYSACCHARIDES

TABLE I Comparison o f backbone c o n f o r m a t i o n a n g l e s i n t h e i s o l a t e d and ' p a c k e d ' 5/1 a n d 5/2 h e l i c a l m o d e l s

Angle

-100+ -161

(a)

-78+

(b)

(d)

-78+

(a) - e [ c

( 1 ) A

"

e t 0

-

Using

9 [ 0

helices 5/2

-121

-148

-119

-111

-99

-98

-97

-92

-22

-81

-61

-98

-6

-98

( 4 )

,c

( 4 )

,c

C

( 5 )

]

, C

(5)A, (l)A'°(4) (4)

(c) - 6 [ C (d)

,o

'Packed' 5/1

-136

-10O>- -161

(c)

(b)

Isolated helices 5/1 5/2

Range

0

{ 1 )

> (4 C

) A

>

C

]

C

( 4 ) A

> (5) ] A

C

(5)' (1),°(4)A, (4)

] A

atom n o t a t i o n i n F i g u r e 2.

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

7.

MOORHOUSE E T A L .

(a)

Xanthan

Gum Conformation

and

Interactions

97

(b)

Figure 5. The 'packed' 5/1 helix viewed (a) perpendicular to and (b) down the helix axis

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

EXTRACELLULAR MICROBIAL POLYSACCHARIDES

Figure 6. Possible hydrogen bonds ( ) that may stabilize the molecule. Some adjoining residues are omitted for clarity, the backbone having solid bonds. See also Table II.

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

Xanthan

MOORHOUSE ET AL.

Gum Conformation

and

Interactions

TABLE I I P o s s i b l e a t t r a c t i v e i n t e r a c t i o n i n the X and 5/2 h e l i c e s

Model

Overshort

5/1

aampestris

3

5/1

Potential Hydrogen bonds

contacts (nm)

non 0

( 2 ) ~ -

°(8a)

D

°(6)

°(5)C

°(2)A

** °(7)B

°(3)B—* °(6) [

o

r

°(3)B

°(2)D 5/2

Η

°(5)Α··· (4)Α (0.195 nm)

°(3)~

Υ

" °(5)C

°(6b)C

> 0

(5)A

°(6)A

* °(5)

°(3)B

' °(5)C

°(3)C

°(5)D

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

]

100

EXTRACELLULAR MICROBIAL POLYSACCHARIDES

P l a c i n g a s i n g l e 5/1 h e l i x i n o u r h e x a g o n a l c e l l r e v e a l s few i n t e r a c t i o n s w i t h i t s nearest neighbors. This suggests that t h e h e l i c e s a r e s l o t t i n g i n t o some g r o o v e t h a t i s w i d e enough t o a c c o m o d a t e them w i t h o u t s t e r i c c l a s h e s . A l t e r n a t i v e l y t h e molec u l e c o u l d be c o n s i d e r e d a s a r i g i d r o d o f p o l y s a c c h a r i d e s u r rounded by a c y l i n d e r o f w a t e r , i n which case v e r y few p o l y s a c c h a r i d e - p o l y s a c c h a r i d e i n t e r a c t i o n s would be a p p a r e n t . Furthermore, a s s u c h a s i t u a t i o n c l o s e l y m i m i c s t h e s o l u t i o n s t a t e , t h e unusual s o l u t i o n p r o p e r t i e s would p r o b a b l y a r i s e from i n t e r a c t i o n s b e t w e e n r e g i o n s o f O r d e r e d ' w a t e r some o f w h i c h may b e t i g h t l y bound t o t h e p o l y s a c c h a r i d e . Current X-ray f i b e r d i f f r a c t i o n t e c h n o l o g y c a n n o t e n a b l e u s t o l o c a t e t h i s amount o f w a t e r ( 1 8 ) , p o s s i b l y NMR s t u d i e s o n s o l u t i o n s may be a b l e t o l o c a t e 'ordered water but without t h d e t a i l t h a t i sometime a v a i l a b l e from d i f f r a c t i o ment f o r p r o l o n g e d p e r i o d c e l l volume c o n s i s t e n t w i t h a s h r i n k a g e i n t h e Bragg s p a c i n g on the e q u a t o r w h i l e t h e f i b e r a x i s d i m e n s i o n r e m a i n s u n a l t e r e d . Apparently the molecular conformation o f the xanthan molecule s u r v i v e s d r y i n g w i t h l i t t l e change a n d a s u b s t a n t i a l q u a n t i t y o f w a t e r w h i c h f i l l s o u t t h e s t r u c t u r e i s n o t f i r m l y bound. W h i l e i t i s p o s s i b l e t o c o n s t r u c t a double h e l i c a l model, u s i n g t h e 5/1 s i n g l e h e l i x a s p r e c u r s o r , i n w h i c h t h e s e c o n d c o a x i a l s t r a n d i s p a r a l l e l t o , a n d r e l a t e d t o , t h e f i r s t b y 180 r o t a t i o n some a p p a r e n t l y u n r e s o l v a b l e o v e r s h o r t i n t e r - s t r a n d contacts exist. I t i s p o s s i b l e t h a t r e l a x i n g t h e summetry s o t h a t the p a r a l l e l c o a x i a l s t r a n d s a r e n o t r e l a t e d b y a 180 f i b e r a x i s r o t a t i o n o r a r e a n t i p a r a l l e l t o one a n o t h e r , c o u l d r e s u l t i n a c c e p t a b l e i n t e r a c t i o n s between c h a i n s . Should t h i s be t h e case i t w i l l s t i l l be n e c e s s a r y t o o b t a i n s u p p o r t i n g e v i d e n c e from other sources t o demonstrate t h e e x i s t e n c e o f double h e l i c e s . N o r m a l l y t h i s would t a k e t h e form a comparison o f t h e model w i t h the X - r a y i n t e n s i t y d a t a f r o m a c r y s t a l l i n e d i f f r a c t i o n p a t t e r n (e.g. 16,17,18) p l u s e v i d e n c e f r o m s o l u t i o n s t u d i e s o f b i - m o l e c u l a r i t y ( e . g . 2 2 ) . We w o u l d s t r e s s however t h a t t h e r e i s no e v i d e n c e o f d o u b l e h e l i c e s e i t h e r i n s o l u t i o n (27) o r t h e s o l i d s t a t e . R e c e n t l y , we h a v e b e e n a b l e t o o b t a i n a d i f f r a c t i o n p a t t e r n t h a t e x h i b i t s i n c r e a s e d c r y s t a l l i n i t y and which h a s been t e n t a t i v e l y i n d e x e d o n a t e t r a g o n a l c e l l i n w h i c h f o u r 5/1 s i n g l e h e l i c e s w i l l p a c k w i t h t h e minimum o f s t e r i c c o m p r e s s i o n . A r e finement u s i n g b o t h s t e r e o c h e m i c a l and X-ray i n t e n s i t y d a t a has not y e t been completed. 1

Conclusions T h i s p r e l i m i n a r y s t u d y shows t h a t t h e o r d e r e d c o n f o r m a t i o n of xanthan i n t h e condensed s t a t e , and p r o b a b l y i n s o l u t i o n , i s r e l a t e d t o t h e 5/1 h e l i x o u t l i n e d h e r e .

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

7.

MOORHOUSE E T A L .

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Gum

Conformation

and Interactions

101

The Interactions t h a t o c c u r i n solution, giving rise t o viscosity effects showing t h e characteristic o f both flexible a n d stiff cross-linked r e g i o n s (4,5) must arise f r o m associations of t h e o r d e r e d 5/1 helical regions. The order/disorder transition s e e n with change o f t e m p e r a t u r e in solution ( 2 3 , 2 4 ) , w o u l d seem l i k e l y t o a r i s e f r o m c o n f o r m a t i o n a l changes p r i m a r i l y w i t h i n t h e s i d e c h a i n a s i t moves away f r o m i t s c l o s e a s s o c i a t i o n w i t h t h e o r d e r e d backbone e i t h e r accompanied b y , o r b e f o r e , c o n f o r m a t i o n a l changes i n t h e b a c k b o n e . T h i s s p r e a d i n g o f t h e 'arms o f t h e p o l y s a c c h a r i d e would cause a n i n c r e a s e d hydrodynamic volume and hence p r o v i d e t h e v i s c o s i t y s t a b i l i t y noted a t e l e v a t e d tempera tures (1,2,3,4). A s s o c i a t i o n , i n s o l u t i o n o f s i n g l e h e l i c e s does n o t r e q u i r e gel formation, a fact tha h e l i c a l xanthan,which doe Weak g e l a t i o n o b s e r v e d temperature probably due t o a n a g g r e g a t i o n phenomenon. I t i s i n t e r e s t i n g t o n o t e t h a t t h e 5/1 h e l i x p r e s e n t s two d i s t i n c t f a c e s ; one h a v i n g t h e s i d e c h a i n s a n d c h a r g e d g r o u p s , the o t h e r e s s e n t i a l l y t h e c e l l u l o s e backbone. As xanthan i n t e r a c t s s y n e r g i s t i c a l l y w i t h t h e 3-1,4 l i n k e d g a l a c t o m a n n a n s l o c u s t b e a n a n d g u a r gums, i t i s p o s s i b l e t h a t t h i s t a k e s p l a c e a t t h e c e l l u l o s e ' g r o o v e ' i . e . b e t w e e n s i m i l a r 8-1,4 l i n k e d g l y c a n s . I t i s t h o u g h t t h a t 'smooth' u n s u b s t i t u t e d r e g i o n s o f t h e g a l a c t o mannan a r e i n v o l v e d i n t h e a s s o c i a t i o n ( 2 3 , 2 5 ) . More d e t a i l e d i n t e r p r e t a t i o n s o f t h i s c o n t i n u i n g w o r k w i l l be p u b l i s h e d e l s e w h e r e ( 2 6 ) . 1

Acknowledgement s We w i s h t o t h a n k D r s . A. J e a n e s a n d P.A. S a n f o r d , U.S.D.A,, P e o r i a a n d D r . I.W. C o t t r e l l , K e l c o , San D i e g o , f o r t h e i r g e n e r ous g i f t s o f s a m p l e s .

Literature Cited 1. Jeanes, A. (1973) In "proceedings of the ACS Conference on Water Soluble Polymers", ed. N.M. Bikales, Plenum Press, New York. pp. 227-242. 2. Jeanes, A. (1974) J . Polymer S c i . , Symp. No. 45, 209-227. 3. McNeely, W.H. and Kang, K.S. (1973) In "Industrial Gums" R.L. Whistler and J.N. BeMiller eds., pp. 473-497, Academic Press, New York. 4. Jeanes, Α., Pittsley, J.E. and Senti, F.R. (1961) J . Appl. Polymer S c i . , 5,519-526. 5. Jeanes, A. and Pittsley, J.E. (1973) J . Appl. Polymer S c i . , 17,1621-1624. 6. Dintzis, F.R., Babcock, G.E. and Tobin, R. (1970) Carbohyd. Res. 13,257-267.

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7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27.

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POLYSACCHARIDES

Jansson, P.E., Kenne, L. and Lindberg, B. (1975) Carbohyd. Res. 45,275-282. Melton, L.D., Mindt, L., Rees, D.A. and Sanderson, G.R. (1976) Carbohyd. Res., 46,245-257. Choy, Y.M. and Dutton, G.G.A. (1973) Can. J. Chem. 51,198-207. Choy, Y.M., Fehmel, F . , Frank, N. and Stirm, S. (1975) J . Virology 16,581-590. Gorin, P . A . J . , and Spencer, J.F.T. (1961) Can. J . Chem. 39, 2282-2289. Gorin, P.A.J. and Spencer, J.F.T. (1963) Can. J . Chem. 41, 2357-2361. Orentas, D.G., Sloneker, J.H. and Jeanes, A. (1963) Can. J . Microbiol., 9,427-430. Lesley, S.M. and Hochster R.M (1959) Can J Physiol 37 513-529. Arnott, S. and Scott, (1972) (Perki ) 324-335. Guss, J.M., Hukins, D.W.L., Smith, P.J.C., Winter, W.T., Arnott, S., Moorhouse, R. and Rees, D.A. (1975) J . Mol. Biol. 95,359-384. Winter, W.T., Smith, P.J.C. and Arnott, S. (1975) J . Mol. Biol. 99,219-235. Moorhouse, R., Winter, W.T. and Arnott, S. (1976) J . Mol. Biol, in press. Smith, P.J.C. and Arnott, S. (1976) Acta Crystallogr., in press. Arnott, S. (1973) Trans. Amer. Crystallogr. Assoc., 9,31-56. Rees, D.A. (1973) In ΜΤΡ International Review of Science: Organic Chemistry Series 1, vol. 7, G.O. Aspinall, ed. 251283. Arnott, S., Fulmer, Α., Scott, W.E., Dea, I.C.M., Moorhouse, R, and Rees, D.A. (1974) J . Mol. Biol., 90,269-284. Morris, E.R. and Rees, D.A. (1976) J . Biol. Chem., in press. Holzworth, G. (1976) J . Biol. Chem., in press. Dea, I.C.M., McKinnon, A.A. and Rees, D.A. (1972) J . Mol. Biol. 68,153-172. Moorhouse, R. and Arnott, S., J . Mol. Biol., in preparation. Morris, E.R. personal communication.

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

8 Infrared and Raman Spectroscopy of Polysaccharides JOHN BLACKWELL Department of Macromolecular Science, Case Western Reserve University, Cleveland, OH 44106

During the last 30 years, infrared spectroscopy has been used to obtain informatio chain conformations of Raman spectra have also been available, and have provided useful complementary data. These techniques have mainly been applied in conjunction with other structural methods, especially x-ray diffraction, where the vibrational data have often given information on hydrogen bonding networks and side-group orientations. This work for polysaccharides can be discussed in two general areas. Firstly, there are the direct structural investigations, which have utilized the identifiable group frequencies. The 0-H, C-H, and carboxyl stretching frequencies, as well as some of the amide modes ,can be identified and their infrared dichroisms determined. Hence, Marrinan and Mann (1) and subsequently Liang and Marchessault (2,3) showed that the four polymorphic forms of cellulose had different spectra in the 0-H stretching region, indicative of different hydrogen bonding in their crystal structures. Based on the dichroisms of the 0-H and C-H stretching bands, these authors discussed the possibilities for hydrogen bonding and selected what they considered the most likely structures. Similarly for chitin, (4,5) the orientation of the amide side chain relative to the fiber axis was determined from the dichroisms of the amide I and II bands. Secondly, known conformations of polysaccharides can often be differentiated by their I.R. and Raman spectra. Apart from the stretching frequencies listed above, most of the bands in polysaccharide spectra are due to complex molecular motions and structural interpretation of their dichroisms is not possible at this time. Nevertheless, despite this lack of understanding, changes in frequency or intensity can be used to follow polymorphic transitions. For example, the transition from cellulose I to cellulose II during mercerization has-been followed by monitoring four intensities in the1500-800cm range (6,7). -1

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T h i s second aspect: i d e n t i f i c a t i o n of known conformations, i s probably the major area f o r p o t e n t i a l s t r u c t u r a l work on polysaccharides using t h i s technique. Raman spectroscopy and the r e c e n t l y developed F o u r i e r transform I.R. method, allow the s p e c t r a of polysaccharides i n s o l u t i o n t o be recorded at r e s o l u t i o n s comparable to the s o l i d s t a t e s p e c t r a . As a r e s u l t , i t i s p o s s i b l e to compare the s o l u t i o n s p e c t r a with those of known s o l i d s t a t e s t r u c t u r e s and hence a s s i g n a conformation to the p o l y saccharide i n s o l u t i o n or i n g e l s , i n a manner analogous to i d e n t i f i c a t i o n of polypeptide conformations i n s o l u t i o n using c i r c u l a r dichroism. In t h i s paper I w i l l review some of the progress we have made i n the l a s t few years i n a n a l y s i s o f a v a r i e t y of p o l y saccharide systems. Ou amylose l e d on to s t u d i e connective t i s s u e glycosaminoglycans and hence t o our present i n t e r e s t i n b a c t e r i a l polysaccharides i n s o l u t i o n . In a d d i t i o n , we have made t h e o r e t i c a l p r e d i c t i o n s of polysaccharide s p e c t r a using normal coordinate a n a l y s i s . Amylose Amylose (a(l,4)-D-glucan) i s the simplest polysaccharide which can be c r y s t a l l i z e d i n d i f f e r e n t chain conformations. P r e c i p i t a t i o n from organic s o l v e n t s leads to the s o - c a l l e d V-amylose s t r u c t u r e , (8,9) where the chains form compact h e l i c e s with s i x glucose residues per t u r n r e p e a t i n g i n 8.0Â. A v a r i e t y of chain packings are p o s s i b l e , depending on the degree of h y d r a t i o n of the presence of organic solvent molecules, but the b a s i c chain conformation i s b e l i e v e d t o be the same. When Vamylose i s maintained at high humidity f o r a p e r i o d of time, conv e r s i o n occurs to one or other of the s t r u c t u r e s found i n n a t i v e s t a r c h , A- and B-amylose, which again a r e b e l i e v e d t o be d i f f e r ent packings of a common chain conformation. The proposed conformation f o r B-amylose (10) i s a more extended 6^ h e l i x , repeating i n 10.4Â. Double h e l i c e s have a l s o been considered, t i l ) but such s t r u c t u r e s w i l l a l s o i n v o l v e more extended chains than occur i n V-amylose. The Raman spectrum o f V-amylose (12) i s shown i n Figure 1. The spectrum f o r B-amylose i s very s i m i l a r , except f o r four small but s i g n i f i c a n t d i f f e r e n c e s , which are shown i n Figure 2 : _ - l i n e s at 946 and 1263cm" f o r V-amylose s h i f t t o 936 and 1254cm~ r e s p e c t i v e l y i n the^B-form, and the r e l a t i v e i n t e n s i t i e s of l i n e s at 1334 and 2940cm are decreased with respect t o t h e i r neighbors (12). Based on our own C-H and 0-H deuterium exchange experiments, three of^the l i n e s i n question can be assigned as f o l l o w s . The 2040cm _ ^ i s probably a CH^ antisymmetric s t r e t c h i n g mode; those at 1334cm and 12£3cm are mixed -CH^OH deformation modes. For the mode at 946cm , from a study of tne s p e c t r a of glucose monomers and oligomers t h i s i s assigned as a l i n k a g e mode,

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

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Raman spectrum of V -amylose in the region 1500-300 cm' (12) 1

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i . e . a complex mode i n v o l v i n g a s i g n i f i c a n t c o n t r i b u t i o n from motion of the g l y c o s i d i c C-O-C. The V - s t r u c t u r e has compact h e l i c e s i n which residues on successive turns ( i . e . residues i and i+6) are l i n k e d by i n t e r turn hydrogen bonds i n v o l v i n g the -CH^OH s i d e chains. On conversion to the B-form, the chain becomes more extended and these i n t e r t u r n hydrogen bonds w i l l be broken. T h i s w i l l probably l e a d to a r e o r i e n t a t i o n of the s i d e chains and the formation of other hydrogen bonds, e.g. to water molecules. Such changes would be l i k e l y to a f f e c t the frequency and i n t e n s i t y of the -CH^OH modes and would account f o r the changes seen. At the same time, expansion of the chain w i l l be e f f e c t e d by r o t a t i o n of the residues about the g l y c o s i d i c l i n k a g e s , which^would f i t i n with the observed frequenc mode. In the s t r u c t u r turn bond i s broken an throug , the f i b e r repeat i s increased by r o t a t i o n of the residues about the g l y c o s e d i c bonds, which i s compatible with the observed Raman changes. Normal coordinate a n a l y s i s of the i s o l a t e d Vamylose chain p r e d i c t s complex deformation modes which are i n accord with the above assignments. (13) Increase i n the f i b e r repeat of the h e l i x to_J0.4Â reduces the frequency of the " l i n k a g e " mode by 4 cm . The above Raman c h a r a c t e r i s t i c s f o r V- and B- amylose can be used to i n t e r p r e t the s p e c t r a of t h i s polymer i n s o l u t i o n . Figure 3 shows the Raman spectrum of amylose i n deuterated DMSO.(12) Only a short region of the spectrum can be recorded, but the s p e c t r a l c h a r a c t e r i s t i c s are those of the B-form, with the observed frequency at 1254cm_^ and r e l a t i v e l y low r e l a t i v e i n t e n s i t y f o r the l i n e at 1334cm . These r e s u l t s are against the presence of the V - h e l i x i n s o l u t i o n , which i s i n t e r e s t i n g s i n c e the V - s t r u c t u r e i s formed when f i l m s are cast from t h i s solvent. This i s not to say that B - h e l i c e s are present i n s o l u t i o n s i n c e we b e l i e v e that random, s o l v a t e d amylose may show the same c h a r a c t e r i s t i c s . However, i t i s l i k e l y that the CH^OH groups are hydrogen bonded to solvent molecules r a t h e r than being involved i n i n t e r t u r n bonds on compact V - h e l i c e s . Glycosaminoglycans We are i n the process of extending t h i s type of work to the glycosaminoglycans of connective t i s s u e , each of which can be prepared as o r i e n t e d f i l m s i n a number of d i f f e r e n t chain conformations, depending on the r e l a t i v e humidity and type of counter ions. In c o l l a b o r a t i o n with E.D.T. Atkins and coworkers at U n i v e r s i t y of B r i s t o l , we have prepared c r y s t a l l i n e f i l m specimens of h y a l u r o n i c a c i d , c h o n d r o i t i n 4- and 6 - s u l f a t e s , and dermatan s u l f a t e . Raman s p e c t r a could not be obtained due to fluorescence of the specimens i n the l a s e r beam. However, using F o u r i e r transform techniques we have been able to record the

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i n f r a r e d s p e c t r a o f the same o r i e n t e d f i l m s (14) as were prepared f o r x-ray work. Figure 4 shows the p o l a r i z e d i n f r a r e d s p e c t r a of c h o n d r o i t i n 6 - s u l f a t e , prepared i n the 8^ h e l i c a l conformation. T h i s polymer approximates t o a repeating d i s a c c h a r i d e of N - a c e t y l D-galactosamine 6 - s u l f a t e and D-glucuronic a c i d , with a l t e r n a t i n g 8(1,4) and 8(1,3) l i n k a g e s , and~has e i g h t d i s a c c h a r i d e s r e p e a t i n g i n three turns, with a r i s e per residue of 9.8Â (15). The s p e c t r a i n Figure 4 show perpendicular dichroism f o r the amide I and I I modes a t 1650 and 1560cm r e s p e c t i v e l y , i n d i c a t i n g that the plane of the amide group i s approximately perpendicular t o the chain a x i s . S i m i l a r l y the antisymmetric and symmetric carboxyl s t r e t c h i n g frequencies a t 1620 and 1420cm respectively, both have s l i g h t perpendicular dichroism and the plane of the carboxyl group i s more n e a r l y perpendicular t o the c h j i n a x i s The bands with p a r a l l e complex C-0 and C-C s t r e t c h i n to that f o r c e l l u l o s e i n the same range, and i s c h a r a c t e r i s t i c of extended chain polysaccharides. We have a l s o prepared c h o n d r o i t i n 4 - s u l f a t e and dermatan s u l f a t e , each i n the 3- conformation, (16,17) and two forms of h y a l u r o n i c a c i d , both 4^ conformations with d i f f e r e n t f i b e r repeats (18,19). These give s i m i l a r r e s u l t s t o those f o r c h o n d r o i t i n 6 - s u l f a t e f o r the amide o r i e n t a t i o n . For the two forms of h y a l u r o n i c a c i d , and c h o n d r o i t i n 4 - s u l f a t e however, the carboxyl symmetric s t r e t c h i n g band has p a r a l l e l dichroism. These conformations a r e l e s s extended than the 8^ form of C6S, and the C-C0Ô bond can be o r i e n t e d so that i t i s more n e a r l y p a r a l l e l t o the chain a x i s . The same band has perpendicular dichroism f o r dermatan s u l f a t e , which i s c o n s i s t a n t with the CI chain f o r the L - i d u r o n i c a c i d residue of t h i s polysaccharide (17). So f a r we have only examined h y a l u r o n i c a c i d prepared i n two d i f f e r e n t conformations, both 4^ with d i f f e r e n t f i b e r repeats. These specimens do not show any s p e c t r a l d i f f e r e n c e s which can be a s c r i b e d t o the d i f f e r e n c e i n conformation. T h i s i s disappoint i n g , but such d i f f e r e n c e s are more l i k e l y when there are l a r g e r d i f f e r e n c e s i n conformation, e.g. between 3-, 8^, and 4- h e l i c e s . These i n v e s t i g a t i o n s are continuing, and w i l l be a p p l i e d t o s o l u t i o n s i f the d i f f e r e n t conformations can be s u c c e s s f u l l y differentiated. Bacterial

Polysaccharides

More r e c e n t l y we have examined the b a c t e r i a l polysaccharide xanthan, working with specimens obtained from Drs. A. Jeanes and P.A. Sandford at U.S.D.A., P e o r i a . T h i s polysaccharide i s b e l i e v e d t o be a repeating p o l y s a c c h a r i d e , the backbone i s a 8 ( l , 4 ) - g l u c a n with a l t e r n a t i n g residues having a t r i s a c c h a r i d e of mannose 6-acetate, g l u c u r o n i c a c i d , and mannose; approximately 50% of the t e r m i n a l mannose residues have a peruvate residue attached at the 4 and 6 p o s i t i o n s .

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

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Figure S. Raman spectrum of amylose in deuterated DMSO solution (Me SO — d ) in the 1500-1200 cm' re gion. ( ) indicates the approximate base scattering by the 2

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Polarized infrared spectra for the 8 conformation of chondroitin 6-sulfate. ( )A ;(—)A (U). S

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This p o l y s a c c h a r i d e has a very i n t e r e s t i n g property i n that the v i s i o s i t y of an aqueous s o l u t i o n undergoes a sudden i n c r e a s e as the temperature r i s e s (20). I t i s argued that the polysacchar i d e has a compact conformation at low temperatures and undergoes a t r a n s i t i o n to an expanded form at a s p e c i f i c temperature, reported at 55°C. This t r a n s i t i o n has been followed by Rees and coworkers (21) by N.M.R., which i n d i c a t e s an ordered conformation below 55°C and a d i s o r d e r e d random c o i l at higher temperatures. These workers have a l s o followed the change by c i r c u l a r dichroism spectroscopy . We have used F o u r i e r transform i n f r a r e d spectroscopy to i n v e s t i g a t e these thermal changes (22). The specimens were d i a l i z e d thoroughly against d i s t i l l e d water p r i o r to r e c o r d i n g the s p e c t r a . The s p e c t r f 1% xantha s o l u t i o t different temperatures are show seen i n the frequencie general broadening at higher temperatures, i n d i c a t i n g development of a l e s s ordered s t a t e . T h i s broadening can be quantized i n a v a r i e t y of ways; one convenient method i s to measure the areas of the peaks above the unresolved background. P l o t s of these " i n t e n s i t i e s " against temperature f o r three of the bands are shown i n Figure 6. A l l three show a sigmoidal t r a n s i t i o n , with midpoint at 40°C, i n d i c a t i n g development of a more random conformation above t h i s temperature. A d d i t i o n of s a l t s to the xanthan s o l u t i o n i s known to p r e vent the t r a n s i t i o n i n the v i s c o s i t y (20). F i g u r e 7 shows the i n f r a r e d s p e c t r a of a 1% xanthan s o l u t i o n i n 1% KC1 over the same temperature range as i n Figure 5. The c o n t r a s t between Figures"5 and 7 i s q u i t e s t r i k i n g i n that the s p e c t r a of the s a l t s o l u t i o n s show very l i t t l e change with temperature. Our observations of a t r a n s i t i o n at 40°C i s p u z z l i n g s i n c e other workers have reported 55°C. We have a l s o performed v i s c o s i t y and CD measurements on s o l u t i o n s of t h i s polysacchar i d e , and observe t r a n s i t i o n s with midpoints of 38° and 40°. I t i s p o s s i b l e that our specimen of xanthan i s d i f f e r e n t from those used by other workers, perhaps due to mutation or degradation, or that we have achieved a lower i o n i c s t r e n g t h when the specimen was d i a l y s e d against water. Acknowlegements T h i s work was supported by N.S.F. Grant No. GB 32405. am indebeted to my c o l l a b o r a t o r s i n Cleveland and B r i s t o l , e x p e c i a l l y J . J . C a e l , J . Southwick, and J.L. Koenig f o r t h e i r part i n the work described above.

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

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In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

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Figure 7. Fourier transform infrared spectra of a 1% solution of xanthan in 1% aqueous potassium chloride solution at 22* 35* 45°, and 55°C (22)

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

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Abstract Progress in several areas is described in the application of vibrational spectroscopy to investigate the structure and confor mation of polysaccharides. Infrared and Raman spectroscopy pro vides information on the orientation of side groups and the type of hydrogen bonds formed in crystalline polysaccharide structures. In addition, spectral characteristics of polysaccharides prepared in different known crystal structures can be used to investigate the conformation in solution. These methods have been applied to investigations of amylose, where differences in the Raman spectra of the V- and B- forms have been interpreted in terms of the change in conformation, and indicate that the V-conformation is not present in solution Fourier transform infrared spectra of oriented crystallin aminoglycans have been amide and carboxyl groups for the various crystal structures. Finally, infrared spectra of xanthan in solution show that an order-disorder transition occurs as the temperature is increased, which is correlated with the sharp increase in viscosity in the same temperature range. Literature Cited 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.

Marinan, H.J. and Mann, J., J. Polymer Sci. (1958), 32, 357. Liang, C.Y. and Marchessault, R.H., J. Polymer Sci., (1959), 37, 385. Marchessault, R.H. and Liang, C.Y., J. Polymer Sci., (1960), 43, 31. Darmon, S.E. and Rudall, K.M., Disc. Farad. Soc., (1950), 9, 215. Carlstrom, D., J. Biophys. Biochem. Cytol.,(1957),3,669. McKenzie, A.W. and Higgins, H.G., Svensk Papperstidn., (1958) 61, 893. Hurtubise, F.G. and Krassig, Η., Anal. Chem., (1960), 32, 177. Rundle, R.F. and French, D., J. Amer. Chem. Soc., (1943), 65, 558. Zobel, H.F., French, A.D., and Hinkle, M.E., Biopolymers, (1967), 5, 837. Blackwell, J., Sarko, Α., and Marchessault, R.H., J. Molec. Biol., (1969), 42, 379. Kainuma, K. and French, D., Biopolymers, (1972), 11, 2241. Cael, J . J . , Koenig, J.L., and Blackwell, J., Carbohydrate res., (1973), 29, 123. Cael, J . J . , Koenig, J.L., and Blackwell, J., Biopolymers, (1975), 14, 1885. Cael, J . J . , Isaac, D.H., Blackwell, J., Koenig, J.L., Atkins, E.D.T., and Sheehan, J.K., Carbohydrate Res. in press. Arnott, S, Guss, J.M., Hukins, D.M., and Mathews, M.B., Science, (1975), 180, 743.

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Isaac, D.H. and Atkins, E.D.T., Nature (London), New Biol. (1973), 244, 252. 17. Atkins, E.D.T. and Isaac, D.H., J . Molec. B i o l . , (1973), 80, 773. 18. Dea, I.C.M., Moorhouse, R., Rees, D.A., Guss, J.M., and Balazs, E.A., Science, (1973), 179, 560. 19. Guss, J.M., Hukins, D.W., Smith, P.J.C., Winter, W.T., Arnott, S., Moorhouse, R., and Rees, D.A., J . Molec. B i o l . , (1975), 95, 359. 20. Jeanes, Α., Pittsley, J . E . , and Senti, Α., J . Appl. Polymer S c i . , (1961), 17, 519. 21. Rees, D.A. and Morris, Ε., (in press). 22. Southwick, J., Koenig, J.L., and Blackwell, J., (in press).

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

9 Nuclear Magnetic Resonance and Mass Spectroscopy of Polysaccharides FRED R. SEYMOUR Baylor College of Medicine, Marrs McLean Department of Biochemistry, Houston, TX 77030

Various physical method determination have been developed. This discussion will be primarily limited to gas-liquid chromatography/mass spectrometry (g.l.c.-m.s.) and nuclear magnetic resonance spectrometry (n.m.r.). The equipment employed for these determinations has either been recently developed, or recently brought to the degree of sophistication necessary for carbohydrate studies. The procedures are rapid compared to previous methods of obtaining analogous data. Though most of the techniques may be applied to any carbohydrate containing compound, this work has been done with extra-cellular polysaccharides. The extracellular polysaccharides have proven to be valuable materials as they are readily obtainable homogeneous polymers which can be produced in relatively large quantities. These large amounts of uniform polysaccharides provide material to compare the g.l.c.-m.s. data to the much less sensitive (in terms of amount required) n.m.r. data. The wide variety of mannans and glucans available provide a considerable range of structures for this correlation. Though data is available from a number of different sources, the selected examples will be chosen from studies in which I have participated. Five general methods have been employed: a) polymer hydrolysis followed by g.l.c.-m.s., b) polymer permethylation followed by hydrolysis and then g.l.c.-m.s., c) recording the polymer's C-13 n.m.r. spectra, d) recording the polymer's P-31 n.m.r. spectra, and e) employing selective hydrolysis combined with high pressure chromatography (h.p.c.) These methods are complimentary in duplication and confirmation of data, but each method yields specific unduplicatable information. G a s - 1 i q u i d chromatography A v a r i e t y o f d e r i v a t i v e s , column p a c k i n g s , and oven

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c o n d i t i o n s have been employed f o r s a c c h a r i d e g . l . c . séparât i o n 0 ) . In o u r h a n d s , t h e p e r a c e t y l a t e d a l d o n o n i t r i l e s (PAAN) have p r o v e n t o be t h e most u s e f u l d e r i v a t i v e s . The b a s i c r e a c t i o n f o r c o n v e r s i o n o f s a c c h a r i d e s t o t h e i r PAAN d e r i v a t i v e s i s w e l l known. D i f f e r i n g g . l . c . c o n d i t i o n s have been r e p o r t e d f o r t h e s e p a r a t i o n o f PAAN d e r i v a t i v e s (_2, 3., k) . We f i n d a most s a t i s f a c t o r y s y s t e m i s t o u s e n e o p e n t y l g l y c o l s u c c i n a t e . The r e s u l t i n g n a r r o w peaks a l l o w good compound s e p a r a t i o n , t h e columns c a n e a s i l y h a n d l e t h e amounts o f m a t e r i a l r e q u i r e d f o r m.s. d e t e r m i n a t i o n s , and t h e base l i n e r e m a i n s f l a t , not a f f e c t i n g t h e h y d r o g e n f l a m e d e t e c t o r o r t h e mass s p e c t r o m e t e r . The a d v a n t a g e s o f t h e PAAN d e r i v a t i z a t i o n p r o c e d u r e a r e , a) i t i s f a s t and e f f i c i e n t , b) t h e a n o m e r i c c e n t e r o f asymmetry i s d e s t r o y e d , each s a c c h a r i d yieldin singl derivative the r e s u l t i n g s t r a i g h t - c h a i a b l e mass s p e c t r a . I t i s assumed t h a t a s u c c e s s f u l n o n - d e g r a d i n g h y d r o l y s i s has p r e c e d e d t h e s a c c h a r i d e d e r i v a t i z a t i o n and g . l . c . - m . s . determination. Carbohydrate h y d r o l y s i s i s e s s e n t i a l l y a subject i n i t s e l f , b u t i t c a n be n o t e d t h a t t h e C-13 n.m.r. d a t a p r o v i d e a u s e f u l n o n - d e s t r u c t i v e c h e c k on g . l . c . - m . s . s t r u c t u r a l determinations which hypothesize a non-degrading h y d r o l y s i s . TABLE 1 R e t e n t i o n Times o f P e r a c e t y l a t e d Aldononîtri l e D e r i v a t i v e s of Aldoses (a) Parent

Aldose

DL-glyceraldehyde D-erythrose D-digitoxose L-rhamnose 2-deoxy-D-r i bose D-ribose L-fucose D-Lyxose D-arabinose D-xylose (a)

Retention Time (min) 1.2 5.6 9.6 10.6 12.2 12.8 12.8 13.6 14.2 15.6

Parent

Aldose

Retention Time (min)

D-allose 2-deoxy-D-glucose D-mannose D-talose 2-deoxy-D-galactose D-glucose D-galactose 5-thio-D-glucose D-glucoheptose N - a c e t y 1 - D - g l u c o s a m i ne

19.0 19.2 19.6 19.6 20.2 21.0 21.8 24.6 26.8 3^.2

3% N e o p e n t y l g l y c o l s u c c i n a t e on 60/80 mesh Chromosorb W i n a p a c k e d g l a s s column 2 mm by 4 f t .

The r e t e n t i o n t i m e s o f 20 s a c c h a r i d e PAAN d e r i v a t i v e s a r e shown i n T a b l e 1. O n l y two p a i r s c a n n o t be r e s o l v e d and o n l y o n e d i f f e r s o n l y i n terms o f s t e r e o c h e m i s t r y (mannose and t a l o s e ) a n d c a n n o t be s e p a r a t e d by g . l . c . - m . s . T h i s a l l o w s o n e r e a s o n a b l e c o n f i d e n c e i n e s t a b l i s h i n g which sugars are not p r e s e n t . Partial

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

116

EXTRACELLULAR MICROBIAL POLYSACCHARIDES

l i s t s o f t h e r e t e n t i o n t i m e s o f t h e s e PAAN d e r i v a t i v e s on d i f f e r e n t c o l u m n s ( p a c k e d L A C - 4 R - 8 8 6 (3) and open t u b u l a r S E - 3 0 (k)) i n d i c a t e t h a t t h e r e t e n t i o n t i m e o r d e r i s t h e same f o r t h e s e compounds. The r e t e n t i o n t i m e s o f t h e s e PAAN d e r i v a t i v e s a p p e a r t o be a f u n c t i o n o f t h e i n t e r a c t i o n between t h e a c e t y l g r o u p s and t h e s t a t i o n a r y p h a s e . The d e g r e e o f a c e t y l - s t a t i o n a r y p h a s e i n t e r a c t i o n i s a p p a r e n t l y d e p e n d e n t on t h e number o f a c e t y l g r o u p s p e r m o l e c u l e , and t h e a v a i l a b i l i t y o f t h e s e g r o u p s t o i n t e r a c t w i t h t h e s t a t i o n a r y phase. F o r t h e PAAN d e r i v a t i v e s o f the u n s u b s t i t u t e d s a c c h a r i d e s ( t r i o s e , t e t r o s e , pentoses, e t c . ) t h e o r d e r o f emergence o c c u r s i n g r o u p s : f i r s t triose (glycera l d e h y d e ) , then t e t r o s e s (e.g. e r y t h r o s e ) , pentoses ( e . g . r i b o s e ) , h e x o s e s ( e . g . m a n n o s e ) , and h e p t o s e ( g l u c o h e p t o s e ) PAAN d e r i v a t ives, i n a d d i t i o n , f o r t e t r o s e s t h e e r y t h r o s e PAAN d e r i v a t i v e ' s r e t e n t i o n t i m e has bee d e r i v a t i v e , f o r pentose t i m e i s s m a l l e s t and t h e x y l o s e PAAN d e r i v a t i v e ' s r e t e n t i o n t i m e i s t h e l a r g e s t C4 , 5)*. F o r each c l a s s o f s t e r e o i s o m e r s ( e . g . t h e p e n t o s e s ) t h e s t e r e o i s o m e r c o n t a i n i n g t h e g r e a t e s t number o f p a i r s o f c i s a c e t y l groups ( o r hydroxyl groups i n t h e u n d e r i v a t i z e d s u g a r ) has t h e s m a l l e s t r e t e n t i o n t i m e , and t h e s t e r e o i s o m e r c o n t a i n i n g t h e s m a l l e s t number o f p a i r s o f c i s a c e t y l g r o u p s t h e largest retention time. I t i s p o s s i b l e t h a t t h e c i s a c e t y l groups promote s a c c h a r i d e c h a i n b e n d i n g , and t h e s e l e s s l i n e a r m o l e c u l e s p r o v i d e l e s s o p p o r t u n i t y f o r a c e t y l groups t o i n t e r a c t w i t h t h e s t a t i o n a r y phase. On t h e b a s i s o f l i m i t e d d a t a , i t a p p e a r s t h a t the replacement o f a f u n c t i o n a l groups u n i f o r m l y changes t h e r e t e n t i o n time o f a s e r i e s o f stereoisomers. For example, t h e 2 - d e o x y - D - g l u c o s e and 2 - d e o x y - D - g a l a c t o s e PAAN d e r i v a t i v e s have r e t e n t i o n t i m e s o f 1 . 7 m i n u t e s l e s s t h a n t h e i r r e s p e c t i v e Dg l u c o s e and D - g a l a c t o s e PAAN d e r i v a t i v e s and t h e 6-deoxy-L-mannose and 6 - d e o x y - L - g a l a c t o s e PAAN d e r i v a t i v e s have r e t e n t i o n t i m e s 9 . 0 m i n u t e s l e s s t h a n t h e i r c o r r e s p o n d i n g mannose and g a l a c t o s e PAAN d e r i v a t i v e s . I t i s p o s s i b l e t h a t on 6-deoxy h e x o s e s u b s t i t u t i o n t h e r e m a i n i n g k a c e t y l g r o u p s have t h e same g e n e r a l a c e t y l - s t a t i o n a r y phase i n t e r a c t i o n a s t h e p e n t o s e s w i t h t h e 6 methoxy g r o u p n o t p a r t i c i p a t i n g . However, i n t h e c a s e o f 2 methoxy h e x o s e s u b s t i t u t i o n ( o r any n o n - t e r m i n a l s u b s t i t u t i o n ) t h e methylene u n i t a c t s as a c h a i n e x t e n d e r w i t h t h e a c e t y l - s t a t i o n a r y phase i n t e r a c t i o n s t i l l a p p r o x i m a t i n g t h a t o f a normal h e x o s e PAAN d e r i v a t i v e . Under t h e s t a n d a r d PAAN d e r i v a t i z a t i o n p r o c e d u r e t h e 5 ~ t h i o D - g l u c o s e r e s u l t s i n a w e l l d e f i n e d g . l . c . peak. However, m.s. shows t h a t t h i s g . l . c . peak i s n o t t h e PAAN d e r i v a t i v e , b u t t h e peracetylated 5-thio-D-glucopyranoside. N-acety1-D-glucosamine y i e l d s t h e c o r r e s p o n d i n g PAAN d e r i v a t i v e w i t h a r e t e n t i o n t i m e much l o n g e r t h a n t h e D - g l u c o s e PAAN d e r i v a t i v e — i n d i c a t i n g i n c r e a s e d N - a c e t y l i n t e r a c t i o n w i t h t h e s t a t i o n a r y phase.

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

9.

SEYMOUR

Mass

NMR

and MS of

117

Polysaccharides

Spectrometry

The ammonia c h e m i c a l i o n i z a t i o n ( c . i . a . ) - m.s. o f t h e above PAAN d e r i v a t i v e s a r e v e r y s i m p l e . The most p r o m i n e n t , a n d u s u a l l y o n l y , m/e peaks a r e M + 18 and M - 5 9 , r e p r e s e n t i n g a d d i t i o n o f t h e ammonium i o n , o r t h e a d d i t i o n o f a p r o t o n and s u c c e s s i v e loss o f a c e t i c a c i d . The d e r i v a t i z a t i o n p r o c e d u r e i s n o r m a l l y d u p l i c a t e d , f i r s t w i t h N-15 h y d r o x y l a m i n e and t h e n w i t h p e r d e u t e r a t e d a c e t i c a n h y d r i d e . The N-15 i n t r o d u c t i o n s h i f t s M by 1 a.m.u. f o r e a c h a l d e h y d e o r i g i n a l l y p r e s e n t a n d t h e d e u t e r i u m s h i f t s M by 3 a.m.u. f o r e a c h h y d r o x y l g r o u p o r i g i n a l l y p r e s e n t . T h e r e f o r e , t h e m o l e c u l a r w e i g h t , number o f h y d r o x y l g r o u p s , and number o f a l d e h y d e g r o u p s p r e s e n t i n a l d o s e s c a n r a p i d l y be e s t a b l i s h e d The e l e c t r o n impac carbon-carbon cleavage o T h i s backbone c l e a v a g e i s e q u a l l y l i k e l y between any c a r b o n s , e x c e p t t h e C-1 and C-2 p o s i t i o n s , and d i f f e r e n t l e n g t h f r a g m e n t s are generated from both ends. The g l y c e r a l d e h y d e PAAN d e r i v a t i v e g i v e s v e r y f e w m/e f r a g m e n t s ; t h e s e same m/e a l s o a p p e a r i n t h e e r y t h r o s e PAAN s p e c t r a w i t h a new s e t o f m/e f r a g m e n t s . As t h e m o l e c u l e i s l e n g t h e n e d , more f r a g m e n t s a r e p o s s i b l e , and c o m p a r i son o f t h e s p e c t r a o f d i f f e r e n t l e n g t h m o l e c u l e s i n d i c a t e s t h e o r i g i n a l c a r b o h y d r a t e p o s i t i o n o f each fragment. The f r a g m e n t a t i o n pathways a r e a l s o i d e n t i f i e d by N-15 n i t r i l e s u b s t i t u t i o n and by d e u t e r o a c e t y l s u b s t i t u t i o n . Upon t h e s u b s t i t u t i o n o f a f u n c t i o n a l g r o u p a t a s p e c i f i c p o s i t i o n i n t h e c a r b o h y d r a t e m o l e c u l e , a l l m/e f r a g m e n t s o r i g i n a t i n g f r o m t h a t p o s i t i o n w i l l be s h i f t e d . T h e r e f o r e , t h e f u n c t i o n a l g r o u p s mass and p o s i t i o n c a n be e s t a b l i s h e d . F o r an a l d o s e PAAN d e r i v a t i v e , a c o m b i n a t i o n o f g . l . c . m.s. u s i n g c . i . a . and e . i . c a n e s t a b l i s h t h e m o l e c u l a r w e i g h t , t h e number o f a l d e h y d e and h y d r o x y l g r o u p s , t h e t y p e and p o s i t i o n o f f u n c t i o n a l g r o u p s , a n d p r o v i d e an e s t i m a t e o f t h e s t e r e o chemistry o f the molecule. Methane c h e m i c a l i o n i z a t i o n ( c . i . m . ) - m.s. o f PAAN d e r i v a t i v e s have been e x a m i n e d and t h o u g h interprétable s p e c t r a a r e o b t a i n e d f o r e a c h compound, i f t h e e . i . a . - m . s . and t h e e . i . m.s. a r e known, l i t t l e a d d i t i o n a l i n f o r m a t i o n i s o b t a i n e d . In g e n e r a l , c . i . m . mass f r a g m e n t s r e s u l t f r o m t h e p r o g r e s s i v e and e x t e n s i v e l o s s o f f u n c t i o n a l groups ( t h e 0 - a c e t y l groups a r e l o s t as a c e t i c a c i d and k e t e n e ) and t h e b a c k b o n e c h a i n r e m a i n s i n t a c t . F o r t h e PAAN d e r i v a t i v e o f N - a c e t y l g l u c o s a m i n e , t h e N - a c e t y l group i s n o t e a s i l y l o s t a n d t h e r e f o r e t h e s p e c t r u m o f t h i s compound i s v e r y s i m i l a r t o t h e c o r r e s p o n d i n g g l u c o s e PAAN d e r i v a t i v e — w i t h each m/e f r a g m e n t d e c r e a s e d by 1 a.m.u. P e r m e t h y l a t i o n G a s - L i q u i d Chromatography/Mass

Spectrometry

P o l y s a c c h a r i d e permethy1 a t i o n , f o l l o w e d by h y d r o l y s i s and

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

118

EXTRACELLULAR MICROBIAL POLYSACCHARIDES

a n a l y s i s o f t h e r e s u l t i n g m e t h y l e t h e r s a c c h a r i d e s has t r a d i t i o n a l l y been employed f o r d e t e r m i n i n g s u g a r - s u g a r l i n k a g e t y p e and degree o f branching. The p r o c e d u r e i s s i m p l e i n c o n c e p t , b u t has proven d i f f i c u l t t o apply. T h i s i s due t o t h e d i f f i c u l t y o f i d e n t i f y i n g and q u a n t i t a t i n g t h e r e a c t i o n p r o d u c t s , o r t h e i r d e r i v a t i v e s , and has been used i n c o n j u n c t i o n w i t h p e r a c e t y l a t e d c y c l i c derivatives or the peracetylated a l d i t o l s . Lance and J o n e s (6) s e p a r a t e d m e t h y l e t h e r s o f x y l o s e PAAN compounds and Dmitrîev e t a l . (2) r e p o r t e d t h e m a j o r m/e f r a g m e n t s o f t h e e . i . m.s. o f s e l e c t e d PAAN d e r i v a t i v e s . The t e t r a - 0 , t r i - 0 , and dî-0-methyl e t h e r s o f D-mannopyranoside were s y n t h e s i z e d and g . l . c . c o n d i t i o n s f o u n d f o r t h e s e p a r a t i o n o f t h e s e compounds ( 7 ) . T h i s g . l . c . s e p a r a t i o n , combined w i t h p r e v i o u s l y developed e f f i c i e n t m e t h y l a t i o d h y d r o l y s i methods allowed the r a p i d p e r m e t h y l a t i o i d e n t i t y o f t h e g . l . c . peak m.s. The a v a i l a b i l i t y o f t h e p u r e m e t h y l e t h e r s o f m e t h y l a-Dm a n n o p y r a n o s i d e and t h e e s t a b l i s h m e n t o f a g . l . c . column c a p a b l e o f r e s o l v i n g t h e PAAN d e r i v a t i v e s , a l l o w e d a p r e c i s e d e t e r m i n a t i o n o f t h e f r a g m e n t a t i o n pathways. Di-O-methyl d e r i v a t i v e s o f m e t h y l α-D-mannopyranoside were s u b j e c t e d t o random p a r t i a l m e t h y l a t i o n and t h e r e s u l t i n g r e a c t i o n m i x t u r e h y d r o l y z e d and d e r i v a t i z e d t o PAAN d e r i v a t i v e s . On g . l . c . a n a l y s i s t h e s e m i x t u r e s gave a s e r i e s o f p e a k s , t h e r e t e n t i o n t i m e s i n d i c a t i n g t h e p o s i t i o n o f t h e c o m b i n e d m e t h y l - 0 - and deuteromethy1-0- e t h e r groups. The p o s i t i o n o f new e t h e r g r o u p s i d e n t i f i e d t h e s e a s d e u t e r o m e t h y 1 g r o u p s , and on c o m p a r i s o n o f t h e e . i . - m . s . o f t h e s e compounds t o t h e e . i . - m . s . o f t h e c o r r e s p o n d i n g n o n - i s o t o p i c a l l y s u b s t i t u t e d PAAN d e r i v a t i v e s , t h e o r i g e n o f each m/e f r a g m e n t c o u l d be e s t a b l i s h e d ( 7 ) . The e x t e n s i v e k n o w l e d g e o f g . l . c . - m . s . a l l o w e d t h e s t r u c t u r e o f s i x t e e n mannans t o be e s t a b l i s h e d i n terms o f l i n k a g e t y p e and d e g r e e o f b r a n c h i n g . T h i s method y i e l d e d e s s e n t i a l l y i d e n t i c a l d a t a f o r s e v e r a l o f t h e mannans. S i x c l a s s e s o f mannans w e r e o b s e r v e d , t h e s e p o l y s a c c h a r i d e s d i f f e r i n g i n b o t h l i n k a g e t y p e s and d e g r e e o f b r a n c h i n g . T h i s d a t a i s summarized i n T a b l e 2. The d a t a i n T a b l e 2 a l l o w s t h e c o n s t r u c t i o n o f an a v e r a g e r e p e a t i n g u n i t f o r each polymer c l a s s . F o r e x a m p l e , D-mannan p r o d u c e d by P a c h y s o l e n t a n n o p h i l u s Y-2460 c a n be e x p r e s s e d a s :

-{Μ - 0 - * ) } -

v

I 3 f

I M - (1+2) - M - (1+2) - M

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

9.

SEYMOUR

NMR

and MS of

119

Polysaccharides

TABLE 2 M o l e P e r c e n t a g e o f M e t h y l a t e d D-mannose Components i n H y d r o s y l a t e s o f P e r m e t h y l a t e d D-mannans ( a ) NRRL Number

M e t h y l e t h e r s o f D-mannose 2,3,4,6-•

3,4,6-

2.5 3.9 26.4 19.2 27.7 22.8

51.1 48.3 42.9 54.1 20.4

Y-1842 Y-2448 Y-2460 Y-2023 YB-2097 YB-1344 (a)

Data t a k e n

2,4,6-

2,3,444.3

44.0

2.1 9.7

25.2

3,42.0 3.9 16.9

2,4-

26.1 23 8

from r e f e r e n c e 8.

As a f i r s t a p p r o x i m a t i o n , i t was assumed t h a t t h e (1+6)l i n k a g e s were e x c l u s i v e l y c o n f i n e d t o t h e mannan b a c k b o n e c h a i n . T h i s a s s u m p t i o n was l a t e r t e s t e d by a c e t o l y s i s ( s e e b e l o w ) . T h i s g . l . c . - m . s . t e c h n i q u e was t h e n a p p l i e d t o g l u c a n s . The g e n e r a t i o n o f r e f e r e n c e compounds was n o t n e c e s s a r y . The mass s p e c t r a a r e n o t a f f e c t e d by s t e r e o c h e m i s t r y c h a n g e s , and t h e c o r r e s p o n d i n g mannose and g l u c o s e m e t h y l - e t h e r s y i e l d i d e n t i c a l s p e c t r a . T h e r e f o r e , each g l u c o s e m e t h y l e t h e r PAAN d e r i v a t i v e g . l . c . peak c o u l d be i d e n t i f i e d by c o m p a r i s o n t o t h e known mannose compounds. On t h e b u t a n e d i o l s u c c i n a t e columns employed f o r m e t h y l - e t h e r s a c c h a r i d e PAAN s e p a r a t i o n t h e r e t e n t i o n times a r e g e n e r a l l y , but not n e c e s s a r i l y , d i f f e r e n t f o r c o r r e s p o n d i n g g l u c o s e and mannose compounds. A g r o u p o f d e x t r a n s , p r e v i o u s l y suspected o f c o n t a i n i n g unusual s t r u c t u r a l f e a t u r e s , was a n a l y z e d by g . l . c . - m . s . and t h e r e s u l t s (9) a r e summarized i n T a b l e 3 . The d a t a i n T a b l e 3 a g a i n a l l o w s t h e c o n s t r u c t i o n o f average r e p e a t i n g u n i t s f o r t h e v a r i o u s p o l y s a c c h a r i d e s . F o r e x a m p l e , f r a c t i o n L o f t h e d e x t r a n p r o d u c e d by L e u c o n o s t o c m e s e n t e r o d i e s NRRL Β-1299 c a n be e x p r e s s e d a s h a v i n g a g e n e r a l repeating unit o f : -{G

- (1+6) - G -

(1+6)}-

I G where "G" i s t h e D - g l u c o p y r a n o s i d e

unit.

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

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120

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TABLE 3 M o l e p e r c e n t a g e s o f M e t h y l a t e d D - g l u c o s e Components i n H y d r o l y z a t e s o f P e r m e t h y l a t e d Dextrans (a) Methyl e t h e r s o f D-glucose

NRRL

Number (b) 2 , 3 , 4 , 6 B-1351 Β-1399 6-1254 Β-1299 B-1355 (a) (b)

S L L S S

5.8 12.8 22.1 39.1 6.9

2,3,483.3 74.5 55.0 26.0 46.9

2,3,6-

2,4,6-

2,3-

2,410.5 5.9

3.4

3,40.3 6.8

19.5 35.0

Data t a k e n f r o m r e f e r e n c e 9 . The d e x t r a n p r o d u c i n g NRRL s t r a i n number. polymer f r a c t i o n s .

11.2

34.9

S and L r e f e r t o

A t t h i s p o i n t i t w i l l be seen t h a t g . l . c . - m . s . has been used t o c o n f i r m t h e u n i q u e p r e s e n c e o f g l u c o s e o r mannose a s t h e a l d o s e u n i t o f a s e r i e s o f d e x t r a n s and a s e r i e s o f mannans. In c o n j u n c t i o n w i t h p e r m e t h l y l a t i o n , g . l . c . - m . s . has been employed f o r e s t a b l i s h i n g t h e g e n e r a l r e p e a t i n g u n i t f o r t h e s e d e x t r a n s and D-mannans. A number o f D-mannans, n o t l i s t e d i n T a b l e 2, were shown t o have e s s e n t i a l l y i d e n t i c a l l i n k a g e t y p e s t o t h o s e shown. I t i s p o s s i b l e , i n p r i n c i p l e , t o perform t h e s e o p e r a t i o n s on a s u b - m i l i g r a m b a s i s . For ease o f m a t e r i a l s h a n d l i n g , a f e w mg o f e a c h p o l y m e r were employed f o r t h e mannan d e t e r m i n a t i o n s , and due t o i n c r e a s e d permethy1 a t i o n d i f f i c u l t y , a p p r o x i m a t e l y 10 t o 15 mg o f d e x t r a n s w e r e u s e d . T h i s data then provided t h e b a s i s f o r comparison w i t h t h e remaining t e c h n i q u e s , w h i c h r e q u i r e l a r g e r amounts o f m a t e r i a l . High-Pressure

Chromatography

P r e v i o u s work has shown t h a t a c e t o l y s i s o f mannans r e s u l t s i n s e l e c t i v e h y d r o l y s i s , w i t h t h e ( 1 + 6 ) - 1 i n k a g e s c l e a v e d much more r a p i d l y t h a n o t h e r s u g a r - s u g a r l i n k a g e s ( 1 0 ) . E m p l o y i n g t h i s s e l e c t i v e h y d r o l y s i s , f o l l o w e d by a d e a c e t y l a t i o n s t e p , yielded a mixture o f oligosaccharides. I t had p r e v i o u s l y p r o v e n p o s s i b l e t o employ h.p.c. t o s e p a r a t e a m i x t u r e o f oligosaccharides. By c a l i b r a t i n g t h e s y s t e m a g a i n s t known o l i g o s a c c h a r i d e s , t h e r e t e n t i o n t i m e s and d e t e c t o r r e s p o n s e s c o u l d be e s t a b l i s h e d . The h.p.c. s y s t e m was t h e n employed t o s e p a r a t e and q u a n t i t a t e t h e a c e t o l y s i s o l i g o s a c c h a r i d e s a c c o r d i n g to degree o f p o l y m e r i z a t i o n ( d . p . ) . An e x a m p l e o f t h i s d a t a f o r t h e mannan o f P a c h y s o l e n t a n n o p h i l u s , NRRL Y-2460 i s shown i n the f o l l o w i n g t a b l e .

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

9.

SEYMOUR

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and MS of Polysaccharides

121

TABLE 4 O l i g o s a c c h a r i d e s f r o m A c e t o l y s i s o f NRRL Y - 2 4 6 0 D-mannan Degree o f Polymerizat ion Mole

ratio

1 10.0

2

3 3.7

4

3.6

11.9

5

6

7

8

1.3

0.7

1.0

0.9

T h i s d a t a i s a n a l y i z e d by making two b a s i c a s s u m p t i o n s , a) o n l y t h e ( 1 + 6 ) - 1 i n k a g e s have been b r o k e n , a n d b) a l l t h e ( l + 6 ) - l i n k ages a r e i n t h e c a r b o h y d r a t e b a c k b o n e . If this iscorrect, t h e n s e q u e n c e s o f ( 1 + 6 ) - 1 i n k a g e s w i l l y i e l d monomers and t h e s i d e c h a i n s w i l l remai backbone s a c c h a r i d e u n i t r e p r e s e n t a s i d e c h a i n non-reducing end group, a b r a n c h i n g end g r o u p , and non-(1+6)-1 i n k e d s a c c h a r i d e s . T h i s d a t a may t h e n be a n a l y z e d t o y i e l d an a v e r a g e r e p e a t i n g u n i t w h i c h c a n be e x p r e s s e d i n t e r m s o f m e t h y l e t h e r s -- an e x a m p l e i s shown b e l o w . TABLE 5 C o r r e l a t i o n o f M e t h y l a t i o n and A c e t o l y s i s Data f o r NRRL Y-2460 D-mannan ( a ) Data S o u r c e

C a l c u l a t e d percentages Tetra

Methylation

26.4

Acetolys i s

23.0

(a)

Data t a k e n

2,3,4-Tri

2.1 10.1

o f methyl e t h e r s

Non2,3, 4 - T r i

Di

42.9

26.1

44.5

23.0

from r e f e r e n c e 8.

I t c a n be seen t h a t t h e a c e t o l y s i s d a t a c l o s e l y p a r a l l e l s the m e t h y l a t i o n data. The m a j o r d i s c r e p a n c y i s t h e amount o f ( l + 6 ) - l i n k a g e s ( 2 , 3 , 4 - t r i - 0 - m e t h y l e t h e r ) as a c e t o l y s i s g e n e r a l l y g i v e s a h i g h e r v a l u e than m e t h y l a t i o n . The (1+6)l i n k a g e a c e t o l y s i s v a l u e comes f r o m t h e amount o f monomeric u n i t s o b s e r v e d by h . p . c , a n d i t i s p o s s i b l e t h a t n o n - ( l + 6 ) l i n k a g e c l e a v a g e c o n t r i b u t e s t o i n c r e a s e t h i s v a l u e . Two general r e s u l t s a r e o b t a i n e d from comparison o f t h i s d a t a : f i r s t l y , the assumption that the (1+6)-1inkages a r e confined t o t h e backbone i s c o n f i r m e d ; s e c o n d l y , .there a p p e a r s t o be a d i s t r i b u t i o n o f s i d e c h a i n lengths around t h e average o f t h r e e s a c c h a r i d e u n i t s p e r s i d e c h a i n . Good agreement between a c e t o l y s i s and m e t h y l a t i o n d a t a was o b t a i n e d f o r f o u r o f t h e s i x mannan t y p e s s t u d i e d . Mannan Y-1842 showed g r e a t d i f f e r -

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

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e n c e s i n d e g r e e o f b r a n c h i n g a s d e t e r m i n e d by m e t h y l a t i o n a n d acetolysis. The d a t a s u g g e s t t h a t a l a r g e number o f ( 1 + 6 ) l i n k a g e s must o c c u r i n t h e s i d e c h a i n s . For p o l y s a c c h a r i d e s c o n t a i n i n g ( 1 + 6 ) - l i n k e d s a c c h a r i d e s , t h e c o r r e l a t i o n o f m e t h y l a t i o n and a c e t o l y s i s d a t a i s a u s e f u l method to e s t a b l i s h t h e p o s i t i o n o f t h e (1+6)-1inkages. I f t h e s e (1+6)1 i n k a g e s f o r m t h e backbone c h a i n , t h e s i d e c h a i n l e n g t h d i s t r i b u t i o n c a n be e s t a b l i s h e d . C-13 nurn.jr.

Spectroscopy

This technique i sa l o g i c a l step a f t e r the c o n s t i t u e n t s u g a r s , l i n k a g e t y p e s , and d e g r e e o f b r a n c h i n g have been e s t a b l i s h e d by g . l . c . - m . s . I principle h carbo i different chemical environment w i l C-13 s p e c t r a l r e g i o n . Th -13 s h i f t s i s d e p e n d e n t on t h e m a g n e t i c f i e l d s t r e n g t h . When compared t o H-1 n.m.r., C - 1 3 n.m.r. g i v e s much b e t t e r s e p a r a t i o n s i n an equivalent f i e l d . In a d d i t i o n , due t o improved r e l a x a t i o n t i m e s , C-13 n.m.r. c a n g i v e q u i t e s h a r p s i g n a l s f o r l a r g e p o l y m e r s . It has been e s t a b l i s h e d t h a t s i m p l e s a c c h a r i d e s ( e . g . m e t h y l a-Dg l u c o s e ) w i l l y i e l d s i x C-13 n.m.r. s a c c h a r i d e p e a k s ; t h e a n o m e r i c c a r b o n i n t h e 95 t o 105 ppm ( r e l a t i v e t o TMS) r e g i o n , t h e C - 2 , C - 3 , C - 4 , and C-5 peaks i n t h e 7 0 - 7 5 ppm r e g i o n , and t h e C-6 peak a t a p p r o x i m a t e l y 60 ppm ( 1 1 ) . On c o n v e r s i o n o f a h y d r o x y l g r o u p t o an a l k y l e t h e r g r o u p , t h e c h e m i c a l s h i f t o f t h e c o r r e s p o n d i n g s a c c h a r i d e carbon i s s h i f t e d d o w n f i e l d ( t o l a r g e r ppm v a l u e s ) . F o r m e t h y l e t h e r f o r m a t i o n t h i s change i n c h e m i c a l s h i f t has been shown t o be a u n i f o r m 10 ppm d o w n f i e l d s h i f t f o r each s a c c h a r i d e carbon p o s i t i o n (12). T h e r e f o r e , a c o n v e n i e n t approach t o p o l y s a c c h a r i d e a n a l y s i s i s t o c o n s i d e r t h e polymer as an agrégation o f i n d e p e n d e n t a l k y l e t h e r m o n o s a c c h a r i d e s . F o r e x a m p l e , m e t h y l a t i o n d a t a ( T a b l e 3) and t h e i m p l i e d g e n e r a l r e p e a t i n g u n i t have been p r e s e n t e d f o r d e x t r a n B-1299 f r a c t i o n S. F o r p u r p o s e s o f C - 1 3 n.m.r. t h i s p r o p o s e d b a s i c u n i t may be c o n s i d e r e d as e q u i v a l e n t t o an e q u a l m o l a r m i x t u r e o f m e t h y l Dm a n n o p y r a n o s i d e ( t h e end g r o u p ) , m e t h y l 2 , 6 - d i - 0 - m e t h y l - D - m a n n o p y r a n o s i d e ( t h e b r a n c h i n g g r o u p ) , and m e t h y l 6-0-methy1-D-mannop y r a n o s i d e ( t h e backbone e x t e n d i n g g r o u p ) . For three saccharides, a maximum o f e i g h t e e n (6x3) s a c c h a r i d e C-13 c h e m i c a l s h i f t s c o u l d be o b s e r v e d . Two l i m i t a t i o n s o f C - 1 3 n.m.r. s h o u l d be r e c o g n i z e d . First, t h e r e l a t i v e l y low s e n s i t i v i t y o f t h e C - 1 3 n u c l e i r e q u i r e s l a r g e s a m p l e s (100 t o 200 mg) w i t h F o u r i e r t r a n s f o r m d a t a p r o c e s s i n g . S m a l l e r s a m p l e s may be u s e d , b u t t h e d a t a a c q u i s i t i o n t i m e s t e a d i l y i n c r e a s e s . Secondly, t h e s i g n a l i n t e n s i t y o f each c l a s s o f c a r b o n n u c l e i i s n o t d e p e n d e n t on t h e t o t a l number o f e a c h species present. C-13 n u c l e i w i t h g r e a t e r d e g r e e s o f f r e e d o m of motion y i e l d l a r g e r s i g n a l s . However, i t has p r e v i o u s l y been shown t h a t f o r s a c c h a r i d e s , t h e c o n t r i b u t i o n o f e a c h c a r b o n

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

NMR and MS of Polysaccharides

9. SEYMOUR

123

s p e c i e s t o t h e C - 1 3 n.m.r. s p e c t r a i s a p p r o x i m a t e l y e q u a l ( 1 3 ) » F o r t h e C - 1 3 n.m.r. s p e c t r a o f d e x t r a n Β-1299 f r a c t i o n S ( F i g . 1 ) , and a n a l a g o u s d e x t r a n s p e c t r a , s e v e r a l p o i n t s become apparent. For the dextrans d e s c r i b e d i n Table 3 , a s e r i e s o f s i x c h e m i c a l s h i f t s ( d e s i g n a t e d a s A t h r o u g h F) a r e p r e s e n t i n each spectrum. T h e s e s i x peaks d o m i n a t e t h e s p e c t r a o f t h e more l i n e a r α-linked d e x t r a n s and r e p r e s e n t t h e c o n t r i b u t i o n o f a methyl 6 - 0 - m e t h y l - D - g l u c o p y r a n o s i d e analog. As p o l y m e r s w i t h g r e a t e r d e g r e e o f b r a n c h i n g were e x a m i n e d , t h e c o n t r i b u t i o n o f t h e o r i g i n a l s i x peaks d e c r e a s e d and o t h e r c h e m i c a l s h i f t s become prominent (14). B-1299 f r a c t i o n S d e x t r a n ( F i g . 1) p r o v i d e s an e x a m p l e o f a h i g h l y b r a n c h e d d e x t r a n w i t h t h e c o n t r i b u t i o n o f t h e o r i g i n a l s i x peaks i n d i c a t e d by l e t t e r s A t h r o u g h F. The 7 0 - 7 5 ppm r e g i o n (B t h r o u g s h i f t s , but o n l y seven a r detection. A p p a r e n t l y a number o f t h e s e c h e m i c a l s h i f t s a r e n o t resolved. In t h e a n o m e r i c r e g i o n t h e e x p e c t e d t h r e e peaks a r e o b s e r v e d , t h e d o w n f i e l d peak r e p r e s e n t i n g t h e ( 1 + 6 ) - 1 i n k e d u n i t . A l l a n o m e r i c p r o t o n s a r e l o c a t e d i n t h e 9 6 t o 101 ppm r e g i o n , d e m o n s t r a t i n g t h a t each o f the observed l i n k a g e s i s a. F o r l i n e a r d e x t r a n t h e 7 5 * 8 5 ppm r e g i o n d i s p l a y s no c h e m i c a l shifts. For dextrans c o n t a i n i n g 1+2, 1+3, o r 1+4-1inkages (as d e m o n s t r a t e d by g . l . c . - m . s . ) t h e 7 5 - 8 5 ppm r e g i o n c o n t a i n s t h e g l y c o s y l l i n k e d c a r b o n s ( C - 2 , C - 3 , o r C - 4 ) w h i c h upon s u b s t i t u t i o n have had t h e i r c h e m i c a l s h i f t s moved d o w n f i e l d f r o m t h e 7 0 - 7 5 ppm r e g i o n . We have o b s e r v e d a c h e m i c a l s h i f t o f 7 6 . 5 ppm f o r a - ( 1 + 2 ) - 1 i n k a g e s , 7 9 . 5 ppm f o r a - ( 1 + 3 ) - 1 i n k a g e s , and 8 1 . 6 ppm f o r a-(1+4)-1inkages (14). T h e o n l y c h e m i c a l s h i f t i n t h e 7 5 - 8 5 ppm

C

Ε

Ε

C

J

78

58

69 I05

PPM Figure 1. C-13 NMR spectra of dextran B-1299 fraction S recorded at 27° in D 0; ppm relative to TMS. Inset (78-69 ppm) recorded at 70°. 2

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

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EXTRACELLULAR MICROBIAL

POLYSACCHARIDES

r e g i o n o f t h e d e x t r a n Β-1299 f r a c t i o n S s p e c t r a i s a t 76.42 ppm, i n agreement w i t h t h e g . l . c . - m . s . d a t a . T h i s n.m.r. method t h e r e f o r e a l l o w s i d e n t i f i c a t i o n and rough q u a n t i t a t i o n o f t h e l i n k a g e t y p e s p r e s e n t by a n o n - d e s t r u c t i v e t e c h n i q u e n o t d e p e n d e n t on h y d r o l y s î s. In g e n e r a l , t h e d e x t r a n C-13 n.m.r. s p e c t r a w e r e r e l a t i v e l y simple, t h i s being e s p e c i a l l y n o t i c a b l e i n the anomeric region. A p r e v i o u s C-13 n.m.r. s t u d y o f p u l u l l a n s o b s e r v e d t h r e e w e l l d e f i n e d a n o m e r i c c h e m i c a l s h i f t s and employed t h i s a s a v e r y p l a u s i b l e argument f o r t h e o r d e r e d r e p e a t i n g - (1+4) - (1+6) - (1+4) g l u c o p y r a n o s i d e s u b u n i t (15)» Many o f t h e s e d e x t r a n s a l s o show s i m p l e C - 1 3 n.m.r. s p e c t r a w h i c h i n t u r n i m p l i e s a b a s i c o r d e r e d repeating sub-unit. Another point o f i n t e r e s i C-13 n.m.r. s p e c t r a on p o l y s a c c h a r i d e s had b r o a peak "sharpened y r a i s i n g t h e t e m p e r a t u r e . A h i g h t e m p e r a t u r e (70 ) i n s e t o f t h e 7 0 - 7 5 ppm r e g i o n i s shown i n F i g u r e 1 . The g e n e r a l s p e c t r u m p r o f i l e r e m a i n s t h e same, b u t e a c h peak i s n a r r o w e r . A number o f t e m p e r a t u r e d e p e n d e n t e f f e c t s were o b s e r v e d , t h e most i n t e r e s t i n g b e i n g t h a t a l l o f t h e c h e m i c a l s h i f t s w e r e tempe r a t u r e d e p e n d e n t , moving d o w n f i e l d on i n c r e a s i n g t e m p e r a t u r e . In a d d i t i o n , d i f f e r e n t c h e m i c a l s h i f t s d i s p l a y e d d i f f e r e n t t e m p e r a t u r e d e p e n d e n c i e s , i n t h e r a n g e o f Δδ/ΔΤ o f 0.01 t o 0 . 0 3 ppm/C° ( r e l a t i v e t o TMS). T h e s e d i f f e r e n t Δδ/ΔΤ e x c l u d e b u l k m a g n e t i c s u s c e p t i b i l i t y as t h e m a j o r f a c t o r and s u g g e s t t h a t t h e m a g n i t u d e o f Δδ/ΔΤ i s s t r u c t u r e r e l a t e d . In f a c t , t h e l a r g e s t Δδ/ΔΤ o b s e r v e d a r e g e n e r a l l y a s s o c i a t e d w i t h c a r b o n s involved i n sugar-sugar linkages (14). I t i s n e c e s s a r y t o c o n s i d e r t h e Δδ/ΔΤ e f f e c t , e s p e c i a l l y when c o m p a r i n g C - 1 3 n.m.r. c a r b o h y d r a t e s p e c t r a i n t h e c l o s e l y packed 7 0 - 7 5 ppm r e g i o n . The c h e m i c a l s h i f t s o f t h e C - 2 , C - 3 , C-4, and C-5 carbons f a l l i n g i n t h i s r e g i o n a r e a p p a r e n t l y d i a g n o s t i c f o r s a c c h a r i d e s o f s p e c i f i c l i n k a g e t y p e s ; however, a t e m p e r a t u r e change o f 50 c a n c a u s e a r e s o n a n c e change s o g r e a t as t o a l l o w c h e m i c a l s h i f t s t o i n t e r c h a n g e p o s i t i o n s . P-31 n_.m.£.

Spectroscopy

A d d i t i o n a l n.m.r. d a t a has been g e n e r a t e d by e m p l o y i n g a F o u r i e r t r a n s f o r m n.m.r. w i t h a P-31 p r o b e . The P-31 n u c l e i a r e r e l a t i v e l y i n s e n s i t i v e t o n.m.r. a n d , a s w i t h C - 1 3 s t u d i e s , l a r g e r amounts o f c a r b o h y d r a t e s were n e c e s s a r y . A variety of e x t r a c e l l u l a r y e a s t 0 - p h o s p h o n o h e x o s a n s were a v a i l a b l e f o r study. T h e s e compounds c a n be d i v i d e d , on a c h e m i c a l b a s i s , i n t o two g r o u p s ( 1 6 ) . Type I i s e x e m p l i f i e d by p o l y ( p h o s p h o r i c d i e s t e r s ) o f D-mannose o l i g o s a c c h a r i d e s . Type I I a r e p o l y s a c c h a r i d e s i n which t h e g l y c o s y l phosphate residues occur as n o n - r e d u c i n g e n d - g r o u p s — e i t h e r a s D-mannose, D - g l u c o s e , o r as d i s a c c h a r i d e s . Many o f t h e mannans and p h o s p h o n o h e x o g l u c a n s

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

9.

SEYMOUR

NMR and M S of Polysaccharides

125

a r e r e l a t e d i n s o f a r a s t h e y a r e p r o d u c e d by t h e same y e a s t s t r a i n u n d e r d i f f e r i n g amounts o f o r t h o p h o s p h a t e i n t h e c u l t u r e m e d i a . The P-31 n.m.r. s i g n a l s f r o m t h e s e O-phosphonohexosans w e r e q u i t e sharp. The n a t i v e polymers a p p a r e n t l y c o n t a i n a l l phosphate g r o u p s a s t h e d i e s t e r , but i s o l a t i o n p r o c e d u r e s can r e s u l t i n p a r t i a l h y d r o l y s i s t o the mono-ester. P-31 n.m.r. p r o v i d e s an e x c e l l e n t method o f s u r v e y i n g f o r t h i s h y d r o l y s i s a s t h e n.m.r. s i g n a l s o f t h e m o n o - e s t e r and t h e d i - e s t e r p h o s p h a t e s a r e w i d e l y s p a c e d , the m o n o - e s t e r f a l l i n g a t a p p r o x i m a t e l y -4. ppm ( r e l a t i v e t o 85% o r t h o p h o s p h o r i c a c i d ) . In g e n e r a l , e a c h O - p h o s p h o n o h e x o g l y c a n gave a s i n g l e s h a r p P-31 s i g n a l , t h e c h e m i c a l s h i f t b e i n g u n i q u e f o r e a c h p o l y m e r . The two t y p e s o f O - p h o s p h o h e x o g l y e a n s can be r e p r e s e n t e d a s : (

M -

M -

M -

Ρ -

Μ )

χ

G

Type I

Type I I

Where M r e p r e s e n t s a m a n n o p y r a n o s i d e u n i t , Ρ r e p r e s e n t s a p h o s p h o d i e s t e r u n i t , and G r e p r e s e n t s a n o n - r e d u c i n g mannose, glucose, o rdisaccharide unit. Though e a c h O-phosphonomannan s t u d i e d has d i s p l a y e d a d i f f e r e n t P-31 c h e m i c a l s h i f t , t h e r e i s no o b v i o u s d i f f e r e n c e between Type I and Type II P-31 n.m.r. s p e c t r a ( s e e T a b l e 6 ) . TABLE 6 P-31 C h e m i c a l S h i f t s f o r O-phosphonomannans and M a t e r i a l s (a) Anomeric sugar phosphate

mannose M

mannose ·' glucose n

galactose

NRRL p r o d u c i n g strain

Related

Orthophosphate d i e s t e r chemical s h i f t

Type I Y-1842 YB-1443 Y-2448 Y-2461

1.94 1.74 1.84 1.72

Type I I Y-411 YB-2079 YB-2194 Y-2579 Y-2023 Y-6493

I.78 1.74 and 1.90 1.07 1.16 1.10 and 1.28 1.06

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

126

EXTRACELLULAR MICROBIAL POLYSACCHARIDES

(a) d a t a

în T a b l e 6 t a k e n

from reference

17.

However, T a b l e 6 does show t h a t a s i g n i f i c a n t change i n t h e c h e m i c a l s h i f t o c c u r s when t h e a n o m e r i c s u g a r p h o s p h a t e i s g l u c o s e ( a t a p p r o x . 1.1 ppm) r a t h e r t h a n mannose ( a t a p p r o x 1.8 ppm). The a n o m e r i c s u g a r p h o s p h a t e i s b e l i e v e d t o be α-linked i n a l l c a s e s and t h e changes i n c h e m i c a l s h i f t a r e t h e r e s u l t o f s t e r e o i s o m e r e f f e c t s a t t h e d i s t a n c e o f s e v e r a l atoms f r o m t h e p h o s p h o r u s atom. R e l a t i v e l y s u b t l e changes i n p o l y m e r s t r u c t u r e a r e r e f l e c t e d i n t h e P-31 c h e m i c a l s h i f t v a l u e s . P o l y m e r s Y-2448 (δ = 1.84 ppm) and Y-1842 (6 = 1.74 ppm) a r e a p p a r e n t l y s t r u c t u r a l l y i d e n t i c a l e x c e p t t h a t f o r Y-1842 t h e s u g a r s a r e a - 1 i n k e d ( n o t t h e s u g a r p h o s p h a t e a n o m e r i c l i n k a g e ) and f o Y-2448 t h e s u g a r s a r e 3-1 i n k e d . O-Phosphonomanna di-saccharide residues a l s For e x a m p l e , i n p o l y m e r s f r o m YB-2097 and Y-2023, where b o t h t y p e s o f r e s i d u e a r e p r e s e n t , two d i e s t e r r e s o n a n c e s a r e observed. In YB-2097 O-phosphonomannan t h e mannose 6 - p h o s p h a t e r e s i d u e s a r e i n a n o m e r i c l i n k a g e w i t h r e s i d u e s o f mannopyranose and 6-0-a-D mannopyranosy1-D-mannopyranose, i n Y-2023 0-phosphomannan t h e l i n k a g e i s t o r e s i d u e s o f D - g l u c o p y r a n o s e and 2-0α-D-mannopyranosy1-D-glucopyranose. P r o t o n - c o u p l e d s p e c t r a f o r t h e d i e s t e r s showed q u a r t e t p a t t e r n s t h a t c o u l d be a n a l y z e d by c o m p u t e r s i m u l a t i o n t o o b t a i n the c o u p l i n g - c o n s t a n t s . These data are i n accord w i t h the i n t e r p r e t a t i o n t h a t most o f t h e l i n k a g e s i n t h e 0-phosphonomannans a r e o f t h e D-mannopyranose 6-(D-mannopyranosy1 p h o s p h a t e ) type. Conclus ions Examples have been p r e s e n t e d t o d e m o n s t r a t e how v a r i o u s forms o f g . l . c , h . p . c , m.s., and n.m.r. a r e employed i n e x t r a c e l l u l a r polysaccharide structure determination. These s t r u c t u r a l d e t e r m i n a t i o n s have p r o v e d f r u i t f u l i n p r o v i d i n g i n s i g h t s i n t o the r e l a t i o n s h i p of a wide v a r i e t y o f e x t r a c e l l u l a r polysaccharides. In t u r n , t h e e x t r a c e l l u l a r p o l y s a c c h a r i d e s have p r o v i d e d m a t e r i a l s t o c o r r e l a t e t h e v a r i o u s s t r u c t u r a l d e t e r m i n a t i o n t e c h n i q u e s , and t h e s e methods may now be a p p l i e d t o more c o m p l e x s a c c h a r i d e c o n t a i n i n g p o l y m e r s .

Literature cited 1. 2.

Dutton, G.G.S., Advan. Carbohyd. Chem. Biochem., (1974) 30, 9-110. Dmitriev, B.A., Backinowsky, L.V., Chizhov, O.S., Zolotarev, B.M., and Kochetkov, N.K., Carbohyd. Res., (1971) 19 432-435.

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

9. SEYMOUR

3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.

17.

NMR and MS of

Polysaccharides

127

Varma, R. Varma, R.S., and Wardi, A.H., J . Chromatogr. (1973) 77, 222-227. Szafranek, J., Pfaffenberger, C.D., and Horning, E.C., Anal. Lett. (1973) 6, 479-492. Seymour, F.R., Chen, E.C.M., and Bishop, S.H., Carbohyd. Res. in press. Lance, E.G., and Jones, J.K.N., Can. J . Chem. (1967) 45, 1995-1998. Seymour, F.R., Plattner, R.D., and Slodki, M.E., Carbohyd. Res. (1975) 44, 181-198. Seymour, F.R., Slodki, M.E., Plattner, R.D., and Stodola, R. Μ., Carbohyd. Res. (1976) 48, 225-237. Seymour, F.R., Slodki, M.E., Plattner, R.D., and Jeanes, Α., Carbohyd. Res. in press Rosenfeld, L . , an 32, 287-298. Perlin, A . S . , Casu, B . , and Koch, H . J . , Can. J . Chem. (1970) 48, 2596-2606. Usui, T., Yamoka, N., Matsuda, K., Tuzimura, K., Sugiyama, H. and Seto, S., J . Chem. Soc. Perkin, I, 1973, 2425-2432. Gorin, P.A.J., Can. J . Chem., (1973) 51, 2375-2383. Seymour, F.R., Knapp, R.D., Bishop, S.H., Carbohyd. Res. in press. Jennings, H . J . , and Smith, I.C.P., J . Am. Chem. Soc. (1973) 95, 606-608. Slodki, M.E., Ward, R.M., Boundy, J.Α., and Cadmus, M.C. in Terui, G. (Ed.), Ρroc. Int. Ferment. Symp. IVth: Ferment. Technol. Today, Soc. Ferment. Technol., Osaka, 1972 pp 597-601. Costello, A.J.R., Glonek, T., Slodki, M.E., Seymour, F.R., Carbohyd. Res. (1975) 42, 23-37.

Acknowledgements T h i s work was s u p p o r t e d , i n p a r t , by a R o b e r t A. W e l c h F o u n d a t i o n G r a n t (Q 2 9 4 ) , a N a t i o n a l S c i e n c e F o u n d a t i o n G r a n t ( B M S - 7 4 - 1 0 4 3 3 ) , and N a t i o n a l I n s t i t u t e s o f H e a l t h G r a n t s (HL-05435, HL-14194, HL-17372). S p e c i a l t h a n k s a r e due t o D r s . Aliène J e a n e s and Morey E. S l o d k i o f t h e N o r t h e r n R e g i o n a l R e s e a r c h L a b o r a t o r y , ARS, USDA, P e o r i a , I l l i n o i s , f o r p r o v i d i n g t h e d e x t r a n s , mannans, and O-phosphonohexosans d e s c r i b e d i n t h i s paper.

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

10 Polysaccharide Polyelectrolytes W. M. PASIKA Chemistry Department, Laurentian University, Sudbury, Ontario, Canada

Macromolecules which possess a large number of some functionality an called polyelectrolytes function aids in the solubilization of the polyelectrolyte substance and is responsible for its unique properties. Although the ionogenic function may be regarded as a salt, dissolution of the polyelectrolyte substance is not comparable to the dissolution of a simple salt. A simple salt such as sodium chloride in solution produces a cation and an anion of comparable size. Each ion has independent mobility. A polyelectrolyte dissolves to yield a polyion and counter ions. The polyion holds a large number of charges in close proximity because they are attached to the macromolecular backbone. Although the polyion has mobility, the individual charges attached to the chain do not. They remain within the domain of the macromolecular c o i l . Not all the gegions or counterions are completely mobile. Anionic polyelectrolytes have positive counter ions whereas cationic polyelectrolytes have negative counter ions. Polyampholytes can acquire either positive or negative charge along the macromolecular backbone depending upon the composition of the solution. P i c t o r i a l l y , one has the following

128

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

10. PAsiKA

Polysaccharide

Polyelectrolytes

129

Because o f f r e e energy r e s t r i c t i o n s , n o t a l l the i o n o g e n i c groups " i o n i z e " . Many e x i s t as i o n p a i r s . A l a r g e number of p o l y s a c c h a r i d e p o l y e l e c t r o l y t e s can be i s o l a t e d from a v a r i e t y o f n a t u r a l s o u r c e s , h e p a r i n , h y a l u r o n i c a c i d , c h o n d r o i t i n and k e r a t i n , t o name a few, are i s o l a t e d from a n i m a l s o u r c e s . The more f a m i l i a r examples s u p p l i e d by the p l a n t w o r l d a r e p e c t i n i c a c i d s , a l g i n a t e s and carageenan. A number of p o l y s a c c h a r i d e p o l y e l e c t r o l y t e s , such as Xanthan, can be o b t a i n e d from nonpathogenic m i c r o organisms (1) . The common c h a r a c t e r i s t i c i s t h a t the macromolecular backbone i s composed o f s a c c h a r i d e r e s i d u e s c a r r y i n g i o n o g e n i c groups. The l a t t e r are more o f t e n than not "Synthetic" polysaccharid o b t a i n e d by s u i t a b l y d e r i v a t i z i n g p o l y s a c c h a r i d e s . The e n s u i n g d i s c u s s i o n w i l l focus on d e r i v a t i z e d d e x t r a n i n an attempt t o i l l u s t r a t e some o f the f a c t o r s which i n f l u e n c e the c h a r a c t e r i s t i c s o f p o l y saccharide p o l y e l e c t r o l y t e s . Viscosity. A l l macromolecular substances i n s o l u t i o n enhance the v i s c o s i t y o f the s o l v e n t c o n s i d e r a b l y . The l a r g e r the m o l e c u l a r weight o r macromolecular s i z e , the g r e a t e r the enhancement. In c h a r a c t e r i z i n g the macromolecular s i z e through the v i s c o s i t y enhancement, i t i s more c o n v e n i e n t l y done w i t h the v i s c o s i t y f u n c t i o n s l i s t e d i n F i g . 1. The dependence o f reduced v i s c o s i t y on c o n c e n t r a t i o n o f n e u t r a l macrom o l e c u l a r s u b s t a n c e s ( i . e . , dextran) i s l i n e a r as d e p i c t e d i n F i g . 1. E x t r a p o l a t i o n o f the v i s c o s i t y d a t a t o " z e r o " c o n c e n t r a t i o n y i e l d s the i n t r i n s i c v i s c o s i t y , which measures the hydrodynamic volume p e r a gram o f macromolecular s u b s t a n c e a t i n f i n i t e dilution. The reduced v i s c o s i t y which p e r t a i n s t o s o l u t i o n s o f f i n i t e c o n c e n t r a t i o n has the same u n i t s o f volume p e r gram o f s u b s t a n c e . P o l y e l e c t r o l y t e s ( i . e . , d e x t r a n s u l f a t e ) i n water do not e x h i b i t l i n e a r reduced v i s c o s i t y curves over the c o n c e n t r a t i o n range t h a t m a c r o m o l e c u l a r subs t a n c e s are u s u a l l y s t u d i e d ( 1%). The reduced v i s c o s i t y curve i s a c o n t i n u o u s l y i n c r e a s i n g f u n c t i o n with d i l u t i o n ( F i g . 2). The c o n t i n u a l i n c r e a s e w i t h d i l u t i o n does n o t o c c u r i n d e f i n i t e l y . At extremely low c o n c e n t r a t i o n s ( 10" ) the reduced v i s c o s i t y f u n c t i o n decreases very r p i d l y with f u r t h e r d i l u t i o n . S h o u l d the d i l u t i n g aqueous s o l v e n t c o n t a i n an e l e c t r o l y t e such as NaC3. e t c . , the reduced v i s c o s i t y a

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

EXTRACELLULAR

η.

ÎL.1 τι

R E L A T I V E

V

\ sp

MICROBIAL POLYSACCHARIDES

VISCOSITY

S P E CIF IC

VISCOSITY

R E D U C E D

VISCOSITY

t,

sp

N

c INTRINSIC

VISCOSITY

n.s

U N I T S O F Usp

ANDQ\]ARE

Figure 1.

dl/g

or

ml/g

Viscosity functions

cone, polymer g/dl Figure 2.

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

10.

PAsiKA

Polysaccharide

Polyelectrolytes

131

curves e x h i b i t maxima a t f i n i t e c o n c e n t r a t i o n s . The l a r g e r the e x t e r n a l s a l t c o n c e n t r a t i o n , the s m a l l e r the reduced v i s c o s i t y v a l u e s and t h e f u r t h e r t o t h e r i g h t t h e maximum reduced v i s c o s i t y v a l u e tends t o appear ( F i g . 2 ) . A l i n e a r dependence o f reduced v i s c o s i t y on p o l y e l e c t r o l y t e c o n c e n t r a t i o n i s o b t a i n e d i n the presence o f a s u f f i c i e n t l y high e x t e r n a l s a l t concentration. The v i s c o s i t y b e h a v i o u r o f p o l y e l e c t r o l y t e s i s governed by the f i r s t , second and t h i r d e l e c t r o v i s c o u s e f f e c t (2) ( F i g . 3 ) . The 1 s t e l e c t r o v i s c o u s e f f e c t a r i s e s because o f the d i f f e r e n c e i n s i z e o f t h e macro i o n and t h e c o u n t e r i o n s . I n an hydrodynamic gradient, the small r a p i d l y than t h e muc s e p a r a t i o n o f t h e c o u n t e r i o n c l o u d from t h e macro i o n occurs. Because t h e two a r e c o u p l e d by a coulombic type i n t e r a c t i o n , t h e l a r g e r macro i o n a c t s as a b r a k e on t h e c o u n t e r i o n movement. This increases the v i s c o s i t y of the s o l u t i o n . I n s o l u t i o n , as t h e l i q u i d f l o w s , macro i o n s w i l l be d r i v e n p a s t each o t h e r because o f t h e hydrodynamic g r a d i e n t . Should the h i g h l y charged macro i o n s pass c l o s e l y , c o u l o m b i c r e p u l s i v e f o r c e s w i l l come i n t o p l a y . The f a s t e r moving macro i o n w i l l d e v i a t e from i t s i n i t i a l l i n e a r pathway. A g a i n , excess energy i s expended and t h e v i s c o s i t y o f t h e medium i s i n c r e a s e d . The l a r g e r the charge on t h e macro i o n , t h e s t r o n g e r w i l l be t h e 2nd e l e c t r o v i s c o u s e f f e c t . The 3 r d e l e c t r o v i s c o u s e f f e c t a r i s e s because o f t h e i n t e r a c t i o n o f t h e charges t h a t a r e a t t a c h e d t o t h e macromolecular backbone. I n t h e case o f a f l e x i b l e m a c r o m o l e c u l a r c o i l , t h i s i n t e r a c t i o n expands t h e c o i l t o an average c o n f o r m a t i o n which m i n i m i z e s t h e r e p u l s i v e i n t e r a c t i o n s . A t t h e new e q u i l i b r i u m c o n f o r m a t i o n ( l a r g e r than t h a t of the n e u t r a l macromolecule), the c o n t r a c t i l e f r e e energy o f t h e m a c r o m o l e c u l a r backbone i s e q u a l t o t h e e x p a n s i v e coulombic f r e e energy a r i s i n g from i o n i zation. The i n c r e a s e d m a c r o m o l e c u l a r c o i l s i z e enhances t h e v i s c o s i t y o f t h e s o l u t i o n . The v i s c o s i t y b e h a v i o u r t o t h e l e f t o f t h e maxima i n F i g . 2 i s p r i m a r i l y due t o t h e 2nd e l e c t r o v i s c o u s e f f e c t , w h i l e t h a t t o t h e r i g h t i s p r i m a r i l y due t o t h e 3 r d e l e c t r o viscous e f f e c t . Not a l l o f the c o u n t e r i o n s o f a p o l y e l e c t r o l y t e a r e f r e e t o move about. The f r e e i o n s form a counteion cloud about t h e p o l y i o n , whereas t h e i m m o b i l i z e d i o n s a r e bound t o a s p e c i f i c s i t e o r p o i n t o f t h e macromolecular backbone. T h i s model was p r e s e n t e d e a r l i e r i n the p o l y e l e c t r o l y t e d i s s o l u t i o n equation.

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

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As t h e p o l y e l e c t r o l y t e s o l u t i o n i s d i l u t e d more and more o f t h e s i t e bound c o u n t e r i o n s a r e r e l e a s e d . T h i s b u i l d s up t h e charge on t h e macro i o n which expands, which i n t u r n i n c r e a s e s t h e reduced v i s c o s i t y . E x p a n s i o n on d i l u t i o n , however, cannot o c c u r i n definitely. When t h e c o n c e n t r a t i o n o f t h e e x t e r n a l i o n s o f t h e s o l u t i o n become e q u a l t o o r g r e a t e r than t h a t of the counterions o f the p o l y e l e c t r o l y t e , i o n i z a t i o n of the p o l y e l e c t r o l y t e ceases. Further d i l u t i o n d e c r e a s e s t h e reduced v i s c o s i t y because e x p a n s i o n o f t h e c o i l has c e a s e d and t h e charged p a r t i c l e s a r e p l a c e d f u r t h e r and f u r t h e r a p a r t , c a u s i n g a r e d u c t i o n i n t h e 2nd e l e c t r o v i s c o u s e f f e c t . This i s t h e o r i g i n o f th curves. Dextran P o l y e l e c t r o l y t e

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A s u f f i c i e n t l y large external s a l t concentration w i l l y i e l d l i n e a r reduced v i s c o s i t y - c o n c e n t r a t i o n plots. L i n e a r i t y , however, does n o t i n s u r e t h a t t h e v i s c o s i t y b e h a v i o u r i s t h a t o f t h e n e u t r a l macromolecule. F i g . 4 shows t h e r e d u c e d v i s c o s i t y b e h a v i o u r o f a B-512 l i n e a r d e x t r a n ( Jjt^"] 0.164 d l / g ) and a b r a n c h e d d e x t r a n B - 7 4 2 ( f j \ J - 0.158 d l / g ) and the s u l f a t e d e r i v a t i v e s d e r i v e d from them. Despite l i n e a r i t y , the reduced v i s c o s i t i e s o f the s u l f a t e s are h i g h e r than t h o s e o f t h e n e u t r a l m o l e c u l e s by a f a c t o r o f about two. The d i f f i c u l t y i n c o l l a p s i n g t h e s u l f a t e macromolecular c o i l t o t h e s i z e o f t h e n e u t r a l macromolecule may stem from one o f two f a c t o r s or a combination o f both. Introduction of the s u l f a t e group may d e c r e a s e t h e f l e x i b i l i t y o f t h e macrom o l e c u l a r backbone. A r i g i d backbone tends t o produce a more extended m a c r o m o l e c u l a r c o n f o r m a t i o n which would e x h i b i t h i g h e r r e d u c e d v i s c o s i t i e s . Alternately, a l t h o u g h s t r o n g l o n g range coulombic i n t e r a c t i o n s have been e l i m i n a t e d by t h e e x t e r n a l s a l t , i t may be t h a t s h o r t range i n t e r a c t i o n s o f t h e i o n p a i r s e x i s t . E f f e c t o f Degree o f S u b s t i t u t i o n . The r e d u c e d v i s c o s i t i e s o f a number o f p o t a s s i u m d e x t r a n s u l f a t e s o f d i f f e r i n g degree o f s u b s t i t u t i o n d e r i v e d from B-742(CnJ]*0.158) a r e shown i n F i g . 5. Increasing the degree o f s u b s t i t u t i o n enhances t h e reduced v i s c o s i t y and s h i f t s t h e p o s i t i o n a t which t h e maximum r e d u c e d v i s c o s i t y appears t o t h e l e f t . I n c r e a s i n g t h e number o f i o n o g e n i c groups produces more charge on t h e macro i o n , c a u s i n g g r e a t e r expansion o f the c o i l . On d i l u t i o n , f u r t h e r i o n i z a t i o n

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

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In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

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In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

10.

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and e x p a n s i o n o c c u r i n each c a s e . The h i g h e r the degree o f s u b s t i t u t i o n , the f u r t h e r must the p o l y e l e c t r o l y t e s o l u t i o n be d i l u t e d t o match the e x t e r n a l s a l t c o n c e n t r a t i o n w i t h the c o u n t e r i o n c o n c e n t r a t i o n of the p o l y e l e c t r o l y t e . S i m i l a r v i s c o s i t y behaviour i s o b s e r v e d f o r l i n e a r d e x t r a n s u l f a t e s and f o r branched and l i n e a r carboxymethyl d e x t r a n s . The t y p i c a l p o l y e l e c t r o l y t e v i s c o s i t y c u r v e s e x h i b i t e d by d e x t r a n s u g g e s t t h a t the macromolecular backbone i s f a i r l y f l e x i b l e and t h a t the c o i l can undergo e x p a n s i o n on a c q u i r i n g c h a r g e . E f f e c t o f M o l e c u l a r Weight. F i g . 6 i n d i c a t e s the e f f e c t of molecula weight potassiu carboxymethyl d e x t r a n reduced v i s c o s i t s u b s t i t u t i o n i s constan weigh v a r i e s from 73,000 t o 135,000. The r e d u c e d v i s c o s i t i e s i n c r e a s e w i t h m o l e c u l a r weight and the c o n c e n t r a t i o n at which the reduced v i s c o s i t y maximum appears i s i d e n t i c a l f o r a l l three molecular weights. I t would appear t h a t the m o l e c u l a r w e i g h t does n o t i n f l u e n c e the e x t e n t o r degree o f i o n i z a t i o n and t h a t the e x p a n s i o n i s d i r e c t l y p r o p o r t i o n a l t o the number o f s u b s t i t u t e d a n h y d r o g l u c o s e u n i t s i n the macromolecule £ ( \sf>/ )>τ*χχ 135,000 m o l e c u l a r weight sample a p p r o x i m a t e l y 2x ( T\* / c )VH*X o f 73,000 m o l e c u l a r weight samplej . T h i s s u g g e s t s t h a t the i n t e r a c t i o n of the i o n o g e n i c groups i s a l o c a l i z e d o r n e a r e s t neighbor i n t e r a c t i o n . S h o u l d i t be o t h e r w i s e , then each charge o f p o l y e l e c t r o l y t e would i n t e r a c t w i t h e v e r y o t h e r , compounding the i n t e r a c t i o n s . The h i g h e r m o l e c u l a r weight macromolecule c a r r y i n g more charge would r e g i s t e r a n o n - p r o p o r t i o n a t e reduced viscosity. The l i n e a r p r o p o r t i o n a l i t y between m o l e c u l a r w e i g h t and the maximum reduced v i s c o s i t y would n o t e x i s t . To show more q u a n t i t a t i v e l y t h a t the same i o n i z a t i o n and e x p a n s i o n p r o c e s s i s o c c u r r i n g w i t h the 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 , the d a t a o f F i g . 6 can be p l o t t e d i n terms o f a r e l a t i v e e x p a n s i o n f a c t o r R vs the c o n c e n t r a t i o n o f p o t a s s i u m carboxy methyl d e x t r a n as i n F i g . 7. The numerator o f R i s the maximum reduced v i s c o s i t y and the denominator i s the r e d u c e d v i s c o s i t y a t a p o l y e l e c t r o l y t e c o n c e n t r a t i o n g r e a t e r than t h a t a t which the maximum v i s c o s i t y appears. The c o i n c i d e n c e o f the l i n e a r p l o t s f o r the t h r e e m o l e c u l a r w e i g h t s i n d i c a t e s an i o n i z a t i o n e x p a n s i o n mechanism t h a t i s i d e n t i c a l f o r the t h r e e p o l y e l e c t r o l y t e samples. o

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In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

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p l o t t e d the reduced v i s c o s i t y c u r v e s f o r a branched and a l i n e a r d e x t r a n s u l f a t e o f i d e n t i c a l degree o f s u b s t i t u t i o n d e r i v e d from d e x t r a n s o f near i d e n t i c a l i n t r i n s i c viscosity(0.16 dl/g). Although both v i s c o s i t y curves e x h i b i t t y p i c a l p o l y e l e c t r o l y t e c h a r a c t e r i s t i c s , there are d i f f e r e n c e s . The r e d u c e d v i s c o s i t y maximum f o r t h e branched p o l y e l e c t r o l y t e appears a t a d i f f e r e n t c o n c e n t r a t i o n than t h a t o f the l i n e a r . I n t h e more c o n c e n t r a t e d r e g i o n , t h e v i s c o s i t y i s h i g h e r f o r t h e branched d e x t r a n p o l y electrolyte. The l i n e a r d e x t r a n p o l y e l e c t r o l y t e the h i g h e r v i s c o s i t y i n t h e more d i l u t e s o l u t i o n s . A v i s c o s i t y c u r v e c r o s s o v e r has o c c u r r e d . Similar b e h a v i o u r o c c u r s when t h e v i s c o s i t y c u r v e s a r e r u n i n aqueous N/2000 KC cross over p o i n t s h i f v i s c o s i t i e s are larger (Fig. 9). The d i f f e r e n c e between t h e two d e x t r a n s r e s t s i n t h e i r s t r u c t u r e . The branched d e x t r a n s u l f a t e c o n t a i n s some t h i r t y p e r c e n t non 1,6 l i n k a g e s , w h i l e t h e l i n e a r c o n t a i n s something l e s s than f i v e p e r c e n t . Identical i n t r i n s i c v i s c o s i t i e s of the neutral macromolecules d i c t a t e t h a t a l a r g e r charge d e n s i t y e x i s t s i n t h e c o i l s o f t h e branched d e x t r a n s u l f a t e . This results i n a g r e a t e r e x p a n s i o n f o r t h e branched p o l y e l e c t r o l y t e than f o r t h e l i n e a r . As d i l u t i o n o c c u r s , t h e branched s p e c i e s reaches i t s l i m i t o f e x p a n s i o n e a r l i e r because o f i t s s t r u c t u r a l makeup. The l i n e a r macroion c o n t i n u e s t o expand i n t h e absence o f structural limitation. Hence t h e c r o s s o v e r . The same f e a t u r e s a r e o b s e r v e d when t h e l i n e a r and branched d e x t r a n s a r e c o n v e r t e d t o t h e c a r b o x y m e t h y l d e r i v a t i v e ( F i g . 10) . E f f e c t o f Nature o f t h e I o n o g e n i c F u n c t i o n . I n F i g . 11 a r e p l o t t e d t h e r e d u c e d v i s c o s i t y c o n c e n t r a t i o n c u r v e s f o r a p o t a s s i u m carboxymethyl d e x t r a n o f degree o f s u b s t i t u t i o n o f 0.21 and a p o t a s s i u m d e x t r a n s u l f a t e o f degree o f s u b s t i t u t i o n o f 0.34. Obviously, the h i g h e r t h e degree o f s u b s t i t u t i o n o f i o n o g e n i c group, t h e l a r g e r t h e reduced v i s c o s i t y does n o t h o l d . The p o t a s s i u m d e x t r a n s u l f a t e o f h i g h e r degree o f s u b s t i t u t i o n e x h i b i t s a lower reduced v i s c o s i t y . S i n c e t h e macromolecular backbone and t h e c o u n t e r i o n are i d e n t i c a l , t h e i n v e r s i o n o b s e r v e d can o n l y be due t o t h e n a t u r e o f t h e i o n o g e n i c group and i t s i n t e r a c t i o n with the counter i o n . Potassium dextran s u l f a t e appears t o i o n i z e l e s s than does p o t a s s i u m c a r b o x y m e t h y l d e x t r a n , a l l o w i n g l e s s e x p a n s i o n o f t h e macro ion.

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

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In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

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E f f e c t o f Counter I o n . Reduced v i s c o s i t y c u r v e s o f d e x t r a n s u l f a t e s w i t h L i , Na, K, and Cs as c o u n t e r i o n s are shown i n F i g . 12. The degree o f s u b s t i t u t i o n i s 1.09 and t h e s o l v e n t i s water. The macromolecular backbone and the i o n o g e n i c group are i d e n t i c a l f o r the f o u r p o l y s a c c h a r i d e p o l y e l e c t r o l y t e s . The o n l y d i f f e r e n c e between them i s the c o u n t e r i o n . To o b t a i n t h i s r e s u l t , i t must be t h a t each a l k a l i m e t a l c a t i o n i n t e r a c t t o a d i f f e r e n t degree w i t h the s u l f a t e function. I n t e r e s t i n g l y , a maximum reduced v i s c o s i t y i s e x h i b i t e d by the cesium d e x t r a n s u l f a t e . This would i n d i c a t e t h a t the cesium c o u n t e r i o n b i n d s v e r y t i g h t l y and the e x t e n t o f i o n i z a t i o n o f t h e s a l t i s much l e s s compared w i t h t h a t f th othe counte ions. S i m i l a r reduce o b s e r v e d f o r L i , Na carboxymethy DS 0.84. A cesium carboxymethyl d e x t r a n reduced v i s c o s i t y c u r v e was n o t o b t a i n e d f o r t h i s sequence. E x c e l l e n t l i n e a r c o r r e l a t i o n i s o b t a i n e d between the reduced v i s c o s i t y a t a p a r t i c u l a r c o n c e n t r a t i o n and the c r y s t a l r a d i u s o f the i o n ( F i g . 13). Charge d e n s i t y o f the i o n does n o t seem t o be a v e r y i m p o r t a n t f a c t o r f o r i f i t were L i s h o u l d be the most t i g h t l y bound, y e t i t i s n o t . L i t h i u m i o n s are the most h y d r a t e d o f the a l k a l i m e t a l s e r i e s . Cesium i s the least. T h i s l a c k o f h y d r a t i o n may be r e s p o n s i b l e f o r the t i g h t b i n d i n g o f the cesium i o n . Ion B i n d i n g . I t i s o b v i o u s from the p r e c e d i n g d i s c u s s i o n t h a t the n a t u r e o f the i n t e r a c t i o n o f the c o u n t e r i o n w i t h the charged s i t e on the macromole c u l a r backbone i s i m p o r t a n t i n d e f i n i n g the p o l y electrolyte solution properties. I t i s a l s o obvious t h a t n o t a l l c o u n t e r i o n s i n t e r a c t w i t h the ionogenic s i t e o f the p o l y i o n i n an i d e n t i c a l manner. At t h i s p o i n t i n time, an a c c u r a t e d e s c r i p t i o n o f p o l y e l e c t r o l y t e i o n b i n d i n g has n o t e v o l v e d . I t i s , however, a c c e p t e d t h a t s i t e b i n d i n g and a t m o s p h e r i c b i n d i n g are the two modes o f i n t e r a c t i o n . Site binding g i v e s r i s e t o an i o n p a i r and a l t h o u g h a wide v a r i e t y of i o n p a i r s can be d e f i n e d and p r o b a b l y can e x i s t i n a p o l y e l e c t r o l y t e s o l u t i o n , f o r our purpose i t i s f r u i t f u l t o t h i n k o f an i o n p a i r as an e n t i t y i n which the c o u n t e r i o n does n o t have m o b i l i t y . In atmos p h e r i c b i n d i n g , the c o u n t e r i o n does have m o b i l i t y and on.average t h e r e i s a s l i g h t l y h i g h e r c o n c e n t r a t i o n o f t h e s e m o b i l e b o u n t e r i o n s i n the v i c i n i t y o f the p o l y i o n than i n the b u l k o f the s o l u t i o n . Ion s e l e c t i v e e l e c t r o d e s a r e r e s p o n s i v e t o f r e e o r m o b i l e c a t i o n s and o f f e r a means o f d e t e c t i n g

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

PASiKA

Polysaccharide

Polyelectrolytes

2.0

\/

1

sp

c

Figure 13.

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

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EXTRACELLULAR MICROBIAL POLYSACCHARIDES

TABLE I . "IONIZATION CONSTANTS" OF SODIUM DEXTRAN SULFATE OF PS 0.487 ~" Polyelectrolyte Cone, g / d l .

Percent Ionization

0.9600 0.7200 0.4800 0.3600 0.2400 0.0960 0.0480 0.0240 0.0120

Ionization Constant K x l O

39.7 52.1 47.0 42.2 47.3 64.4 66.1 72. 81.

5.7 9.3 4.4 2.3 1.7 2.5 1.4

10 +

2

3 cone

4 5 x 10

6

7

8

9

10

Figure 14.

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

3

10.

PASiKA

Polysaccharide

Polyelectrolytes

143

their concentrations. I f i t i s assumed t h a t t h e sodium i o n o f an i o n p a i r does n o t i n t e r a c t w i t h t h e sodium i o n e l e c t r o d e and t h a t o n l y t h e m o b i l e i o n s do, then t h e p e r c e n t i o n i z a t i o n o f t h e p o l y e l e c t r o l y t e can be c a l c u l a t e d . The p e r c e n t i o n i z a t i o n s h o u l d i n c r e a s e on d i l u t i o n o f t h e p o l y e l e c t r o l y t e i f t h e d i l u t i o n t h e o r y and t h e e x p l a n a t i o n o f t h e reduced v i s c o s i t y curves i s c o r r e c t i n p r i n c i p l e . The p e r c e n t i o n i z a t i o n f o r a sodium d e x t r a n s u l f a t e o f DS 0.48 has been p l o t t e d as a f u n c t i o n o f p o l y e l e c t r o l y t e c o n c e n t r a t i o n i n F i g . 14. I t i s seen that the percent i o n i z a t i o n increases with d i l u t i o n , p a r a l l e l i n g t h e reduced v i s c o s i t y c u r v e s o f polyelectrolytes i ous media. Interestingly r e m i n i s c e n t o f the percent i o n i z a t i o n curve o f a c e t i c a c i d , s u g g e s t i n g t h a t i t might be p o s s i b l e t o c a l c u l a t e an i o n i z a t i o n c o n s t a n t f o r t h e sodium d e x t r a n sulfate. Values f o r the " i o n i z a t i o n constant" o f the sodium d e x t r a n s u l f a t e a t d i f f e r e n t p o l y e l e c t r o l y t e concentrations are given i n Table I . Allowing f o r the s c a t t e r i n t h e raw d a t a ( F i g . 1 4 ) , i t would appear t h a t w i t h i n l i m i t s t h e v a l u e o f Κ i s c o n s t a n t . The raw d a t a f o r t h e t h r e e most d i l u t e s o l u t i o n s which do n o t appear t o be s c a t t e r e d i n d i c a t e more c o n c l u s i v e l y t h e c o n s t a n c y o f K. In the event t h a t the l a t t e r c o u l d be o b t a i n e d f o r p o l y e l e c t r o l y t e s , then by analogy t o a c e t i c a c i d , where t h e c o u n t e r i o n ( t h e proton) i s c e r t a i n l y s i t e bound, i t c a n be argued t h a t t h e sodium i o n , o r c o u n t e r i o n , i s s i t e bound. An i o n i z a t i o n c o n s t a n t f o r p o l y e l e c t r o l y t e s would a l l o w s i m p l e q u a n t i f i c a t i o n o f t h e i n t e r a c t i o n o r i o n p a i r i n g o f c o u n t e r i o n s w i t h t h e macro i o n .

Literature Cited. 1.

Jeanes, Α., J. Polymer S c i . , Symposium Series (1974) 45, 209.

2.

Conway, B.E. and Dobry-Duclau, A. in "Rheology", ed. Eirich, F.R., 3, 89, Academic Press, N.Y. (1960).

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

11 Some Rheological Properties of G u m Solutions JOHN H. ELLIOTT Research Center, Hercules Inc., Wilmington, DE 19899

End-use applications of water-soluble polymers including extracellula are almost exclusivel properties which they confer upon the final system. A rather detailed knowledge of the rheological behavior of aqueous solutions of such polymers is essential for selection of the most suitable gum for a given end use. This paper w i l l review some general rheological properties of aqueous gum solutions, including suitable experimental instrumentation. Supermolecular structure may be present in certain gum solutions, which gives rise to time dependent rheological behavior. Finally the use of rheological data in selecting gums for specific end uses w i l l be illustrated. Rheological Background In this paper, we shall be concerned primarily with data obtained in viscometric or simple shear flows (1). Here there is a non-zero velocity compo nent in only one direction in the medium. Familiar examples are the flows in capillary, concentric cylinder, and cone and plate instruments. The simplest case is that of the Newtonian l i q u i d , where the shear stress, S (dynes/cm. ) is directly propor tional to the shear rate, γ (sec. ); the constant of proportionality being the viscosity, η (poise), 2

-1

S = ηγ

(1)

Here, t h e v i s c o s i t y i s a c o n s t a n t independent o f s h e a r rate. Gum s o l u t i o n s show t h i s b e h a v i o r a t h i g h d i l u 144

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

11.

ELLIOTT

145

Rheological Properties of Gum Solutions

tions. As t h e c o n c e n t r a t i o n (or m o l e c u l a r weight) i s i n c r e a s e d t o t h e p o i n t where entanglement o c c u r s ( 2 0 , however, t h i s s i t u a t i o n no l o n g e r p r e v a i l s and t h e v i s c o s i t y becomes a f u n c t i o n o f t h e shear r a t e , d e c r e a s i n g w i t h i n c r e a s i n g shear r a t e . This i s called p s e u d o p l a s t i c i t y o r shear t h i n n i n g . The complete p s e u d o p l a s t i c c u r v e i s shown i n F i g u r e 1. I t s h o u l d be emphasized t h a t w i t h i n t h e time s c a l e o f conven t i o n a l l a b o r a t o r y measurements, p s e u d o p l a s t i c i t y i s a r e v e r s i b l e phenomenon. There a r e t h r e e p r i n c i p a l regions o f t h i s l o g η v s . l o g γ curve. A t v e r y low shear r a t e s , t h e v i s c o s i t y i s Newtonian. T h i s zero shear o r f i r s t Newtonian v i s c o s i t y , η , i s a f u n c t i o n o f t h e m o l e c u l a r w e i g h t M and c o n c e n t r a t i o n C of the polymer. I t ha p o l y m e r - s o l v e n t system relationship(2 0

η

0

« c

3

Μ ·

5

3

(2)

4

4

The v a r i a t i o n o f η w i t h Μ · has been w e l l e s t a b l i s h e d f o r polymer m e l t s (_3) · As t h e shear r a t e i s i n c r e a s e d , a d e c r e a s e i n v i s c o s i t y i s observed. A f t e r a r e l a t i v e l y short t r a n s i t i o n r e g i o n , l o g η becomes l i n e a r i n l o g γ. T h i s i s t h e s o - c a l l e d power law r e g i o n and may c o v e r many decades i n s h e a r r a t e . T h i s i s g e n e r a l l y des c r i b e d by t h e f o l l o w i n g e q u a t i o n s 0

S = K(y)

n

and

1 1

η = K(^) "

(3a)

1

(3b)

The s l o p e o f t h e l o g η v s . l o g γ l i n e i n t h i s r e g i o n i s n-1. I f n i s one, t h e l i q u i d i s Newtonian; i f n i s l e s s t h a n one, i t i s p s e u d o p l a s t i c ; i f n i s g r e a t e r than one, t h e system i s d i l a t a n t o r shear t h i c k e n i n g . T h i s b e h a v i o r i s g e n e r a l l y o b s e r v e d i n systems c o n t a i n i n g a h i g h volume f r a c t i o n o f s o l i d s . The power law was c o n s i d e r e d as an e m p i r i c a l r e l a t i o n s h i p f o r many y e a r s ; however, S c o t t - B l a i r ( 4 ) has g i v e n a simple t h e o r e t i c a l d e r i v a t i o n based on t h e c o n c e p t o f t h e b r e a k i n g o f " l i n k a g e s " by s h e a r . As t h e shear r a t e i s i n c r e a s e d a n o t h e r t r a n s i t i o n zone i s o b s e r v e d , f o l l o w e d by a Newtonian r e g i o n , t h e i n f i n i t e shear o r second Newtonian v i s c o s i t y , nooT h i s r e g i o n i s o b s e r v e d a t v e r y h i g h shear r a t e s and i s very d i f f i c u l t t o study e x p e r i m e n t a l l y . The v a l u e of n appears t o show o n l y s l i g h t dependence on m o l e c u l a r w e i g h t , i n c o n t r a s t t o η and may be o r d e r s o f magnitude lower than η (5) . The η region i s of l i t t l e p r a c t i c a l importance and i s r a r e l y o b s e r v e d . O

Q

0

0

0

0

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

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EXTRACELLULAR MICROBIAL POLYSACCHARIDES

The f o l l o w i n g i s a q u a l i t a t i v e r a t i o n a l i z a t i o n o f the g e n e r a l p s e u d o p l a s t i c curve shown i n F i g u r e 1(60 . In the n r e g i o n , the time s c a l e o f the measurement i s s u f f i c i e n t l y l o n g t h a t s t r u c t u r e o r entanglements a r e not d i s r u p t e d and Newtonian f l o w i s o b s e r v e d . As the shear r a t e i s i n c r e a s e d , the time s c a l e becomes s h o r t e r and the p o l y m e r i c u n i t s cannot r e l a x . Struc t u r e i s broken down and the polymer m o l e c u l e s tend t o become o r i e n t e d i n t h e f l o w d i r e c t i o n . These e f f e c t s i n c r e a s e w i t h i n c r e a s i n g shear r a t e , g i v i n g r i s e t o power law b e h a v i o r . As t h e shear r a t e i s i n c r e a s e d t o v e r y h i g h v a l u e s , breakdown and o r i e n t a t i o n have gone as f a r as p o s s i b l e and a f u r t h e r i n c r e a s e i n shear r a t e does n o t a f f e c t them The f l o w i s then Newtonian, the η region The η and powe region importan i n c h a r a c t e r i z i n g aqueous gum systems. Some g e n e r a l i z a t i o n s can be made about b e h a v i o r i n these r e g i o n s . F i g u r e 2 shows l o g η v s . l o g γ p l o t s f o r xanthan gum i n aqueous s o l u t i o n . A t the h i g h e r c o n c e n t r a t i o n s , 2500 and 1500 ppm., the η r e g i o n l i e s a t shear r a t e s below 10~2 sec."" . T h i s region i s q u i t e apparent, however, a t the lower c o n c e n t r a t i o n s , 500 and 250 ppm. I t i s a l s o a p p a r e n t t h a t as the polymer c o n c e n t r a t i o n i s i n c r e a s e d , the t r a n s i t i o n from η t o non-Newtonian f l o w o c c u r s a t lower v a l u e s o f the shear r a t e . The power law s l o p e a t h i g h e r c o n c e n t r a t i o n s i s q u i t e steep and not v e r y s e n s i t i v e t o c o n c e n t r a t i o n . As c o n c e n t r a t i o n i s r e d u c e d , t h i s s l o p e becomes l e s s s t e e p , and, w h i l e not shown i n F i g u r e 2, a t v e r y low c o n c e n t r a t i o n s Newtonian b e h a v i o r i s o b s e r v e d . S o l u t i o n s o f polymers, h a v i n g a l o n g - c h a i n branched s t r u c t u r e , w i l l show a lower η than a l i n e a r polymer o f the same weight average m o l e c u l a r w e i g h t . T h i s has been e x t e n s i v e l y s t u d i e d i n the case o f l i n e a r and l o n g - c h a i n branched p o l y e t h y l e n e s . A c l a s s i c study i n t h i s f i e l d i s t h a t o f Busse and Longworth (7_) . The e f f e c t o f s a l t s on the r h e o l o g i c a l p r o p e r t i e s o f aqueous gum s o l u t i o n s i s a m a t t e r o f c o n s i d e r a b l e p r a c t i c a l importance. The p r e s e n c e o f s a l t s markedly lowers the v i s c o s i t y o f d i l u t e s o l u t i o n s o f p o l y e l e c t r o l y t e s ; i n f a c t , we have o b s e r v e d a d e c r e a s e o f o v e r t h r e e decades i n the η v a l u e o f a 2500 ppm. s o l u t i o n of a polyacrylamide, having a n i o n i c f u n c t i o n a l i t y , i n g o i n g from d i s t i l l e d water t o a 2.2% b r i n e . T h i s e f f e c t may be l a r g e l y a t t r i b u t e d t o the p o l y e l e c t r o l y t e e f f e c t , t h a t i s , the i o n i c s t r e n g t h o f the medium r e d u c e s t h e r e p u l s i o n between a d j a c e n t c h a r g e s on t h e polymer c h a i n . T h i s r e s u l t s i n a conformaQ

σ ο

σ

0

1

0

0

0

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

11.

ELLIOTT

Rheological

Properties

of Gum

Solutions

LOWER NEWTONIAN REGION

LOG SHEAR RATE Figure 1. Viscosity as a function of shear rate for a pseudoplastic

American Chemical Society Library 1155 16th St., N.W.

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; Washington, O.C. 20036 ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

147

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EXTRACELLULAR MICROBIAL POLYSACCHARIDES

t i o n a l change from an extended c o n f i g u r a t i o n toward t h a t o f a random c o i l . In g e n e r a l , s a l t s o l u t i o n s a r e p o o r e r s o l v e n t s f o r w a t e r - s o l u b l e p o l y s a c c h a r i d e s than d i s t i l l e d water. As a consequence, t h e v i s c o s i t i e s o f d i l u t e s o l u t i o n s o f t h e s e polymers and t h e i r i n t r i n s i c v i s c o s i t i e s a r e lower i n s a l t s o l u t i o n s than i n pure water. It i s f r e q u e n t l y o b s e r v e d , however, t h a t c o n c e n t r a t e d s o l u t i o n s show h i g h e r v i s c o s i t i e s when s a l t s a r e p r e s e n t . Tager ( 8 J has c a r r i e d o u t e x t e n s i v e s t u d i e s o f t h e v i s c o s i t i e s of concentrated solutions of organic s o l u b l e polymers i n good and poor s o l v e n t s . In t h e case o f p o l a r polymers, v i s c o s i t i e s i n poor s o l v e n t s may be s e v e r a l decades h i g h e r t h a n those i n good s o l vents. T h i s i s a consequenc r e l a t i v e l y strong supermolecula polymer m o l e c u l e s i n t h e p o o r e r s o l v e n t . The same c o n s i d e r a t i o n s a r e a p p l i c a b l e t o w a t e r - s o l u b l e gums. I t i s apparent from t h e e a r l i e r d i s c u s s i o n t h a t a r e a l i s t i c r h e o l o g i c a l c h a r a c t e r i z a t i o n o f gum s o l u t i o n s r e q u i r e s t h e d e t e r m i n a t i o n o f i t s v i s c o s i t y as a f u n c t i o n o f shear r a t e o v e r a t l e a s t s e v e r a l decades o f shear r a t e . C o n c e n t r i c c y l i n d e r o r cone and p l a t e rheometers, c o v e r i n g a wide range o f shear r a t e s , a r e the most s u i t a b l e i n s t r u m e n t s . In our own work, when the v i s c o s i t y - s h e a r r a t e c u r v e was needed o v e r more than s i x decades o f shear r a t e , i t was n e c e s s a r y t o use t h r e e d i f f e r e n t i n s t r u m e n t s , t h e Weissenberg rheogoniometer, t h e Haake R o t o v i s c o , and t h e H e r c u l e s Hi-Shear V i s c o m e t e r , t o c o v e r t h e low, i n t e r m e d i a t e and h i g h shear r a t e r a n g e s , r e s p e c t i v e l y . Excellent agreement between i n s t r u m e n t s was found i n t h e r e g i o n s of o v e r l a p . Gum s o l u t i o n s show e l a s t i c as w e l l as v i s c o u s properties. These a r e r e a d i l y determined by imposing a s i n u s o i d a l s t r a i n o f s m a l l amplitude upon t h e sample, f o r example, u s i n g a cone and p l a t e i n s t r u m e n t . The r e s u l t i n g s t r e s s wave i s s i n u s o i d a l and has t h e same f r e q u e n c y as t h e imposed s t r a i n wave (9) . I t i s , however, o u t o f phase w i t h t h e s t r a i n wave, t h e phase a n g l e , δ, l y i n g between 0° and 9 0 ° . T h i s may be r e s o l v e d i n t o a component i n phase w i t h t h e s t r a i n , from which t h e dynamic modulus, G', may be c a l c u l a t e d . The s t r e s s wave component i n q u a d r a t u r e w i t h t h e imposed s t r a i n y i e l d s t h e dynamic v i s c o s i t y , η * . In and near t h e η r e g i o n , η and t h e v i s c o s i t y i n s t e a d y shear a r e i n good agreement when t h e s t e a d y shear i s p l o t t e d a g a i n s t γ and η' a g a i n s t t h e f r e q u e n c y , ω, i n radians/second, i . e . , considering ω i n o s c i l l a t i o n equal t o γ i n steady shear(10). A t higher frequen1

0

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c i e s η- g e n e r a l l y i s lower than the c o r r e s p o n d i n g steady shear v i s c o s i t y . F i g u r e 3 shows η, η , and G' f o r a 2% s o l u t i o n o f sodium c a r b o x y m e t h y l c e l l u l o s e (CMC) i n water. It i s seen t h a t η' l i e s somewhat below η. G increases with f r e q u e n c y w h i l e η' d e c r e a s e s , which i s the expected behavior. S u p e r m o l e c u l a r s t r u c t u r e , which i s o f t e n p r e s e n t i n gum s o l u t i o n s , g i v e s r i s e t o a v a r i e t y o f r h e o l o g i c a l phenomena. In c o n t r a s t w i t h p s e u d o p l a s t i c b e h a v i o r , t h e s e e f f e c t s a r e time dependent. The most common i s t h i x o t r o p y , which has been d e f i n e d as a reversible gel-sol transition. I t i s observed e x p e r i m e n t a l l y as a d e c r e a s e i n v i s c o s i t y w i t h time a t a c o n s t a n t shear r a t e c o s i t y value i s reached v i s c o s i t y w i l l r i s e t o i t s o r i g i n a l v a l u e , as the s t r u c t u r e i n the system r e f o r m s . A d i f f e r e n t and w i d e l y used method o f c h a r a c t e r i z i n g t h i x o t r o p y , developed some y e a r s ago by Green and Weltman(11), i n v o l v e s programmed i n c r e a s e s i n shear r a t e from r e s t t o a h i g h v a l u e (the up c u r v e ) , f o l l o w e d by a r a p i d d e c r e a s e back t o z e r o shear r a t e (the down c u r v e ) . A t y p i c a l example o f such a shear r a t e - s h e a r s t r e s s c u r v e i s shown i n F i g u r e 4. The a r e a o f the l o o p i s a measure o f the work p e r u n i t volume p e r second f o r t h i x o t r o p i c breakdown, under the c o n d i t i o n s o f the experiment. The e x t r a p o l a t e d i n t e r c e p t of the down c u r v e on the shear a x i s can be c o n s i d e r e d as a y i e l d stress. I t must be emphasized t h a t i f the programmed r a t e o f i n c r e a s e i n shear r a t e used i n o b t a i n i n g the up c u r v e i s changed, the a r e a o f the h y s t e r e s i s l o o p and the v a l u e o f the e x t r a p o l a t e d y i e l d s t r e s s w i l l , i n g e n e r a l , be d i f f e r e n t . The concept o f a y i e l d s t r e s s i s v e r y u s e f u l i n the r h e o l o g i c a l c h a r a c t e r i z a t i o n o f systems h a v i n g supermolecular s t r u c t u r e . I t was proposed some time ago by Bingham(12). Whether o r not i t r e a l l y e x i s t s has been debated i n the i n t e r v e n i n g y e a r s . Bingham's original definition is 1

1

(S - σ ) 0

= ηγ

(4)

where the shear s t r e s s , S, must exceed the y i e l d v a l u e , σ , b e f o r e f l o w can o c c u r . T h i s i s shown i n F i g u r e 5. In p r a c t i c e , t h i s type o f b e h a v i o r i s never s t r i c t l y observed. The e x p e r i m e n t a l f l o w c u r v e does not i n t e r s e c t the a b s c i s s a s h a r p l y but c u r v e s i n toward the o r i g i n , as shown by the d o t t e d l i n e . A s t r a i g h t por t i o n o f the c u r v e , a t h i g h e r shear r a t e s , however, may be e x t r a p o l a t e d t o the a b s c i s s a t o g i v p a v a l u e o f σ . 0

0

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

EXTRACELLULAR MICROBIAL POLYSACCHARIDES

2 % C M C - 7 H 4 F IN W A T E R

CO

3 _J 10 3 Û ο 2 ~ ol0

3

2 Ο Ο CO

10

10"

10

10

10*

10*

S H E A R R A T E ( S E C * ) OR F R E Q U E N C Y ( R A D I A N S / S E C ) 1

Figure 3. Dynamic viscosity and modulus and steady shear viscosity as functions of frequency or shear rate for 2.0% CMC in water

Figure 4. Hysteresis loop treatment. Stress vs. shear rate for a thixotropic ma terial—arrows indicate in creasing and decreasing shear rate

SHEAR S T R E S S

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Perhaps what may be o p e r a t i o n a l l y c o n s i d e r e d as a y i e l d s t r e s s i s m e r e l y the p r e s e n c e o f a r e l a x a t i o n time which i s v e r y much g r e a t e r than the time s c a l e o f the e x p e r i m e n t a l measurement. There i s a fundamental d i f f e r e n c e between steady s h e a r and dynamic measurements i n the case o f systems e x h i b i t i n g a time dependent r h e o l o g i c a l r e s p o n s e . The t o t a l s t r a i n , t o which the sample i s s u b j e c t e d i n s t e a d y shear, i s d e t e r m i n e d by the shear r a t e and the time t h a t i t i s a p p l i e d . The t o t a l s t r a i n can thus be v e r y l a r g e , l e a d i n g t o e x t e n s i v e s t r u c t u r e breakdown and polymer o r i e n t a t i o n . Dynamic measurements, on the o t h e r hand, are c a r r i e d out a t low s t r a i n a m p l i t u d e s . Under t h e s e c o n d i t i o n s , t h e r e i s l i t t l e o r no s t r u c t u r e breakdown o r polyme t i e s o f the sample ar Dynamic and s t e a d y shear measurements supplement each o t h e r t o g i v e a more complete r h e o l o g i c a l c h a r a c t e r i zation. I t must be remembered, however, t h a t the r h e o l o g i c a l s t a t e o f the system may be q u i t e d i f f e r e n t i n the two t y p e s of measurements. Rheological

Properties

i n End

Uses

Jeanes (13) has r e c e n t l y p u b l i s h e d an e x t e n s i v e r e v i e w on the a p p l i c a t i o n s o f e x t r a c e l l u l a r m i c r o b i a l polysaccharide-polyelectrolytes. In t h i s p a p e r , r h e o l o g i c a l p r o p e r t i e s i n end uses w i l l be i l l u s t r a t e d by s e v e r a l examples w i t h which the w r i t e r has had f i r s t - h a n d experience. These, u n f o r t u n a t e l y , have not been i n v o l v e d p r i m a r i l y w i t h e x t r a c e l l u l a r m i c r o b i a l p o l y s a c c h a r i d e s ; however, they do i l l u s t r a t e the a p p l i c a t i o n o f r h e o l o g i c a l c h a r a c t e r i z a t i o n t o end uses. Food Systems Sodium c a r b o x y m e t h y l c e l l u l o s e , often c a l l e d c e l l u l o s e gum o r CMC, i s a w i d e l y used component o f food systems. I t may a c t as a suspending agent, t h i c k e n e r , p r o t e c t i v e c o l l o i d , humectant, and t o c o n t r o l the c r y s t a l l i z a t i o n o f some o t h e r component. CMC i s c l a s s i f i e d by the Food and Drug A d m i n i s t r a t i o n under " s u b s t a n c e s t h a t are g e n e r a l l y r e c o g n i z e d as safe" (Gras). CMC i s p r e p a r e d by the r e a c t i o n o f a l k a l i c e l l u l o s e w i t h sodium c h l o r o a c e t a t e and i s a polyelectrolyte. Important parameters i n c h a r a c t e r i z i n g CMC are the average degree o f p o l y m e r i z a t i o n (DP), the average number of a n h y d r o g l u c o s e u n i t s per m o l e c u l e ; and the average degree of s u b s t i t u t i o n (DS),

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EXTRACELLULAR MICROBIAL POLYSACCHARIDES

the average number o f c a r b o x y m e t h y l groups per anhydroglucose u n i t . I t was e a r l y r e c o g n i z e d i n our r h e o l o g i c a l c h a r a c t e r i z a t i o n o f CMC s o l u t i o n s and g e l s t h a t samples h a v i n g the same nominal c h e m i c a l c o m p o s i t i o n and s o l u t i o n v i s c o s i t y c o u l d show markedly d i f f e r e n t r h e o l o g i cal properties. E a r l i e r work showed t h a t CMC p r e p a r e d under c o n d i t i o n s g i v i n g more u n i f o r m s u b s t i t u t i o n gave pseudoplastic solutions. I f t h e s e c o n d i t i o n s were not followed, t h i x o t r o p i c solutions r e s u l t e d . This i s p a r t i c u l a r l y t r u e f o r the lower DS l e v e l s . Our work l e d t o the c o n c l u s i o n t h a t a v e r y s m a l l q u a n t i t y o f unsubstituted c r y s t a l l i n e c e l l u l o s e residues, e x i s t i n g as f r i n g e m i c e l l e s , a c t as c r o s s - l i n k i n g c e n t e r s and enable a three-dimensiona N i j h o f f was g r a n t e patent(16 of CMC, h a v i n g low DS, t o form unctuous g e l s f o r low c a l o r i e spreads. T h i s prompted a study o f the r h e o l o g i c a l p r o p e r t i e s o f unctuous m a t e r i a l s . I t was found t h a t when such m a t e r i a l s (e.g., b u t t e r , mayonnaise and ointments) were s u b j e c t e d t o an imposed s i n u s o i d a l s t r a i n , which was g r e a t e r than the l i n e a r v i s c o e l a s t i c l i m i t , the r e s u l t i n g s t r e s s wave was not s i n u s o i d a l , but i n many c a s e s approached a square w a v e ( 1 7 ) . S i m i l a r o b s e r v a t i o n s have been r e p o r t e d by Komatsu, e t a l . (18). Our r e s u l t s were i n t e r p r e t e d i n terms o f a m o d i f i e d Bingham body, c o n s i s t i n g o f an e l a s t i c , a f r i c t i o n a l and a v i s c o u s element connected i n s e r i e s . The r e s p o n s e o f t h i s model t o s t e a d y shear and t o imposed s i n u s o i d a l shear has been c a l c u l a t e d ( 1 9 ) , and the model has p r o v e d t o be u s e f u l i n c h a r a c t e r i z i n g s t r u c t u r e d systems(20). Komatsu e t a l . i n t e r p r e t e d t h e i r e x p e r i m e n t a l r e s u l t s u s i n g the Casson e q u a t i o n , which w i l l be d i s c u s s e d l a t e r . F i g u r e 6 shows the e f f e c t o f DS on the s t e a d y shear p r o p e r t i e s o f 5% CMC s o l u t i o n s and g e l s . Curve A f o r the sample h a v i n g a DS o f 0.7 i s t y p i c a l o f a v i s c o e l a s t i c system, and t h i s was c o n f i r m e d by dynamic measurements. Curve Β f o r a sample o f DS 0.4 shows a s t e e p e r r i s e i n the s t r e s s and a l s o a s t r e s s o v e r s h o o t . The Curve C f o r an e x p e r i m e n t a l sample h a v i n g a DS o f 0.18 shows the v e r y sharp peak and r a p i d s t r e s s decay, c h a r a c t e r i s t i c o f unctuous systems. Curve D shows dynamic measure ments on the DS 0.18 sample. The square n a t u r e o f the s t r e s s curve i s obvious. T a b l e I i l l u s t r a t e s the t y p e s o f CMC used i n a number o f food p r o d u c t s . The c o n n e c t i o n between the t y p e used i n a g i v e n system and the s o l u t i o n r h e o l o g i c a l p r o p e r t i e s d i s c u s s e d above w i l l be a p p a r e n t . The y i e l d s t r e s s , as a u s e f u l r h e o l o g i c a l con c e p t , was d i s c u s s e d e a r l i e r . It i s frequently d i f f i -

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

ELLIOTT

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Figure 5. Stress vs. shear rate for a Bingham body

SHEAR STRESS (S-*OO|f)

0 Figure 6.

10

20

Solutions

30

40

50

Five percent CMC in water; effect of degree of substitution on stress response

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c u l t t o e s t a b l i s h even s e m i - q u a n t i t a t i v e l y , u s i n g Bingham's r e l a t i o n s h i p (equation 4 ) . The Casson equation, - σ ^ = A γ*

(5)

0

which was d e r i v e d f o r s u s p e n s i o n s , has p r o v e d t o be v e r y u s e f u l i n d e t e r m i n i n g s m a l l v a l u e s o f σ from a plot of against γ^(24). As an example, c n o c o l a t e m i l k i s f o r m u l a t e d witK~~about 0.03% κ-carrageenan which p r e v e n t s s e t t l i n g o f t h e cocoa. The κc a r r a g e e n a n causes a c o n s i d e r a b l e i n c r e a s e i n t h e v i s c o s i t y o f t h e m i l k - s u g a r system and t h e e x i s t e n c e o f a y i e l d s t r e s s has been p o s t u l a t e d Plots of S vs γ c o u l d n o t be e x t r a p o l a t e however, t h e Casso l i n e a r f o r values of between zero and one and p e r mit a r e l i a b l e e x t r a p o l a t i o n . ρ

Friction

Reduction

R h e o l o g i c a l b e h a v i o r , d i s c u s s e d so f a r , has been c o n f i n e d t o systems i n l a m i n a r f l o w . The phenomenon o f drag r e d u c t i o n i s o b s e r v e d o n l y i n t u r b u l e n t f l o w . The t r a n s i t i o n from l a m i n a r t o t u r b u l e n t f l o w i n a p i p e o c c u r s when t h e Reynolds Number, R, f o r t h e system becomes g r e a t e r t h a n about 2000 R

=

QZSL

(6)

y

d i s the pipe diameter, ν i s the l i n e a r v e l o c i t y of the f l u i d h a v i n g a d e n s i t y , p , and v i s c o s i t y y . R i s d i m e n s i o n l e s s when s e l f c o n s i s t e n t u n i t s a r e used, and i s the r a t i o o f i n e r t i a l t o v i s c o u s f o r c e s i n the fluid. Note t h a t t h e symbol f o r v i s c o s i t y has been changed from η t o y , which i s commonly used i n engineering. The phenomenon o f f r i c t i o n r e d u c t i o n has been known f o r some time, t h e f i r s t s c i e n t i f i c d e s c r i p t i o n h a v i n g been g i v e n by Toms i n 1948 (2!5) and i t i s o f t e n r e f e r r e d t o as t h e Toms e f f e c t . When s m a l l q u a n t i t i e s (10-500 ppm.) o f a h i g h m o l e c u l a r weight polymer a r e added t o a l i q u i d i n t u r b u l e n t f l o w , t h e r e i s a d r a m a t i c r e d u c t i o n i n t h e power n e c e s s a r y t o m a i n t a i n the same f l o w r a t e . When t h e same c o n c e n t r a t i o n o f polymer i s added t o t h e l i q u i d i n l a m i n a r f l o w , t h e only e f f e c t observed i s a s l i g h t i n c r e a s e i n v i s c o s i t y , which may be s c a r c e l y d e t e c t a b l e . I f p r e s s u r e drop i s measured a l o n g a tube, t h e f l u i d v e l o c i t y o r Reynolds

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TABLE I SOME USES OF CMC IN FOODS FOOD PRODUCT

PROPERTY CONFERRED BY CMC

DP

REFERENCE

CMC TYPE S**) DS

BAKED GOODS

WATER RETENTION, CONTROL OF BATTER VISCOSITY

H, M

0.7

NO

21 , 22, 23

DOUGHNUTS

GREASE HOLDOUT

H M

0.7

NO

21. 22. 23

STARCH SYSTEMS, WHIPPED TOPPINGS

INHIBITION OF SYNERESIS

H M

0.7

NO

21 , 22

SYRUPS, BEVERAGES, JUICES

THICKENER, VISCOSIFIER

H M

0.7

NO

21, 22, 23

ICE CREAM

TEXTURE, BODY, CONTROL OF SUGAR AND ICE CRYSTALLIZATION

H M

0.7

NO

21 , 22, 23

PET FOODS (SEMIMOIST)

BINDER

H M

0.7

NO

21 . 22, 23

CONFECTIONS

CONTROL OF SUGAR CRYSTALLIZATION

L

0.7

NO

21

LOW CALORIE SYRUPS

VERY

LOW CALORIE SPREADS

UNCTUOUSNES

*) H.M.L, HIGH, MEDIUM, OR LOW VISCOSITY

**) S, UNIFORM SUBSTITUTION

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22

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156

number b e i n g h e l d c o n s t a n t , t h e p e r c e n t r e d u c t i o n , % FR, i s g i v e n by %FR = 100 ; .

A P

A p N

o" ΔΡ

\ - 100

(1 -

POLYSACCHARIDES

friction

i

(7)

0

where Δ Ρ i s t h e p r e s s u r e drop f o r t h e pure l i q u i d and ΔΡ i s t h e p r e s s u r e drop f o r t h e same l i q u i d c o n t a i n i n g a low c o n c e n t r a t i o n o f polymer. Percent f r i c t i o n r e d u c t i o n s as h i g h as 70-80% have been o b s e r v e d . The importance o f t h i s e f f e c t t o those i n d u s t r i e s where l a r g e q u a n t i t i e s o f l i q u i d s must be pumped i s o b v i o u s . E x t e n s i v e r e s e a r c h s t u d i e s have a l s o been c a r r i e d by U n i t e d S t a t e s and f o r e i g Many w a t e r - s o l u b l e polymers m i c r o b i a l p o l y s a c c h a r i d e s have been t e s t e d as f r i c t i o n r e d u c t i o n a d d i t i v e s (2*8 ) . The mechanism by which t h e s e polymers produce f r i c t i o n r e d u c t i o n i s n o t c l e a r l y understood. In g e n e r a l , h i g h m o l e c u l a r weight and a l i n e a r s t r u c t u r e g i v e a more e f f i c i e n t polymer. The diameter o f t h e t e s t s e c t i o n i s a v e r y important parameter; g r e a t e r f r i c t i o n r e d u c t i o n s u s u a l l y a r e o b s e r v e d i n s m a l l e r diameter t u b e s . I t i s known t h a t these a d d i t i v e s t h i c k e n the v i s c o u s sub-layer a t the pipe w a l l . V i s c o e l a s t i c e f f e c t s almost c e r t a i n l y p l a y an e f f e c t and i t may be t h a t e l a s t i c energy s t o r a g e by the polymer m o l e c u l e i n t e r a c t s w i t h t h e s m a l l , energy d i s s i p a t i n g , turbulent eddies. Polymer s u p e r m o l e c u l a r s t r u c t u r e may a l s o p l a y a r o l e and i t has been sug g e s t e d t h a t t h i s c o u l d be a s i g n i f i c a n t f a c t o r i n t h e d i a m e t e r e f f e c t (2_9). The shear i n t u r b u l e n t f l o w can degrade the polymer m o l e c u l e s . Guar gum and sodium c a r b o x y m e t h y l c e l l u l o s e a r e more shear r e s i s t a n t b u t l e s s e f f e c t i v e f r i c t i o n r e d u c e r s than p o l y - ( e t h y l e n e oxid e ) and p o l y - ( a c r y l a m i d e s ) (27) . The f i e l d o f f r i c t i o n r e d u c t i o n i s v e r y a c t i v e , and t h e f o l l o w i n g r e v i e w s a r e recommended(24,30,31). 0

Flow Through Porous Media T h i s i s a n o t h e r s i t u a t i o n i n which t h e f l o w i s not l a m i n a r . The b a s i c e q u a t i o n f o r f l o w through porous media i s Darcy's law: Q A

_ k ΔΡ " μ Δ1

Q i s t h e f l o w r a t e i n cm.^/sec. through 9

s e c t i o n a l area o f A

(8) a cross

ΔΡ

(cm/) , ^ j - i s the pressure

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

11.

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Rheological Properties of Gum Solutions

157

g r a d i e n t i n atmospheres/cm., μ i s t h e v i s c o s i t y i n c e n t i p o i s e , and k i s t h e p e r m e a b i l i t y i n d a r c i e s . In cgs u n i t s , p e r m e a b i l i t y has t h e u n i t s cm. and one d a r c y e q u a l s 9 . 8 7 x l 0 - cm. . The d a r c y i s t h e commonly used u n i t o f p e r m e a b i l i t y i n g e o p h y s i c a l work. The r a t i o k/μ i s the m o b i l i t y . T h i s i s a most i m p o r t a n t parameter i n porous media s t u d i e s , and w i l l be d i s c u s s e d i n terms o f the use o f polymer s o l u t i o n s as m o b i l i t y b u f f e r s i n enhanced o i l r e c o v e r y . The economics o f enhanced o i l r e c o v e r y t e c h n i q u e s a r e now f e a s i b l e , i n view o f t h e energy s h o r t a g e and t h e h i g h c o s t o f imported p e t r o l e u m . The importance o f the m o b i l i t y i n such an o p e r a t i o n a r i s e s from t h e f a c t t h a t i f one l i q u i d , e.g., o i l , i s t o be pushe e.g., water o r a polyme have an e q u a l o r lower m o b i l i t y t o p r e v e n t f i n g e r i n g o r water b r e a k t h r o u g h , which would bypass r e c o v e r a b l e o i l i n the f o r m a t i o n ( 3 2 ) . C o n s i d e r a t i o n o f Darcy's e q u a t i o n i n d i c a t e s two mechanisms by which a d d i t i o n o f a polymer t o water can lower the m o b i l i t y . I t can i n c r e a s e the v i s c o s i t y and/or i t can lower the perme a b i l i t y o f the porous medium t o t h e aqueous s o l u t i o n . The l a t t e r i s brought about by polymer a d s o r p t i o n o r entrapment. In g e n e r a l , both mechanisms appear t o be o p e r a t i n g , however, one o r t h e o t h e r tends t o predominate. The f l o w o f a polymer s o l u t i o n t h r o u g h a porous medium i s n o t a l a m i n a r o r v i s c o m e t r i c f l o w and u n u s u a l e f f e c t s may be o b s e r v e d . F o r example, p o l y ( a e r y l a m i d e ) s o l u t i o n s show p s e u d o p l a s t i c b e h a v i o r i n viscometric flows. In porous media, however, such s o l u t i o n s appear t o e x h i b i t d i l a t a n t p r o p e r t i e s (33^) . Indeed a p o l y ( a e r y l a m i d e ) s o l u t i o n i n water showed b o t h p s e u d o p l a s t i c and d i l a t a n t r e s p o n s e s , depending upon t h e f l o w r a t e { 3 4 ) . As a polymer m o l e c u l e moves t h r o u g h the p o r e s o f a porous medium, i t i s s u b j e c t e d t o a c c e l e r a t i o n s and decelerations. These, t o g e t h e r w i t h the s t r e t c h i n g d e f o r m a t i o n which o c c u r s as i t p a s s e s t h r o u g h a f i n e p o r e , i n t r o d u c e e l a s t i c and r e l a x a t i o n e f f e c t s which are absent i n v i s c o m e t r i c f l o w s . Thus the f l o w b e h a v i o r o f polymer s o l u t i o n s i n a porous medium cannot be p r e d i c t e d from v i s c o m e t r i c measurements, but, i n g e n e r a l must be d e t e r m i n e d i n the s p e c i f i c porous medium o f i n t e r e s t . Xanthan gum i s b e i n g c u r r e n t l y t e s t e d f o r use i n m o b i l i t y c o n t r o l f o r enhanced o i l r e c o v e r y . Available i n f o r m a t i o n i n d i c a t e s t h a t i t o p e r a t e s t o lower m o b i l i t y p r i m a r i l y by i n c r e a s i n g v i s c o s i t y . As shown 9

2

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

EXTRACELLULAR MICROBIAL POLYSACCHARIDES

158

i n F i g u r e 2, xanthan s o l u t i o n s show p s e u d o p l a s t i c b e h a v i o r down t o q u i t e low c o n c e n t r a t i o n s . This i s d e s i r a b l e , g i v i n g low v i s c o s i t i e s a t t h e h i g h shear r a t e s encountered d u r i n g i n j e c t i o n , but s i g n i f i c a n t l y h i g h e r v i s c o s i t i e s when moving t h r o u g h t h e o i l b e a r i n g f o r m a t i o n , where shear r a t e s a r e i n t h e range o f 0.1 t o 10 s e c . " . 1

Literature Cited (1) (2)

(3) (4) (5) (6) (7) (8) (9) (10) (11)

(12) (13) (14) (15) (16) (17) (18) (19) (20)

Middleman, S., "The Flow of High Polymers", p. 8, Interscience, New York, 1968. Peterlin, Α . ,"Non-NewtonianViscosity and the Macromolecule", p 225 Advances in Macromolecular Chemistry, Volum Press, New York Reference 1, p. 172. Scott-Blair, G. W., Rheol. Acta (1965) 4, 53. Reference 1, p. 101. Lenk, R. S., "Plastics Rheology", p. 12, Wiley Interscience, New York, 1968. Busse, W. F., and Longworth, R., J. Polymer S c i . (1962) 58, 49. Tager, Α. Α . , Rheol. Acta (1974) 13, 831. Ferry, J. D . , "Viscoelastic Properties of Polymers", p. 12, Wiley, New York, 1970. Bueche, F., "Physical Properties of Polymers", p. 220, Interscience, New York, 1962. Green, H . , and Weltman, R. Ν . , Ind. Eng. Chem., Anal. Ed. (1943) 15, 201. Also Green, H . , "Industrial Rheology and Rheological Structures", Wiley, New York, 1949. Bingham, E. C . , "Fluidity and Plasticity", p. 217, McGraw-Hill, New York, 1922. Jeanes, Α . , J. Polymer S c i . (1974) Symposium No. 45, 209. Ott, Ε., and E l l i o t t , J . Η., Makromol. Chem. (1956) 18/19, 352. deButts, Ε. Η., Hudy, J. Α . , and Elliott, J . Η., Ind. Eng. Chem. (1957) 49, 94. Nijhoff, G. J. J., U.S. Patent 3,418,133. E l l i o t t , J . H . , and Ganz, A. J., J. Texture Studies (1971) 2, 220. Komatsu, Η., Mitsui, T . , and Onogi, S., Trans. Soc. Rheol. (1973) 17:2, 351 E l l i o t t , J . H . , and Green, C. E., J. Texture Studies (1972) 3, 194. E l l i o t t , J . H . , and Ganz, A. J., Rheol. Acta (1974) 13, 1178.

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

11.

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Rheological Properties of Gum Solutions

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(21) Ganz, A. J., Food Product Develop. (1969) 3 (6), 65. (22) Batdorf, J . Β . , "Industrial Gums", R.L. Whistler, Ed., Academic Press, New York, 1959. (23) Glicksman, Μ.,"GumTechnology in the Food Industry", Academic Press, New York, 1969. (24) Scott-Blair, G. W., Rheol. Acta (1966) 5, 184. (25) Toms, Β. Α . , "Proceedings International Rheological Congress, Holland 1948", p. II-135, North Holland Publishing Co., Amsterdam, 1949. (26) L i t t l e , R. C., Hansen, R. J., Hunston, D. L., Kim, Ο. Κ., Patterson, R. L., and Ting, R. Υ . , Ind. Eng. Chem., Fundam. (1975) 14, 283. (27) Van der Meulen J. H J., Appl Sci Res (1974) 29, 161. (28) Hoyt, J. W., Polymer Letters (1971) 9, 851. (29) E l l i o t t , J . H . , and Stow, F. S. Jr., J. Appl. Polym. S c i . (1971) 15, 2743. (30) Gadd, G. Ε., "Encyclopedia of Polymer Science and Technology", Vol. 15, H. F. Mark, Chairman Editorial Board, Ν. M. Bikales, Executive Editor, Interscience-Wiley, New York, 1971. (31) Lumley, J. L., J. Polym. S c i . (1973) Macromolecular Reviews, 7, 263. (32) Collins, R. Ε., "Flow of Fluids Through Porous Materials", p. 196, Reinhold, New York, 1961. (33) Burcik, E. J., Producers Monthly (1967) 31, No. 3 27. (34) Jones, W. Μ., and Davies, Ο. Η., Nature Phys. Sci. (Nov. 13, 1972) 240, 46.

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

12 Rheology of Xanthan G u m Solutions P. J. WHITCOMB General Mills Chemicals, Inc., 2010 E. Hennepin Ave., Minneapolis, MN 55413 B. J. EK and C. W. MACOSKO Dept. of Chemical Engineering and Material Science, University of Minnesota, Minneapolis, MN 55455

Xanthan gum is a charide produced by fermentation with the bacterium "Xanthomonas campestris". Adding ½% of t h i s biopolymer increases water's v i s c o s i t y by a factor of 100,000 at low shear r a t e s ; yet at high shear r a t e s , the factor i s reduced to 10. This remarkable shear t h i n n i n g ability ( p s e u d o p l a s t i c i t y ) can be used to great advantage. In fact the main use of xanthan gum is rheology c o n t r o l . In the past most data on xanthan gum rheology has been taken over a l i m i t e d shear rate range and i s relative, not absolute. Figure 1 presents t y p i c a l shear stress v s . shear rate data. Only two decades of shear rate are covered and the shear rate i s given as rpm, r e l a t i v e u n i t s . The use of arithmetic scales for t h i s p l o t make it difficult to resolve s o l u t i o n properties. Figure 2 presents a typical v i s c o s i t y v s . shear rate p l o t . The use of l o g scales aids i n i n t e r p r e t a t i o n of the flow curves. However shear rate covers only two decades and i s again reported as rpm. While r e l a t i v e data i s useful for comparisons under s p e c i f i e d c o n d i t i o n s , there are many advantages to having absolute data, where the units have p h y s i c a l s i g n i f i c a n c e . Absolute data i s independent of the instrument or geometry used to gather the data. R e l a t i v e data i s not. This means that a very broad shear rate range can be covered by compiling r e s u l t s , obtained i n absolute u n i t s , from several instruments. Shear rate overlap between instruments insures the integrity of the data, since a systematic error due to a p a r t i c u l a r instrument will be detected. With absol u t e data, c o n s t i t u t i v e or e m p i r i c a l r e l a t i o n s h i p s can be used to model the dependence of v i s c o s i t y on shear r a t e . Such models are e s s e n t i a l to p r e d i c t flow 160

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

WHITCOMB ET AL.

Rheology

of Xanthan

Gum

Solutions

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EXTRACELLULAR MICROBIAL POLYSACCHARIDES

behavior, design equipment, and i n t e r p r e t rheology i n terms o f molecular s t r u c t u r e . I t i s t h e purpose o f t h i s work t o c o l l e c t a b s o l u t e data and e v a l u a t e i t i n t e r m s o f an a p p r o p r i a t e model. Description A commercial p r o d u c t , G A L A X Y X B X a n t h a n gum ( l o t D 5 3 5 3 A ) , was u s e d f o rt h i s s t u d y . The c o m m e r c i a l p r o d u c t was p u r i f i e d f u r t h e r u s i n g a m o d i f i c a t i o n o f a p r o c e d u r e b y J e a n e s (l) : T h e gum i s h y d r a t e d i n a water-ethanol mixture, c e n t r i f u g e d , p r e c i p i t a t e d and washed. T h e w e t gum i s t h e n d r i e d a n d g r o u n d , A schematic o f t h e p u r i f i c a t i o F i g u r e 3. Analysi gums a r e g i v e n i n T a b l e I , T h e v i s c o s i t y o f t h e p u r i f i e d gum i s s l i g h t l y l e s s t h a n t h a t o f t h e c o m m e r c i a l g u m , s e e F i g u r e k. The r e m a i n d e r o f t h i s paper d e a l s o n l y w i t h t h e p u r i f i e d gum, TABLE

1

ANALYSIS Commercial Product S o l u t i o n O.D, kOO NM Water

- Wt. %

Nitrogen Protein Ash

- Wt, % MFB -

Wt, % MFB

- Wt, % MFB

Sodium

- Wt. % MFB

Phosphorus

- W t , % MFB

Purified Gum

.07

.19 10,6

1 3 .0

0.67

0 .69

3.8

3 .9

13.8

7 .9

k.h

1 .9

0.32

0 .23

A l l s o l u t i o n s used i n t h i s study were prepared f r o m gum h y d r a t e d i n d i s t i l l e d w a t e r . The s o l u t i o n s w e r e p r e p a r e d b y s p r i n k l i n g t h e g r o u n d x a n t h a n gum onto t h e s i d e s o f a v o r t e x formed i n a high speed blender. A l l gum c o n c e n t r a t i o n s a r e r e p o r t e d o n a moisture free basis.

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

WHITCOMB E T A L .

Rheology

of Xanthan

Gum

Solutions

95% eTWAMOU

IL moisi

Figure 3.

Purification procedure

4+

••

•

4470 Ppri COflHEftciAL XAHIHOH

•

4470 PPM PUfUHfcP )j

2 +

S

ot

•—I—

-4

-z

H— Ο

UOC, 5H&AP- £ATe, <&C

A

Figure 4.

Commercial vs. purified viscosity

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

EXTRACELLULAR MICROBIAL POLYSACCHARIDES

164

I n t r i n s i c v i s c o s i t y was d e t e r m i n e d u s i n g a Cannon-Ubbelohde c a p i l l a r y d i l u t i o n v i s c o m e t e r , number 598 - s i z e 1 0 0 . A l l o t h e r r h e o l o g i c a l d a t a was t a k e n on e i t h e r a W e i s e n b e r g R h e o g o n i o m e t e r (2) o r a Rheom e t r i c s M e c h a n i c a l S p e c t r o m e t e r (3_) u s i n g c o n e a n d plate fixtures. The cone and p l a t e geometry w i t h i t s a s s o c i a t e d e q u a t i o n s i s p i c t u r e d i n F i g u r e 5. The primary advantage o f t h i s geometry i s that shear stress and s h e a r r a t e a r e n e a r l y c o n s t a n t t h r o u g h o u t t h e sample. T h i s means t h a t g r a p h i c a l d i f f e r e n t i a t i o n o f t h e d a t a i s n o t needed t o o b t a i n t h e s h e a r s t r e s s .vs. s h e a r r a t e c u r v e i n a b s o l u t e u n i t s (k). The i n s t r u ments used p r o v i d e s t e a d y r o t a t i o n a l speeds which c a n be v a r i e d t o c o v e r a v a r y i n g cone a n g l e expanded. The shear s t r e s s τ was c a l c u l a t e d from t h e m e a s u r e d t o r q u e o n t h e s t a t i o n a r y member o f t h e cone and p l a t e . The s h e a r r a t e dependent viscosity, η(γ), was d e t e r m i n e d f o r each s h e a r r a t e . The combi n a t i o n s o f cones a n d i n s t r u m e n t s u s e d a r e shown i n T a b l e I I . N o t e how t h e s h e a r r a t e r a n g e s o v e r l a p . 1

2

TABLE I I INSTRUMENT INSTRUMENT

CONE ANGLE RAD, Β

RANGES CONE RADIUS CM. R

SHEAR RATE SEC^ 1

Ϋ MAX.

MIN. WEISSENBERG

MECHANICAL SPECTROMETER

0.1

3.6

5.95 x 10"" *

1U8

0.06

3.6

9.92 x

2l*7

0.1

3.6

10~

0.0k

3.6

0.01

1.25

1

10"

h

koo

2

10

0.25 10-

1

3

kooo

E v e r y cone a n d i n s t r u m e n t c o m b i n a t i o n was c a l i b r a ted w i t h a Newtonian o i l , d i o c t y l phthalate. A l l data was t a k e n a t r o o m t e m p e r a t u r e , w h i c h v a r i e d b e t w e e n 25°C a n d 29°C, The x a n t h a n d a t a was n o t c o r r e c t e d f o r temperature. Figure 6 presents the calibration data c o r r e c t e d t o 25°C, Observe t h e approximate range covered by t h e Rheogoniometer and Mechanical Spectro-

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

12.

WHITCOMB ET AL.

Rheology of Xanthan Gum Solutions

STEADY FLOW

fi

Ϋ «= ϋύ/β

SHEAR RATE SHEAR STRESS

2k*2ïlè

Figure 5.

-2-1

-ι

Flow between a cone and plate

1

1

ο

ι tf*

Figure 6.

SHEAR VISCOSITY

àHtAR B W ,

» t. St*"

1

Dioctyl phthakte (corrected to 25°C)

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

165

166

EXTRACELLULAR MICROBIAL POLYSACCHARIDES

meter. The f o l l o w i n g c o r r e c t i o n (5.) log Where

η η

2

n

5

=

l o g

η

- Viscosity - Viscosity

5

τ

ΔΤ d

2

e q u a t i o n was u s e d

η

τ

d +

d

^°S

η

corrected a t T°C

f o r temperature

(ΔΤ) t o 25°C

(T-25)°C

- S l o p e o f l o g η v s . Τ b e t w e e n 25°C and T°C.

Data D a t a was t a k e n e a c h o f f o u r c o n c e n t r a t i o n s o f t h e p u r i f i e d gum; 1 0 , 0 0 0 , 2 , 0 0 0 , 1 , 0 0 0 a n d kkf p p m . T h i s d a t a was f i r s t p l o t t e d as s h e a r s t r e s s v s . s h e a r r a t e t h e n as v i s c o s i t y v s . shear rate, I n t h e graphs o f shear stress the dashed l i n e r e p r e s e n t s t h e shear s t r e s s p l o t o f water. I n t h e graphs o f v i s c o s i t y t h e base l i n e a t 10~ poise i s t h e v i s c o s i t y of water, A l lgraphs a r e in absolute units. The s h e a r s t r e s s p l o t s o f t h e 10,000 a n d 2,000 ppm s o l u t i o n s a r e s h o w n i n F i g u r e 7. N o t i c es i x d e c a d e s o f s h e a r r a t e a r e c o v e r e d f o r t h e 1 0 , 0 0 0 ppm solution. At very low shear rates t h e curve f o r t h e 1 0 , 0 0 0 ppm s o l u t i o n s e e m s t o b e f l a t t e n i n g o u t . I t appears that t h i s s o l u t i o n has a d e f i n i t e y i e l d stress, τ , n e a r 13 d y n e / c m . T h e 2 , 0 0 0 ppm s o l u t i o n s h o w s n o evidence o f a y i e l d stress. The v i s c o s i t y p l o t s o f t h e 1 0 , 0 0 0 a n d 2,000 ppm s o l u t i o n s a r e s h o w n i n F i g u r e 8, The y i e l d s t r e s s o f t h e 1 0 , 0 0 0 ppm s o l u t i o n i s n o t a s e v i d e n t o n a v i s cosity plot. However, Newtonian r e g i o n s a r e more r e a d i l y i d e n t i f i e d on a p l o t o f v i s c o s i t y t h a n on one of shear s t r e s s . T h e v i s c o s i t y c u r v e o f t h e 2,000 ppm s o l u t i o n appears t o be f l a t t e n i n g toward a c o n s t a n t value at low shear rates, This Newtonian value i s known a s t h e z e r o s h e a r r a t e v i s c o s i t y , ηο· F i g u r e 9 shows t h e s h e a r s t r e s s v s , s h e a r rate p l o t f o r t h e 1 , 0 0 0 a n d hkj p p m s o l u t i o n s . There a r e no i n d i c a t i o n s o f a y i e l d s t r e s s . The v i s c o s i t y v s , shear rate curves, Figure 10, f o r these concentrations appear t o be a p p r o a c h i n g a Newtonian r e g i o n a t t h e l o w end o f t h e s h e a r r a t e range covered. These concen t r a t i o n s a r e e x p e c t e d t o go N e w t o n i a n a t low shear r a t e s , a s d i d t h e 2,000 ppm s o l u t i o n , T h e i n t r i n s i c v i s c o s i t y o f t h e p u r i f i e d gum w a s determined from a p l o t o f inherent v i s c o s i t y 2

2

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

12.

WHITCOMB E T A L .

Rheology

of Xanthan

Gum

Solutions

ο · zooo

···

-4

-3

-2-

-I

Figure 8.

•

Ο

•

I

Xanthan gum rheology

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

167

168

EXTRACELLULAR MICROBIAL POLYSACCHARIDES

Figure 9.

Xanthan gum rheology

it

Θ · 447 Pm

G

Θ ©0

-2-1— -2.

Θ

— -—-— ·>-

t

Figure 10.

»

Xanthan gum rheology

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

*

h

12.

WHITCOMB ET AL.

Rheology

of Xanthan

Gum

Solutions

extrapolated t o zero concentration. This i s i l l u s t r a t e d i n F i g u r e 11 a n d y i e l d s an v i s c o s i t y o f 35.70 d e c i l i t e r s / g m . Discussion

and

169

determination intrinsic

Conclusions

There appears t o be a c r i t i c a l concentration b e t w e e n 2 , 0 0 0 a n d 1 0 , 0 0 0 ppm a b o v e w h i c h a y i e l d s t r e s s exists. I d e n t i f y i n g and measuring y i e l d stress i s important for p r e d i c t i n g t h e long term s t a b i l i t y o f suspensions, Concentrations below t h e c r i t i c a l value w i l l have a Newtonian r e g i o n a t very l o w shear r a t e s , This r e g i o n i s c h a r a c t e r i z e d by t h e zero shear rate v i s c o s i t y , r)o. A l Newtonian region a t h i s r e g i o n t h e s o l u t i o n v i s c o s i t i e s w i l l go b e l o w that o f their solvent, water. This region i s charac t e r i z e d by t h e i n f i n i t e shear r a t e v i s c o s i t y , . Many m a t h e m a t i c a l e x p r e s s i o n s have been proposed to model t h e p s e u d o p l a s t i c behavior e x h i b i t e d by xanthan. The most w i d e l y used i s t h e power l a w o f O s t w a l d (6) . η = Κ f ~ n

1

The p o w e r l a w i s a p p e a l i n g b e c a u s e o f i t s s i m p l i c i t y , t h e r e a r e o n l y t w o a d j u s t a b l e p a r a m e t e r s n a n d Κ. K, t h e v i s c o s i t y a t 1 s e c "" * m e a s u r e s c o n s i s t e n c y a n d n , t h e f l o w i n d e x , m e a s u r e s p s e u d o p l a s t i c i t y . Many e m p i r i c a l and a n a l y t i c a l s o l u t i o n s f o r complex flows have been worked o u t u s i n g t h e power law. Examples b e i n g l a m i n a r a n d t u r b u l e n t p i p e f l o w , (7.) a n n u l a r f l o w (8_) , f l o w t h r o u g h p o r o u s m e d i a (9.) » m i x i n g c h a r a c t e r i s t i c s a n d h e a t t r a n s f e r p r o b l e m s (10_) t o c i t e a few. The m a i n d i s a d v a n t a g e o f t h e power l a w i si t s f a i l u r e i n t h e r e g i o n s o f v e r y l o w s h e a r , rio o r τ , a n d very high shear, . More s o p h i s t i c a t e d models c S n account f o r t h e Newtonian regions i n high and l o w shear rate regions. However, f o r t h e xanthan c o n c e n t r a t i o n s s t u d i e d t h e l o g data i s l i n e a r f o r s e v e r a l decades o f shear r a t e . T a b l e I I I shows t h e power l a w f i t o f o u r xanthan data. The h i g h d e t e r m i n a t i o n i n d e x and t h e broad range o f shear r a t e f i t f o r each c o n c e n t r a t i o n v e r i f y t h e u t i l i t y o f t h i s model, 1

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

170

EXTRACELLULAR MICROBIAL POLYSACCHARIDES

TABLE I I I POWERLAW

CONSTANTS

Κ CONC. (DYNE*SEC) η PPM /CM DIMENSIONLESS 2

10,000

35

2,5xKf

2

2.5xlO

1

6.25xlO

3.U

.39

l.OxlO""

1,000

0 98

Λ9

U.TlxlO

kkj

- 1

.98

+ 3

.23

2,000

f

DETERMINATION INDEX

1

γ RANGE, SEC"" MAX. MIN.

U.OxlO*

2

+ 2

.97 .99

0.23

Examination of Table I I I reveals Κ increases with increasing concentration while η decreases. T h i s means t h a t t h e h i g h e r t h e c o n c e n t r a t i o n o f xanthan t h e thicker the s o l u t i o n . However, h i g h e r c o n c e n t r a t i o n s a r e more p s e u d o p l a s t i c , have a l o w e r η v a l u e , so a t h i g h shear r a t e s a l l c o n c e n t r a t i o n s , a t l e a s t t h o s e o f 1 0 , 0 0 0 ppm and l e s s , a p p r o a c h t h e v i s c o s i t y o f w a t e r . The power l a w f i t f o r t h e 1 0 , 0 0 0 a n d 1 , 0 0 0 ppm s o l u t i o n s a r e shown i n F i g u r e 12. Note t h e d i f f e r e n c e i n Κ and η f o r the two c o n c e n t r a t i o n s , see T a b l e I I I . Power law d a t a i s v e r y v a l u a b l e i n e v a l u a t i n g s o l u t i o n p r o p e r t i e s and solving p r a c t i c a l problems. A n o t h e r type o f t h e o r y has been used t o e x p l o r e intrinsic viscosity. Assuming the conformation of a x a n t h a n m o l e c u l e c a n be a p p r o x i m a t e d by a c y l i n d r i c a l rod, i t i s possible to estimate i t s characteristic length. U s i n g t h e t h e o r y o f K h a l i k a n d B i r d (11.) r o d l e n g t h c a n be d e t e r m i n e d by t h e f o l l o w i n g e x p r e s s i o n : T.3

=

[η]

(US)

L [η]MW D Ν -

(MW)

( I n (L/D)

)

Rod l e n g t h Intrinsic viscosity Molecular weight Rod d i a m e t e r A v o g a d r o number

T h i s e x p r e s s i o n can be s o l v e d by i t e r a t i o n i f r o d l e n g t h i s t h e o n l y unknown. The m o l e c u l a r w e i g h t o f x a n t h a n h a s e s t i m a t e d t o b e i n t h e r a n g e o f l . U t o 3.6 χ 10 ( D i n t z i s et a l (12)) , The i n t r i n s i c viscosity was m e a s u r e d t o be 35.7 d e c i l i t e r s / g m . We h a v e 6

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

12.

WHiTCOMB E T A L .

ο

1

1

1

'

Rheology of Xanthan Gum Solutions

4 — ι — ι — ι — ι — \ — ι — Η .ά>5 .ΟΙΟ Λ ~ '' ' J. ~

171

1 — ι — I — ι — ι — I — " — I .οΊ* *™ Jill

Figure 11.

Intrinsic viscosity

Figure 12.

Power lawfitof data

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

172

EXTRACELLULAR MICROBIAL POLYSACCHARIDES

v

v

estimated the diameter of a xanthan r o d t o be i n t h e r a n g e o f l 6 t o kOA the length calculation i s quite i n s e n s i t i v e t o diameter, 9

TABLE CALCULATED MOLECULAR WEIGHT GM/MOLE l.k

χ

10

6

l.U

χ

10

6

3.6 χ

10

3.6 χ

10

IV LENGTH

DIAMETER 1

[η] ML/GM

LENGTH MICRONS

16

3570

0,73

6

16

3570

1.01

6

ho

3570

0.96

Table IV gives the r e s u l t s of the r o d length calculation. I t would appear that a xanthan " r o d " has a l e n g t h b e t w e e n 0.7 a n d 1.0 m i c r o n s . This i s i n g o o d a g r e e m e n t w i t h H o l z w a r t h s (l_3) membrane c h r o m a tography measurements. He f o u n d t h a t e s s e n t i a l l y a l l x a n t h a n p a r t i c l e s c a n p a s s t h r o u g h a membrane w i t h 1.0 m i c r o n p o r e s b u t a r e b l o c k e d by a membrane w i t h 0.8 micron pores. f

Nomenclature D Κ L M MWn Ν O.D. R Τ β t η Ποη

οο-

[η]

Rod d i a m e t e r , (microns) Power l a w c o n s t a n t , i n t e r c e p t Rod l e n g t h (microns) Torque (gm-cm) Molecular Weight Power l a w c o n s t a n t , s l o p e A v o g a d r o number Optical Density Cone r a d i u s (cm) T e m p e r a t u r e (°C) Cone a n g l e (radians) Shear r a t e (sec ) Viscosity (poise) Zero shear r a t e v i s c o s i t y (poise) (poise) I n f i n i t e shear rate v i s c o s i t y Intrinsic viscosity (deciliters/gm) - 1

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

WHITCOMB E T A L .

12.

τ τ oo

Rheology of Xanthan Gum Solutions

173

2

1

2

Shear s t r e s s (dyne/cm ) Yield stress (dyne/cm ) Angular speed (radians/sec , 1 2

y

Literature Cited (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13)

Jeanes, A., P i t t s l e y , J.E., Senti, F.R., J. Applied Polymer S c i , (1961), 5, p. 519-526. Van Wazer, J.R., et al, "Viscosity and Flow Measurement", p. 113-116, Wiley, Ν.Υ. 1963. Macosko, C.W., S t a r i t a , J., S.P.E. Journal,(1971), 27, p. 38-42. Middleman, S., "The Wiley Willey, S.J. Ph.D. Thesis, University of Minnesota, 1976. Ostwald, W., Kolloid-Zeitschrift, (1925), 36, p. 99-117. Metzner, A . B . , Reed, J.C., A.I.Ch.E. J., (1955), 1, p. 434-440. Mishra, P., Mishra, I., A.I.Ch.E. J.,(1976), 22, p. 617-619. Sheffield, R.E., Metzner, A . B . , A.I.Ch.E. J., (1976), 22, p. 736-744. Wilkinson, W.L., "Non-Newtonian Fluids", Pergamon, New York, 1960. Abdel-Khalik, S . K . , B i r d , R.Β., Biopolymers, (1975), 14, p. 1915-1932. D i n t z i s , E . R . , Babcock, G.Ε., Tobin, R., Carbohydrate Research. (1970), 13, p. 257-267. Holzwarth, G . , "Polysaccharide from Xanthomonas Campestris: Rheology, Solution Conforma t i o n , And Flow Through of Petroleum Chemistry, A.C.S., New York Meeting, April 4-9, 1976.

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

13 Synergistic Xanthan Gels I. C. M . D E A

and

E . R.

MORRIS

Unilever Research, Colworth/Welwyn Laboratory, Sharnbrook, Bedford, MK44, 1LQ, Great Britain

Although showing considerable evidence of strong i n t e r molecular i n t e r a c t i o n gel. On mixing with anothe bean gum, however, f i r m rubbery g e l s are formed at low polymer l e v e l s , t y p i c a l l y around 1% t o t a l p o l y s a c c h a r i d e , with most e f f e c t i v e xanthan u t i l i s a t i o n a t locust-bean gum: xanthan r a t i o s of about 3:1 (1/2/3.)· The molecular o r i g i n of the synergism has u n t i l r e c e n t l y (4_,5_,(5) remained obscure. In t h i s paper we w i l l attempt to answer two questions:1) What i s the mechanism o f formation o f these mixed g e l s ? 2) Why should two p o l y s a c c h a r i d e s of such d i v e r s e o r i g i n s interact: T e c h n o l o g i c a l Relevance o f B i o l o g i c a l Function Many o f the i n d u s t r i a l uses o f p o l y s a c c h a r i d e s r e s t s o l e l y on t h e i r water b i n d i n g c a p a c i t y and high v i s c o s i t y a t low concentrations. I n c r e a s i n g l y , however, more s o p h i s t i c a t e d a p p l i c a t i o n s depend on d e t a i l e d molecular s t r u c t u r e , and e x p l o i t i n v i t r o the s p e c i f i c f u n c t i o n o f the p o l y s a c c h a r i d e i n v i v o . T h i s i s p a r t i c u l a r l y t r u e i n g e l l i n g systems. Thus agar, carrageenan, f u r c e l l a r a n , p e c t i n and a l g i n a t e a l l have a s t r u c t u r a l r o l e i n nature, which g i v e s r i s e d i r e c t l y to t h e i r g e l l i n g behaviour. A l g i n a t e , f o r example, i s the major s t r u c t u r a l p o l y s a c c h a r i d e of Brown Seaweed. Chemically i t i s a block co-polymer of D-mannuronic and L - g u l u r o n i c a c i d , i n which the homopolymeric polyguluronate sequences are capable o f forming very strong i n t e r molecular c r o s s - l i n k s , while polymannuronate or a l t e r n a t i n g sequences show f a r l e s s tendency t o a s s o c i a t e (5-11). The r e l a t i v e amount o f the v a r i o u s block types i s under enzymic cont r o l a t the polymer l e v e l (12), p r o v i d i n g s u b t l e b i o l o g i c a l cont r o l o f the mechanical p r o p e r t i e s o f d i f f e r e n t p a r t s of the p l a n t at d i f f e r e n t stages of maturation. These s t r u c t u r a l v a r i a t i o n s are r e f l e c t e d d i r e c t l y i n the g e l a t i o n p r o p e r t i e s of the p o l y -

174

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

13.

DEA AND MORRIS

Synergistic

Xanthan

Gels

175

saccharide i n v i t r o . Thus a l g i n a t e e x t r a c t e d f r o m g r o w i n g fronds g i v i n g a p p r e c i a b l y l e s s r i g i d g e l s than m a t e r i a l from mature s t i p e s . Knowledge o f t h e n a t u r a l r o l e o f i n d u s t r i a l l y i m p o r t a n t polysaccharides can therefore provide valuable i n s i g h t i n t o t h e i r e f f e c t i v e commercial u t i l i s a t i o n . Thus a n y u n d e r s t a n d i n g we c a n g a i n o f t h e b i o l o g i c a l u t i l i t y o f x a n t h a n s y n e r g i s m may w e l l be of i n t e r e s t from a t e c h n o l o g i c a l a s w e l l a s an academic s t a n d point . Polysaccharide

Gel Structure

An u n d e r s t a n d i n g o f t h e mechanism o f s y n e r g i s t i c g e l a t i o n i s perhaps best approache s i n g l e p o l y s a c c h a r i d e system s t a b l e h a l f - w a y house between t h e s o l i d s t a t e , w i t h m o l e c u l e s i n r e g u l a r ordered conformations packed together w i t h l i t t l e hydrat i o n , and the s o l u t i o n s t a t e , w i t h e x t e n s i v e l y hydrated polymer m o l e c u l e s i n random c o n f o r m a t i o n s . The s t r u c t u r a l i n t e g r i t y o f polysaccharide gels i s maintained by intermolecular a s s o c i a t i o n i n t o l o n g , s t r u c t u r a l l y r e g u l a r j u n c t i o n zones, i n which the m o l e c u l e s a d o p t t h e same o r d e r e d c o n f o r m a t i o n a s i n t h e s o l i d state. These j u n c t i o n z o n e s a r e t h e r e f o r e e s s e n t i a l l y c r y s t a l l i n e , a l t h o u g h t h e y may o n l y i n v o l v e t w o p o l y m e r c h a i n s , a n d a r e terminated t y p i c a l l y by an i n t e r r u p t i o n i n the regular covalent s t r u c t u r e (e.g. i n a l g i n a t e t h e o c c u r r e n c e o f a mannuronate residue would terminate a s s o c i a t i o n o f polyguluronate sequences). Such j u n c t i o n s a r e h e l d t o g e t h e r b y a r e g u l a r a r r a y o f n o n c o v a l e n t i n t e r m o l e c u l a r b o n d s , whose e n e r g y o f f s e t s t h e l o s s o f conformational entropy i n forming such a r i g i d assembly, and whose c o - o p e r a t i v e a c t i o n e l e v a t e s t h e l i f e t i m e o f t h e j u n c t i o n s to a macroscopic timescale. The j u n c t i o n z o n e s a r e t h e n l i n k e d b y r e g i o n s o f t h e m o l e c u l e which are s t r u c t u r a l l y incapable o f forming stable a s s o c i a t i o n s , or a r e prevented from doing so by network c o n s t r a i n t s . These n o n - a s s o c i a t e d r e g i o n s p r e s u m a b l y m a i n t a i n e s s e n t i a l l y t h e same disordered conformation as i n s o l u t i o n , and s o l u b i l i s e the g e l network by extensive h y d r a t i o n . Thus b o t h a s s o c i a t i n g a n d n o n associating molecular regions are e s s e n t i a l f o r g e l a t i o n , too much a s s o c i a t i o n l e a d i n g t o p r e c i p i t a t i o n , a n d t o o l i t t l e preventing formation o f a cohesive network. Aggregation o f R i g i d

Structures

I n g e l s o f c a r r a g e e n a n o r a g a r t h e p r i m a r y mechanism o f i n t e r m o l e c u l a r a s s o c i a t i o n i s b y double h e l i x formation. F u r t h e r development o f t h e g e l network, however, i n v o l v e s a s s o c i a t i o n o f h e l i c e s i n t o l a r g e r a g g r e g a t e s ( 1 4 - 1 6 ) . The extent o f aggregation increases as e l e c t r o s t a t i c r e p u l s i o n b e tween t h e m o l e c u l e s d e c r e a s e s , b e i n g g r e a t e s t f o r t h e n e u t r a l

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

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EXTRACELLULAR MICROBIAL POLYSACCHARIDES

agarose h e l i x . Aggregation of r i g i d r o d - l i k e species i s a h i g h l y favourable process, s i n c e , u n l i k e t h e formation of ordered j u n c t i o n s between f l e x i b l e m o l e c u l e s , l i t t l e l o s s o f c o n f o r m a t i o n a l e n e r g y i s i n v o l v e d . M o r e o v e r , above a c r i t i c a l c o n c e n t r a t i o n , a l i g n m e n t o f e x t e n d e d s t r u c t u r e s becomes a g e o m e t r i c a l n e c c e s s i t y (17) p r o v i d i n g a n a d d i t i o n a l d r i v e t o a g g r e g a t i o n . S i m i l a r considerations apply t o thea s s o c i a t i o n of poly s a c c h a r i d e c h a i n s w h i c h , w h i l e n o t t o t a l l y r i g i d , have s e v e r e l y r e s t r i c t e d m o b i l i t y about t h e g l y c o s i d i c l i n k a g e s between a d j a c e n t r e s i d u e s , a n d t h e r e f o r e t e n d t o f a v o u r e x t e n d e d conford i n a t i o n s c l o s e t o t h a t f o u n d i n t h e s o l i d s t a t e . L o c u s t - b e a n gum f a l l s i n t o t h i s category. Chemically i t i s a galactomannan, w i t h a ρ 1-4 l i n k e d mannan b a c k b o n e a n d l i n k e d galactose subs t i t u e n t s , which occur i Fractions of varying galactos c o m m e r c i a l l o c u s t - b e a n gum s a m p l e s b y u t i l i s i n g t h e g r e a t e r s o l u b i l i t y o f t h e more h i g h l y s u b s t i t u t e d c h a i n s (4)· A s o u t l i n e d i n F i g u r e 2, t h e s o l i d s t a t e c o n f o r m a t i o n o f t h e mannan c h a i n i s an extended, t w o - f o l d , r i b b o n - l i k e s t r u c t u r e , v i r t u a l l y i d e n t i c a l t o t h a t o f c e l l u l o s e , s i n c e b o t h h a v e a 1-4 d i e q u a t o r i a l l y l i n k e d hexopyranose backbone, and d i f f e r o n l y i n t h e o r i e n t a t i o n o f 0(2). Under normal c o n d i t i o n s t h e r e i s no evidence o f a g g r e g a t i o n of galactomannan molecules i n s o l u t i o n . F r e e z i n g and thawing c o n c e n t r a t e d l o c u s t - b e a n gum s o l u t i o n s , h o w e v e r , y i e l d s s t a b l e g e l s whose g e l s t r e n g t h i n c r e a s e s w i t h d e c r e a s i n g g a l a c t o s e content. On f r e e z i n g , t h e f o r m a t i o n o f i c e c r y s t a l s must p r o g r e s s i v e l y r a i s e t h e c o n c e n t r a t i o n o f polymer i n t h e remaining u n f r o z e n s o l u t i o n , t o t h e p o i n t where a l i g n m e n t o f t h e c h a i n s becomes s t e r i c a l l y e s s e n t i a l , u n t i l f i n a l l y t h e c h a i n s p a c k together as i nthe s o l i d state. Once f o r m e d t h e c h a i n - c h a i n c o n t a c t s b e t w e e n t h e S m o o t h u n s u b s t i t u t e d mannan b a c k b o n e r e g i o n s a p p e a r t o be s u f f i c i e n t l y e n e r g e t i c a l l y f a v o u r a b l e t o h o l d t h e m o l e c u l e s t o g e t h e r on thawing. I n t h e r e s u l t a n t g e l network, t h e s u b s t i t u t e d h a i r y regions presumably a c t as t h e s o l u b i l i s i n g i n t e r c o n n e c t i n g regions which prevent p r e c i p i t a t i o n of the associated chains. Xanthan i s n o t alone i n showing synergism w i t h galactoman nan s, b u t s h a r e s t h i s p r o p e r t y w i t h b o t h a g a r a n d c a r r a g e e n a n . F o r b o t h o f t h e s e i t h a s b e e n e s t a b l i s h e d t h a t t h e mechanism o f g e l a t i o n i n v o l v e s a s s o c i a t i o n o f u n s u b s t i t u t e d backbone r e g i o n s of t h e galactomannan, i n an ordered conformation, w i t h t h e r i g i d , o r d e r e d , h e l i c a l s t r u c t u r e o f t h e p o l y s a c c h a r i d e (19). We must t h e r e f o r e c o n s i d e r w h e t h e r a s i m i l a r mechanism o p e r a t e s f o r xanthan - galactomannan i n t e r a c t i o n s . 1

1

Xanthan N a t i v e

1

Conformation

An e s s e n t i a l r e q u i r e m e n t o f t h i s m o d e l i s a r i g i d , r e g u l a r , o r d e r e d s t r u c t u r e w i t h w h i c h t h e mannan c h a i n c a n a l i g n . Until

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

13.

DEA AND MORRIS

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Xanthan

Gels

177

recently no such r i g i d conformation was suspected for xanthan. Spectroscopic and rheological studies now show, however, that the native conformation of the molecule i s a r i g i d rod, which i s only melted out to the expected random c o i l under conditions of very high temperature and low ionic strength (4*6,20,21). The orderdisorder transition i s conveniently monitored as a sharp sigmoidal discontinuity i n a single-wavelength optical rotation (Figure 3). Detail of the ordered structure i s s t i l l the subject of X-ray studies (22), but i t i s known to involve the charged t r i saccharide side-chains aligning with the cellulose backbone of the xanthan molecule, i n a 5-fold h e l i c a l structure. The solution properties of xanthan are entirely consistent with extensive orientation and aggregation of the r i g i d molecular rods (20-21), analogous to the previousl carrageenan double helices Molecular Origin of Xanthan Synergism The strength of interaction between xanthan and galacto— mannans i s closely correlated with the degree of substitution of the mannan chain. Guar gum, i n which the ratio of mannose to galactose i s close to 2:1 does not gel with xanthan i n any concentration, although a slight viscous interaction i s observed ( l ) . Soft, f a i r l y weak gels are obtained with gum tara, where the mannose to galactose ratio i s around 3:1, as against 4 Ï1 i n locust—bean gum, which gives far stronger and more r i g i d gels. Even greater enhancement of gel properties i s found for hot water soluble locust-bean gum fractions i n which the ratio i s 5 î1 or more. Thus, once more, unsubstituted regions of the mannan backbone are implicated i n junction formation. Optical rotation studies of synergistic xanthan gelation provide strong evidence that the native ordered xanthan conformation i s present i n the mixed gel. As shown i n Figure 4y the characteristic sigmoidal curve which accompanies the orderdisorder transition persists i n the presence of galactomannan, and i s essentially complete before the onset of gelation. This interpretation i s confirmed by X-ray studies on oriented films prepared from synergistic xanthan gels (23), which show v i r t u a l l y the same diffraction features as for xanthan alone. We therefore conclude that xanthan — galactomannan gels are crosslinked by cooperative association of unsubstituted mannan regions i n a regular ribbon-like conformation, to the native ordered xanthan structure, as outlined schematically i n Figure 5· Biological U t i l i t y Xanthan i s the extracellular polysaccharide from Xanthomonas campestris which causes blight i n cabbage crops. Other related Xanthomonas species are parasitic upon a wide variety of other plants, and a l l appear to synthesise ordered polysaccharides 5

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

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EXTRACELLULAR MICROBIAL POLYSACCHARIDES

Figure 1. Schematic for a galactomannan such as locust bean gum. (O) 1-^4 linked β-Ό-mannopyranose residues; (Φ) α-Ό-gahctopyranose residues.

Figure 2.

Schematic

05*

-isoh

' /Λ,ϊν^.,.... -2501-

'

80

60 TEMPERATURE

40 «C

^

20 KELTROL 365nm

Figure 3.

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

Synergistic

DEA AND MORRIS

COOL

-ΛΑΑΑΑ

HEAT RANDOM COIL

Xanthan

Gels

GALACTOMANNAN >

XANTHAN NATIVE CONFORMATION

ΛΑΑΑΑ MIXED GEL

Figure 5

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

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180

POLYSACCHARIDES

capable o f s y n e r g i s t i c i n t e r a c t i o n (24). Xanthan i s a complex m o l e c u l e ( 2 5 , 2 6 ) , a n d a l a r g e number o f s t e p s a r e i n v o l v e d i n i t s b i o s y n t h e s i s T27)» I t w o u l d be s u r p r i s i n g (28,29) i f t h e amount o f g e n e t i c i n f o r m a t i o n i n v o l v e d were s t o r e d s i m p l y t o d i s c h a r g e the r e l a t i v e l y t r i v i a l f u n c t i o n s w h i c h have so f a r been proposed for extracellular bacterial polysaccharides. The e x i s t e n c e o f a n ordered n a t i v e xanthan s t r u c t u r e , and i t s a f f i n i t y f o r s p e c i f i c sequences i n p l a n t p o l y s a c c h a r i d e m o l e c u l e s a l s o argue f o r a sophisticated biological function. To e x p l o r e t h i s f u r t h e r we h a v e e x a m i n e d t h e scope a n d s p e c i f i c i t y o f xanthan synergism. Enhancement o f m i c r o c r y s t a l l i n e c e l l u l o s e g e l s (7) shows t h a t x a n t h a n c a n i n t e r a c t w i t h g l u c a n a s w e l l a s w i t h mannan s e q u e n c e s . I n d e e d a v e r y s t r o n g i n t e r a c t i o n i s observe p o l y s a c c h a r i d e from Amorphophallu l i n k e d l i n e a r c o n t a i n s b o t h g l u c o s e a n d mannose r e s i d u e s . This m a t e r i a l shows a b o u t t h e same s t r e n g t h o f i n t e r a c t i o n w i t h t h e a g a r d o u b l e h e l i x a s d o e s l o c u s t — b e a n gum. W i t h x a n t h a n , h o w e v e r , i t s i n t e r a c t i o n i s v e r y much s t r o n g e r , a n d i n d e e d r e c o g n i s a b l e g e l s a r e formed a t t o t a l p o l y s a c c h a r i d e c o n c e n t r a t i o n s as l o w a s

0.05$. There i s e v i d e n c e t h a t a t t h i s c o n c e n t r a t i o n x a n t h a n c a n b i n d d i r e c t l y t o t h e c e l l w a l l s o f l i v i n g p l a n t t i s s u e (21,30331)· I t t h e r e f o r e a p p e a r s t h a t s y n e r g i s m w i t h g a l a c t o m a n n a n s may be a c o - i n c i d e n t a l by-product o f a n a t u r a l r o l e which i n v o l v e s i n t e r a c t i o n w i t h c e l l u l o s i c m a t e r i a l s on t h e c e l l w a l l s u r f a c e s o f t h e h o s t p l a n t . S u c h i n t e r a c t i o n s may p e r h a p s be i n v o l v e d i n r e c o g n i t i o n o f appropriate s i t e s f o r eventual c o l o n i s a t i o n by the b a c t e r i a , o r i n p r e p a r a t i o n o f t h e c e l l surface f o r attachment o f the p a r a s i t e .

Abstract Although neither xanthan nor locust-bean gum will gel alone under normal conditions, mixed gels can be formed at total poly saccharide concentrations well below 1%. Chemically locust-bean gum i s a galactomannan, with a mannose to galactose ratio of around 4:1. The 1,6 linked galactose residues occur i n long blocks, ('hairy regions'), interspersed by unsubstituted B 1,4 mannan backbone. Gelation occurs by co-operative association of these 'smooth regions' with the xanthan molecule i n its ordered conformation, while the 'hairy regions' act as connecting seg ments which solubilise the gel network and prevent precipitation. Xanthan shares this synergistic behaviour with the ordered conformations of carrageenan, furcellaran, and agar, but i s unique in showing a marked preference for interaction with B 1,4 glucose containing polysaccharides (including derivatised cellu lose) rather than mannan. This suggests a possible biological role for the polymer i n substrate recognition by the synthesising bacterium, Xanthomonas campestris.

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

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DEA AND MORRIS

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Xanthan

Gels

181

Literature Cited 1. "Xanthan Gum, a Natural Biopolysaccharide for Scientific Water Control", Kelco Co., San Diego, California, 1972. 2. Rocks, J.K. Food Technol. (1971). 25, 476-483. 3. Federal Register, U.S. Government Printing Office, Washington D.C., Xanthan Gum, Section 121.1224 5376-5377, March 19th. 1969. 4. Dea, I.C.M. & Morrison, A. Advan. Carbohyd. Chem. Biochem. (1975). 31, 241-312. 5. Rees, D.A. Biochem. J. (1972). 126, 257-273. 6. Morris, E.R. i n "Molecular Structure and Function of Food Carbohydrate". (Eds Birch G.G & Green L.F.) 125-130 Applied Science Publisher 7. Rees, D.A. Advan. Carbohyd. Chem. Biochem. (1969). 24, 267-332. 8. Morris, E.R., Rees, D.A. &Thom,D. J. Chem. Soc. Chem. Commun. (1973). p. 245. 9. Grant, G.T., Morris, E.R., Rees, D.A., Smith, P.J.C. & Thom, D. FEBS Lett. (1973). 32, 195-197. 10. Morris, E.R., Rees, D.A., Sanderson, G.R. &Thom,D. J . Chem. Soc. Perkin II. (1975). pps. 1418-1425. 11. Morris, E.R., Rees, D.A. &Thom,D. In preparation. 12. Madgwick, J., Haug, A. & Larson, B. Acta Chem. Scand. (1973). 27, 3592-3594. 13. Rees, D.A. i n "Biochemistry of Carbohydrates". (Ed. Whelan, W.J.) 1-42, Butterworths, London, 1975. 14. Rees, D.A., Steele, I.W. & Williamson, F.B. J . Polymer S c i . (C). (1969). 28, 261-276. 15. McKinnon, Α.Α., Rees, D.A. & Williamson, F . B . , J. Chem. Soc. Chem. Commun. (1969). pps. 701-702. 16. Arnott, S., Fulmer, Α., Scott, W.E., Dea, I.C.M., Moorhouse, R. & Rees, D.A. J. Mol. Biol. (1974). 90, 269-284. 17. Flory, P . J . Proc. Roy. Soc. ser. A. (1956). 234, 50-73. 18. Baker,C.W.& Whistler, R.L. (1975). Carbohyd. Res. 45, 237-243. 19. Dea, I.C.M., McKinnon, Α.Α., & Rees, D.A. (1972). J . Mol. Biol. 68, 153-172. 20. Morris, E.R. This Symposium. 21. Morris, E.R., Rees, D.A., Young, G., Walkinshaw, M. & Darke, A. J. Mol. Biol. Submitted. 22. Moorhouse, R. This Symposium. 23. Moorhouse, R. Personal Communication. 24. Schuppner, H.R. J r . Australian Patent, 401,434. (1966). 25. Jansson, P.E., Kenne, L . & Lindberg, B. Carbohyd. Res. (1975). 45, 275-282. 26. Melton, L.D., Mindt, L., Rees, D.A. & Sanderson, G.R. Carbohyd. Res. (1976). 46, 245-257.

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27. Sutherland, I.W. This Symposium. 28. Sutherland, I.W. Advan. Microbiol. Physiol. (1972). 8, 143-213. 29. Sutherland, I.W. & Norval, M. Biochem. J. (1970). 120, 567-576. 30. Leach, J.G., Lilly, V.G., Wilson, H.A. & Purvis, M.R. J r . Phytopathology. (1975). 47, 113-120. 31. Lesley, S.M. & Hochster, R.M. Canad. J. Physiol. (1959), 37, 513-529.

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

14 X a n t h a n Gum—Acetolysis as a T o o l for the Elucidation of Structure

C. J. LAWSON and K. C. SYMES Tate and Lyle Ltd., Group Research and Development, P.O. Box 68, Reading, U.K.

The development of microbial gums is now moving at an ever increasing pace and it appears likely products will be available upon those found in many plant gums, but also of a novel nature to be exploited in as yet undeveloped applications. The most successful microbial gum to date is undoubtedly xanthan gum produced by Xanthomonas campestris, and this polymer is now commanding a market of several thousand tons per annum. The market position for xanthan gum has been developed through the unique physical properties which it shows, which are exploited for example in oil recovery and food applications. These properties are briefly, high viscosity, extreme pseudoplasticity stability to extremes of pH, salt tolerance and synergistic gelation in the presence of locust bean gum. The above properties are of course dictated by the primary, secondary and tertiary structures of the gum and it is necessary to determine these if any real understanding of the relationship between function and structure is to be obtained. Early reports on the structure of xanthan gum, presented the repeating unit as being made up of glucose, glucuronic acid, mannose and the substituents pyruvate and acetate, in a 14 or 16 residue repeating unit. (1) (2) A repeating unit as large as this is unusual as most microbial gums have tri, tetra or pentasaccharide repeats. Also some of the chemical evidence was somewhat ambiguous, for example the assignment of the pyruvate as being linked to a glucose residue when it could equally have been associated with mannose. More recently two papers have been published, revising the structure and proposing a new pentasaccharide repeating unit containing the same sugar residues as before. We now provide further supporting evidence for the revised structure and suggest an approach to a rapid and convenient qualitative analysis of aspects of covalent structure of this and similar polysaccharides. The interest of Tate and Lyle in microbial gums was originally connected only with microbial alginate, (3) but as a natural consequence of involvement with gums generally, it was decided to examine 183

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EXTRACELLULAR MICROBIAL

POLYSACCHARIDES

the possibilities of d e v e l o p i n g other m i c r o b i a l p o l y s a c c h a r i d e s for i n c o r p o r a t i o n into a possible range of p r o d u c t s .

O n e c a n d i d a t e for e x a m i n a t i o n was

Xanthomonas campestrls as the properties shown by xanthan gum were c o n s i d e r e d to be c o m p l e m e n t a r y to m i c r o b i a l a l g i n a t e . T r a d i t i o n a l l y , three b a s i c lines of a p p r o a c h c a n be a d o p t e d in the e l u c i d a t i o n of p o l y s a c c h a r i d e structure.

These are m e t h y l a t i o n a n a l y s i s ,

p e r i o d a t e o x i d a t i o n a n d i s o l a t i o n of fragments w h i c h c a n be c h a r a c t e r i s e d ; the last a p p r o a c h o n l y b e i n g of use when the p o l y s a c c h a r i d e s have a r e p e a t ing u n i t .

The first two a p p r o a c h e s had b e e n reported in the previous papers

a n d therefore the third was the l o g i c a l c h o i c e .

A c e t o l y s i s was used because

aqueous a c i d hydrolysis often g i v e s a c i d i c oligomers from u r o n i c a c i d c o n t a i n i n g p o l y s a c c h a r i d e s a n d these are more d i f f i c u l t to c h a r a c t e r i s e than neutral fragments.

Also a

g i v e a c o m p l e m e n t a r y resul consideration.

A c e t o l y s i s i s , p r a c t i c a l l y , a r e l a t i v e l y straightforward

process performed a t room temperature in a p p r o x i m a t e l y two days using commonly a v a i l a b l e reagents.

A sample of xanthan fermentation broth

o b t a i n e d in b a t c h c u l t u r e of NRRL B1459 was t a k e n .

P u r i f i e d x a n t h a n gum

was r e c o v e r e d after b a c t e r i a l c e l l s were r e m o v e d using high speed c e n t r i f u g a t i o n a n d trypsin d i g e s t i o n , by a l c o h o l p r e c i p i t a t i o n . The c a r e f u l l y d r i e d gum was shaken w i t h the a c e t o l y s i s mixture o f reagents used b y M o r g a n a n d O ' N e i l l , (4) in studies on desulphated carrageenan.

λ-

The a c e t o l y s a t e was then poured into w a t e r , the a c e t y l a t e d

products e x t r a c t e d into c h l o r o f o r m , a n d d e a c e t y l a t e d using m e t h a n o l i c sodium m e t h o x i d e in the usual w a y .

T h e p a l e y e l l o w syrup o b t a i n e d in

h i g h y i e l d r e v e a l e d , on c h r o m a t o g r a p h i c e x a m i n a t i o n , a number o f spots in a d d i t i o n to the e x p e c t e d m o n o s a c c h a r i d e s .

The p r o d u c t was then

r e s o l v e d into a c i d i c a n d neutral fractions b y separation on ion e x c h a n g e resin in the a c e t a t e f o r m .

(5)

A s h o p e d , the major proportion o f o l i g o -

m e r i c m a t e r i a l was in the neutral f r a c t i o n .

The oligomers Β to Ε were then

o b t a i n e d in a p u r i f i e d state from the neutral f r a c t i o n by a c o m b i n a t i o n of c e l l u l o s e c o l u m n a n d t h i c k paper c h r o m a t o g r a p h y .

(Figure 1) (Figure 2)

A t this p o i n t in the work it was l e a r n e d from Professor Rees o f results o b t a i n e d b y his group (6) a n d o f Professor Lindbergs group (7) proposing the r e v i s e d structure o f xanthan gum w h i c h has b e e n m e n t i o n e d e a r l i e r . The r e v i s e d r e p e a t i n g unit is based upon a c e l l u l o s i c b a c k b o n e w i t h t r i s a c c h a r i d e side c h a i n s o c c u r r i n g on a l t e r n a t e g l u c o s e residues.

Analysis

o f the a c e t o l y s i s oligomers was therefore c o n t i n u e d in order to a s c e r t a i n , whether they were consistent w i t h the a b o v e structure. T h e structure o f o l i g o s a c c h a r i d e C w i l l be used as a n e x a m p l e o f the a p p r o a c h a d o p t e d , a n d the other o l i g o s a c c h a r i d e s w i l l o n l y be m e n t i o n e d for the purpose o f m e n t i o n i n g s p e c i f i c points of d i f f e r e n c e in their a n a l y s i s . This o l i g o s a c c h a r i d e was found to consist of glucose a n d mannose in a 2:1 r a t i o after hydrolysis a n d g l c o f the d e r i v e d a l d i t o l a c e t a t e s .

This was

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

14.

LAWSON A N D SYMES

Structure

Elucidation

of Xanthan

Gum

185

XANTHAN G U M Ac 0/AcOH/H S0 2

2

4

t ACETYLATED

PRODUCTS NoOMe/MeOH

PRODUCTS Acetate resin

NEUTRAL

FRACTION

Cellulose column/PC OLIGOSACCHARIDES B,C,D&E

ACIDIC FRACTION

electrophoresis ALDOBIOURONIC ACID A

Figure

1.

Figure

The

acetolysis of gum

xanthan

2. Xanthan acetolysate neutral oligosaccharides

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

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186

POLYSACCHARIDES

c o n f i r m e d by α c o l o r î m e t r î c assay o f the r a t i o o f carbohydrates to g l u c o s e w h i c h a l s o showed that 5 0 % g l u c o s e was lost on r e d u c t i o n with b o r o h y d r i d e . T h e o l i g o s a c c h a r i d e was therefore shown to be t r i s a c c h a r i d e h a v i n g g l u c o s e as the r e d u c i n g m o i e t y .

T h e mass spectrum of the T . M . S . ether o f r e d u c e d

C h a d ions a t 451 as e x p e c t e d for fission of the substituted terminal hexose a n d 525 from the a l d i t o l m o i e t y , thus p r o v i d i n g e v i d e n c e that the t r i s a c c h a r i d e is not b r a n c h e d .

Further more the series o f fragments o b t a i n e d a t

m / e ratios 1 0 3 , 205 a n d 3 0 7 were those p r e d i c t e d from a 4 - l i n k e d r e d u c e d g l u c o s e residue a n d this was c o n f i r m e d w i t h a deuterium l a b e l l i n g e x p e r i ment.

(Figure 3)

The r e d u c e d o l i g o s a c c h a r i d e was then c o n v e r t e d into

the p a r t i a l l y m e t h y l a t e d a l d i t o l a c e t a t e s o f its c o m p o n e n t sugars w h i c h w e r e a n a l y s e d b y gas c h r o m a t o g r a p h y .

The retention times o f the three

resulting peaks were c o m p a r e substitution pattern of the lished as e i t h e r 2-substituted mannose or 3-substituted g l u c o s e .

(Figure 4)

The s e q u e n c e o f sugar residues in the t r i s a c c h a r i d e a n d their a n o m e r i c c o n f i g u r a t i o n was then c l e a r l y shown by the use o f the e n z y m e sidase.

a-manno-

This e n z y m e c l e a v e d the sugar into mannose a n d e e l I o b î o s e d e m o n -

strating that it is i n d e e d

a-mannosyl eel I o b i ose o f the structure s h o w n .

(Figure 5) U s i n g a s i m i l a r a p p r o a c h the structures o f the other mannose c o n t a i n i n g o l i g o s a c c h a r i d e s were e l u c i d a t e d .

In the case o f the mannose c o n t a i n i n g

d î s a c c h a r î d e (E) the position of the mannosyl substituent was d e t e r m i n e d using the l e a d t e t r a a c e t a t e o x i d a t i o n method d e s c r i b e d b y Perl i n . (8) O n hydrolysis o f the o x i d i s e d d î s a c c h a r î d e , arabinose was d e t e c t e d , s h o w i n g that mannose was l i n k e d to O 3 . (Figure 6) The b r a n c h e d t r i s a c c h a r i d e (B) was u n e x p e c t e d l y resistant to the a c t i o n of both

a-mannosidase

and

β - g l u c o s î d a s e presumably through s t e r i c h i n d e r a n c e o f a d j a c e n t hexoses on O 3 a n d O 4 a n d therefore p a r t i a l a c i d hydrolysis was used for this f a c e t o f the structural i n v e s t i g a t i o n .

The a c i d i c d î s a c c h a r î d e (A) was shown to

c o n t a i n g l u c u r o n i c a c i d a n d mannose in r o u g h l y e q u a l proportions a n d was assumed to be the a l d o b i u r o n i c a c i d p r e v i o u s l y i s o l a t e d from the g u m . O l i g o s a c c h a r i d e D was shown to be c e 11 o b i ose by c o - c h r o m a tography w i t h a n a u t h e n t i c sample on paper a n d gas c h r o m a t o g r a p h y .

(Figure 7)

A l l o f the sugars in the n e w l y proposed r e p e a t i n g unit o f the p o l y s a c c h a r i d e w i t h the s i n g l e e x c e p t i o n o f the terminal mannose residue a r e represented in a t least one o f the o l i g o m e r s , a n d our results are e n t i r e l y consistent w i t h the r e v i s e d s t r u c t u r e .

(Figure 8)

It is possible that the c o v a l e n t structures o f gums p r o d u c e d under d i f f e r e n t c o n d i t i o n s may vary i n some w a y , for e x a m p l e , a f t e r c h e m i c a l treatment.

(10)

T h e r e is e v i d e n c e a l s o that structural v a r i a t i o n may o c c u r

in gums from d i f f e r e n t species o f xanthomonas (9).

V a r i a t i o n in structure

is l i k e l y to be a s s o c i a t e d w i t h v a r i a t i o n in p h y s i c a l properties a n d it is possible that a range o f xanthan gum types c o u l d be d e v e l o p e d to g i v e a

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

14.

Structure

LAWSON AND SYMES

Elucidation

of Xanthan

187

Gum

CH OTMS 2

TMSOHEX .

HEX -

307(308) -OTMS

_205

ITMSO-

103 CH OTMS 2

451 (452)

451

Figure 3. Mass spectroscopy of the perO-trimethyhilyl ether of the derived glycitol from oligosaccharide C (Figures in parentheses are after NaBD reduction) h

[Man]1or >3[Glc]1->

[GlclH CH OAc

ÇH OAc

2

2

-Ο Me

+

AcO-OMe

OR

-OAc CH OMe 2

CH OMe 3

Ο Me MeO-OAc -OMe CH OMe

CH OMe

2

2

•2[Man]1->

•UGlucitol)

Figure 4. Partially methylated alditol acetates possible from gas chromato graphic evidence

oc-Mannosidase Ψ MANNOSE +

CELL0BI0SE

Figure 5. Action of a-mannosidase on oligosaccharide C

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

188

EXTRACELLULAR MICROBIAL POLYSACCHARIDES

-OHpH

OH

|Pb(OAc)

4

O H C ^ Î Î )

OA^CHO

H CT 3

HOH C 2

HO? OH Η Figure 6.

OH

Lead tetraacetate oxidation of disaccharide Ε

Arabinose

A.

p-P-GlcAp-(l->2)-P-Mang

B.

B-D-Glcp-(B4)-D-Glcp

i< - D - M a n p

C.

B - D - G l c p - ( l-*4> - D - G l c p

3

Î l I a. - D - M a n p

D. Figure 7. Oligosaccharides from acetolysis of xanthan gum

Ε.

p-D-Glcp-(l->4)-D-Glcp

* -D-Manp-(l->3) - D - G I cp

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

14.

LAWSON

AND

SYMES

Structure

Elucidation

of Xanthan

Ε

189

Gum

C

- ——ι

• GA«

11

D

1

— M-

»GA-

•Μ ι

M

JL

1

I

!

•M

-,

G-4i

— .

> GA-

A Figure 8.

GA<

-M

Β

Location of acetolysis oligosaccharides in the repeating unit of xanthan gum

Figure 9.

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

190

EXTRACELLULAR MICROBIAL

POLYSACCHARIDES

range o f products analogous to other gums in the plant s e r i e s , such as the alginates and carrageenans. H a v i n g c h a r a c t e r i s e d the f i v e fragments o f xanthan gum as d e s c r i b e d , it was r e a l i s e d that a means o f c o m p a r i n g a t least some facets of the c o v a l e n t structural features o f d i f f e r e n t samples might be o f f e r e d , w h i c h c o u l d be a c c o m p l i s h e d in a r e l a t i v e l y short p e r i o d o f t i m e .

F i v e samples

o f gum were therefore o b t a i n e d n a m e l y (1) X a n t h a n gum p r o d u c e d by Tate a n d L y l e (2) K e l t r o l (3) a sample o f gum c o n t a i n i n g c u l t u r e broth treated w i t h a l k a l i at pH 12.2 at 8 3 ° C as d e s c r i b e d by Patton (4)

Xanthomonas

p h a s e o l i p o l y s a c c h a r i d e o b t a i n e d from D r . P. Sandford N R R L Peoria a n d (5) A gum from Xanthomonas juglandis o b t a i n e d from D r . D . MRE Porton.

El I w o o d o f

Small samples o f the a b o v e gums were treated as a l r e a d y

d e s c r i b e d a n d the a c e t o l y s a t e l a t i o n a n d separation o f neutra s a c c h a r i d e s were then e x a m i n e d on paper c h r o m a t o g r a p h y .

(Figure 9)

A l l of the mannose c o n t a i n i n g o l i g o s a c c h a r i d e s were r e v e a l e d in s i m i l a r proportion in e a c h p o l y s a c c h a r i d e .

C e l l o b i o s e is shown i n c o m p l e t e l y r e -

s o l v e d a n d it appears to v a r y s l i g h t l y in its p r o p o r t i o n .

A slow m o v i n g

sequence of spots c a n be seen w h i c h are p r o b a b l y higher neutral o l i g o s a c c h a r i d e s or possibly c h a r g e d oligomers w h i c h h a v e come through the resin treatment.

The latter a p p e a r in a p p r o x i m a t e l y the same proportion

in e a c h p o l y s a c c h a r i d e .

A faster m o v i n g spot thought to be xylose c a n be

seen in the chromatogram of the Xanthomonas phaseoli p o l y s a c c h a r i d e .

This

latter sugar is possibly a s s o c i a t e d w i t h the x a n t h a n - l i k e p o l y s a c c h a r i d e but there is as y e t n o p o s i t i v e e v i d e n c e for this.

The sample o f gum from the

Xanthomonas juglandis has not o n l y the a d d i t i o n of x y l o s e , but a l s o r h a m nose.

The e v i d e n c e for this is m u c h more c o n c l u s i v e than the e v i d e n c e for

the i d e n t i t y o f x y l o s e in Xanthomonas phaseol » a n d the two sugars c a n be removed by f r a c t i o n a t i o n w i t h c e t a v l o n showing c o n c l u s i v e l y that the x a n t h a n - l i k e p o l y s a c c h a r i d e does not c o n t a i n rhamnose or x y l o s e .

The

sugars d o h o w e v e r a p p e a r to be present as p o l y s a c c h a r i d e s as they are n o n dial ysable. F i n a l l y , a l t h o u g h not s h o w n , the proportions o f the a c i d o l i g o s a c c h a r ides a p p e a r e d s i m i l a r in a l l samples a n d d o m i n a t e d by the a l d o b i u r o n i c a c i d , as shown by paper chromatography o f the unseparated a c e t o l y s a t e s in oligomers l o c a t e d using neutral l e a d a c e t a t e s p r a y . In c o n c l u s i o n , w h i l e a l l findings are n e c e s s a r i l y q u a l i t a t i v e , the a c e t o l y s i s a p p r o a c h d e s c r i b e d , provides a method for c o m p a r i n g samples o f xanthan gum a l t h o u g h no information is a v a i l a b l e from the terminal mannose unit or the pyruvate a n d a c e t a t e substituents.

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

14.

LAWSON

A N D SYMES

Structure

Elucidation

of Xanthan

Gum

191

Literature Cited 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

Sloneker, J.H. & Jeanes, A . Canad J Chem (1962) 40 2066 Siddiqui, I.R. Carbohyd Res (1967) 4 284 Imrie, F . K . E . British Patent 1331771 Morgan, K . & O'Neill, A.N. Canad J Chem (1959) 37 1201 Blake, J . D . & Richards, N.G. Carbohyd Res (1970) 14 375 Melton, L . D . (The Late), Mindt, L., Rees, D . A . & Sanderson, G.R. Carbohyd Res (1976) 46 245 Jansson, P.E., Kenne, L . & Lindberg, B. Carbohyd Res (1975) 45 275 Perlin, A . S . Anal Chem (1965) 27 396 Evans, C . E . Unpublished Results Patton, J . T . (1970) Unite

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

15 Variation

in

Xanthomonas

C h a r a c t e r i z a t i o n of

campestris

Xanthan

NRRL

B-1459:

P r o d u c t s of D i f f e r i n g

Pyruvic Acid Content

P. A. SANDFORD, J. E. PITTSLEY, C. A. KNUTSON, P. R. WATSON, M. C. CADMUS and A. JEANES Northern Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture, Peoria, IL 61604

Xanthan, the exocellula Xanthomonas campestris NRRL B-1459 now produced i n d u s t r i a l l y i n both the United States (1) and Europe (2), has numerous applications i n food and nonfood industries QS, 4). Xanthan i s composed of g-glucose (Glc), Ç-mannose (Man), ancT J}-glucuronic acid (GlcA) i n the r a t i o o f 2Ï2:1 (5-7) and of varying amounts o f pyruvic and acetic acid (4, 5). Early structural analyses (<8, 9) and more recent studies (TO-T3) indicate that xanthan consists o f repeating pentasaccharide units (Figure 1). Upon a c e l l u l o s i c backbone, trisaccharide side chains composed of 3-D-Man(l-*4)-$-DGlcA(l->2)-α-D-Man are g l y c o s i d i c a l l y linked to alternate glucose units at the 3-0-position. Acetic acid i s attached as an ester to the 6-0-position of the internal mannose of the side chain (10) and pyruvic acid i s condensed as a ketal with terminal mannose units (10, 1£, 15). Recently, various substrains have been found (16-18) m c e r t a i n stock cultures of the bacterium Xanthomonas campestris NRRL B-1459 that produce xanthan d i f f e r i n g i n y i e l d , v i s c o s i t y , various solution properties, and i n pyruvic acid and acetyl content. These preliminary studies suggested that differences i n pyruvic acid content were the main cause of these observed variations. Therefore, we re-examined the solution properties of two xanthan samples of d i f f e r i n g pyruvic acid con tent at lower polysaccharide concentration and also examined xanthan samples o f intermediate pyruvic acid content. Most xanthan applications are based on i t s unusual Theological proper t i e s (5_, 19, 20); therefore, the d i f f e r i n g rheological behavior of xanthans ο ι d i f f e r i n g pyruvic acid content has p r a c t i c a l significance.

192

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

15.

SANDFORD ET AL.

Characterization of Xanthan Products

193

Experimental Materials The standard reference samples are those described previously (16) as PS-L and PS-Sm but they now are given the designation HPXan (for high pyruvate xanthan) and LPXan (for low pyruvate xanthan), respectively. Laboratory P u r i f i c a t i o n o f Xanthan The production and recovery of xanthan from broth were as previously reported (16). Xanthan from commercial sources was purified i n a similarTashio The y i e l d of p u r i f i e d xantha sources was 70-85%. Viscosity Measurements Calibration of Viscometers. Standard o i l s o f known v i s c o s i t y were used to calibrate viscometers. Viscosity Measurement at Xanthan Levels Above 0.25%. Viscosity measurements were made with a cone-plate micro viscometer (Wells-Brookfield, Model RVT, 4.7 mm diameter and 1.565° angle cone) at 25° C and 1 rpm unless otherwise indicated. Dispersions f o r viscosity-concentration curves were prepared by volumetric, s e r i a l d i l u t i o n , although the same results were obtained from individually prepared dispersions. Salt effects were observed by incremental addition o f small amounts o f s o l i d s a l t to homogeneous, completely dispersed solutions of the polysaccharide. Readings usually were made after three revolutions, or when the values had become constant. Viscosity Measurement at Xanthan Levels At or Below 0.1%. A Brookfield viscometer (modeTlVT) f i t t e d with an"TJltra-low (UL) adapter (Couette-type stainless-steel c e l l ) was used to measure the v i s c o s i t y o f d i l u t e solutions. Viscosity values (therefore shear rates) at 3.0 rpm with the UL adapter were closest to those obtained with either the cone-plate viscometer at 1 rpm or the LVT spindle (No. 3) at 30 rpm. Viscosity vs Temperature. In the polysaccharide range of 0.25 to 2%, a Brookfield viscometer (model LVT) f i t t e d with a No. 4 spindle was used to measure v i s c o s i t i e s (30 rpm). Dispersions i n an 8-mm (inside diameter) tube, i n which a thermocouple was placed i n the dispersion to measure temperature, were heated i n an o i l bath over the temperature range of 2° to 95° C (above 95° C bubbles appear which lead to e r r a t i c readings). In the polysaccharide range of 0.1% or below, the UL adapter was placed

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

194

EXTRACELLULAR MICROBIAL

POLYSACCHARIDES

i n an aluminum cylinder (1-cm walls) t o distribute heat evenly. Water was placed between UL adapter cup and the aluminum cylinder to assure heat transfer. The aluminum cylinder was heated with e l e c t r i c a l resistance heating tape connected to a Variac. Temp erature was measured with a thermocouple placed i n the aluminum cylinder. Viscosity vs pH. The v i s c o s i t y o f 0.5% dispersions at various pH s was measured with the cone-plate viscometer (1.0 rpm, 25° C) as previously described (16). f

Intrinsic Viscosity Size 75 Cannon semimicr viscometer (Canno Instrument Co.) were used to measure r e l a t i v I n t r i n s i c v i s c o s i t i e s , expressed as d e c i l i t e r s per,gram (dl/g), were determined by extrapolation of plots o f r e l " vs C to zero concentration (C, g/100 ml). C n

A n a l y t i c a l Measurements The method o f Duckworth and Yaphe (21) was used for pyruvate determination. Xanthan (3-5 mg) was hydrolyzed 3 hr at 100° i n 2 ml 1 Ν HC1, neutralized with 2 ml 2 Ν Na C0 , and diluted to 10 mT with water. A 2-ml aliquot was pipetted into a quartz cuvette with a 1-cm l i g h t path, and 1 ml o f 1 Ν aqueous triethanolamine buffer and 50-yl NADH solution (10 mg per ml o f 1% NaHCO-τ) were added. Absorbance (A) was measured at 340 nm and 4 μΐ lactate dehydrogenase (4,000 units per ml) were added. Absorbance was measured again after 5 min, and at 5-min intervals u n t i l stable. Percent pyruvate was calculated by the equation: 2

3

ο, ιχ^„„* - 5 X 88 X 100 (A i n i t i a l - A f i n a l ) X 3.05 ο pyruvate Sample wt X 6.22 X 1,000 Λ

where 88 i s the molecular weight of pyruvic acid, 3.05 i s solution volume, 6.22 i s the extinction coefficient of NADH, and 5 i s a d i l u t i o n factor. Q-Acetyl was determined by the hydroxamic acid method (22). Component sugars i n xanthan were determined by radiochromatographic analysis o f an acid hydrolysate after reduction with %-sodium borohydride (23). g-Mannose and J}- glucose content of xanthan was independently checked by gas chromatography of t h e i r a l d i t o l acetates (24). D-Glucuronic acid was also assayed by the carbazole method (2ΊΓ). Neutral equivalent weights were determined by t i t r a t i n g [with standardized KOH (0.1 M)] decationized solutions (0.01 to 0.1%) (5).

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

15.

SANDFORD

E T AL.

Characterization

of Xanthan

Products

195

Results General Properties The p r e c i p i t a t i o n and rehydration behavior of xanthan products d i f f e r c h a r a c t e r i s t i c a l l y with pyruvic acid content of the product. During p r e c i p i t a t i o n with ethanol (2 volumes, also KC1, 1%), xanthan high i n pyruvate (>4%) comes out of solution as a cohesive stringy precipitate that tends to wind around the s t i r r e r . Under i d e n t i c a l conditions, xanthan low i n pyruvate (2.5 to 3.5%) usually precipitates as a less cohesive particulate material that does not wind on the s t i r r e r . B r i e f heating of dispersions o f xanthan low i n pyruvate (e.g., 0.1 to 1%, 3 min at 95° C) causes p r e c i p i t a t i o n behavior with alcohol d KC1 t chang t that l i k xanthan high i n pyruvat form, both pyruvate types when freeze dried are white fibrous products. Freeze-dried (K-salt form) HPXan products character-i s t i c a l l y take longer to réhydrate than low-pyruvate samples. Apparently i t i s more d i f f i c u l t for water to completely penetrate into HPXan. Dispersions of LPXan are generally clearer than HPXan, which tend to have some opalescence, but this difference may relate to the removal of c e l l s and debris i n our i s o l a t i o n procedure. A n a l y t i c a l Measurements In Table I , the a n a l y t i c a l results of HPXan and LPXan are l i s t e d and compared to that expected f o r various theoretical xanthan structures o f d i f f e r i n g pyruvate content. When HPXan i s compared to LPXan only the amount of pyruvic and Q-acetyl appear to d i f f e r s i g n i f i c a n t l y , and perhaps the difference i n Q-acetyl i s not s i g n i f i c a n t . The amount of g-glucose, D-mannose, and g-glucuronic acid i n both HPXan and LPXan are nearly i d e n t i c a l . The major compositional difference between these two types of xanthan i s i n pyruvate content. The neutral equivalent weights of HPXan and LPXan are consistent with t h e i r g-glucuronic and pyruvic acid content. HPXan compares favorably to the theoretical repeat unit depicted i n Figure 1, i n which there i s an average of one pyruvic acid ketal on every other terminal g-mannose unit; LPXan i s more l i k e the theoretical repeat unit that has one pyruvate on every fourth terminal g-mannose i n the side chain. V i s c o s i t y Measurements Viscosity vs Polysaccharide Concentration. When compared at polysaccharide concentrations o f 1% or above, the v i s c o s i t y o f LPXan i s equal to or s l i g h t l y higher than HPXan (see Figure 2). At polysaccharide levels at and below 0.5%, LPXan i s generally less viscous than HPXan (Figure 2). Thus, the v i s c o s i t y / concentration curves of HPXan and LPXan cross near the 0.5%

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

Type

Low

LPXan

High^ Lowl/

Theory-2

0

2.4

4.6

8.7

2.5

4.4

g/100 g

.

4.8

4.6

4.5

4.3

3.7

4.5

g/100 g

g-Acetyl

39.8

37.6 38.7

35.6

37.7

37.0

g/100 g

g-Glucose

39.8

37.6 38.7

35.6

42.9

43.4

g/100 g

g-Mannose

Pyruvic acid k e t a l on every other terminal D-mannose i n side chain. 4/ —' Pyruvic acid k e t a l on every fourth terminal g-mannose i n side chain. — No pyruvic acid k e t a l on terminal g-mannose i n side chain.

— Pyruvic acid k e t a l on every terminal g-mannose i n side chain.

21

— Assume monosaccharide repeat u n i t as i n Figure 1.

Theory-4

Theory-3

Max-/

Theory-1

B. Theoretical^/

High

HPXan

Experimental

Sample

21.5

20.8

20.3

19.2

19.3

19.5

g/100 g

Acid

g-Glucuronic

theoretical repeat units of d i f f e r i n g pyruvic acid content.

904

745

639

506

790

633

Weight

Equivalent

Neutral

neutral equivalent weight values of HPXan and LPXan to that of

Comparison of pyruvic acid, a c e t y l , monosaccharide content, and

Pyruvic Acid

TABLE I

15.

SANDFORD

E TA L .

Characterization

of Xanthan

197

Products

Figure 1. Structure of extracellular polysaccha ride of Xanthomonas campestris according to Jansson et al (10). Linkages denoted by indicates pyruvic acid is not linked to every terminal Ώ-mannose.

Pyruvic

101 0.01

1 I I I I 0.05 0.1 0.5 1.0 5.0 Polysaccharide concentration, % (w/w)

I

Figure 2. Viscosity vs. polysaccharide concentra tion. Viscosity of aqueous dispersions of the potas sium salt-form of high-pyruvate xanthan (HPXan) (4.4% pyruvate) andlow-pyruvate xanthan (LPXan) (2.5% pyruvate) were measured at 1 rpm (3.84 sec' ) and25°C. 1

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

198

EXTRACELLULAR MICROBIAL POLYSACCHARIDES

polysaccharide l e v e l . At polysaccharide concentrations above 0.251, both pyruvate types can be p a r t i a l l y removed from solution as a g e l by centrifugation (100,000 X g, 60 min). Under similar conditions but at lower concentrations (0.1%) polysaccharide o f either pyruvate type i s not removed by centrifugation. Viscosity vs Shear Rate. Both HPXan and LPXan display recoverable snear-rate thinning at the polysaccharide concentrations tested. As shown i n Figure 3, the v i s c o s i t y decreases with increasing shear rate. At the 1% l e v e l , HPXan shows s l i g h t thixotropic behavior; i . e . , previously sheared xanthan gives lower v i s c o s i t y values which recover with time. LPXan consistently shows s l i g h t antithixotropic behavior at the 1% l e v e l . At lower concentrations of both types thixotrop antithixotrop i observed. V i s c o s i t y vs Temperature. Figure 4 i l l u s t r a t e s the t y p i c a l v i s c o s i t y behavior of HPXan and LPXan dispersions (1%) when measured at various temperatures. Vastly different results are found with the presence of KC1. When no added s a l t i s present, the v i s c o s i t y of both pyruvate types starts to drop with increasing temperature. When the temperatures o f the dispersions reach about 50° C both types display v i s c o s i t y changes i n the opposite direction (see curves C and D i n Figure 4). With HPXan, a dramatic r i s e i n v i s c o s i t y i s seen, while with LPXan a s l i g h t decrease i s seen. With continued heating of the samples, HPXan s v i s c o s i t y reaches a maximum around 70° after which the v i s c o s i t y decreases rapidly. I f the v i s c o s i t y of the heated samples i s rechecked on cooling, the same v i s c o s i t y peak i s observed at the temperatures 50-70°; i n f a c t , this effect can be repeated over and over with alternating heating and cooling. In rechecking the v i s c o s i t y of LPXan while i t i s cooling (see curve D, Figure 4), i t too now displays a sharp v i s c o s i t y peak i n the 50-80° temperature range that was not seen i n the i n i t i a l heating curve. On reheating, LPXan displays the v i s c o s i t y peak i n the 50-80° temperature range, as i s observed with HPXan. The LPXan must be heated above ^60° before i t displays the v i s c o s i t y peak seen at temperatures between 70-80° C. However, extended heating at 60° for 8 hr d i d not produce the v i s c o s i t y peak. Heating LPXan dispersions at 95° f o r 3 min does produce the v i s c o s i t y peak at temperatures o f 70-80°. 1

Heating LPXan alters i t s r e a c t i v i t y with s a l t . Figure 4 shows the effect of added s a l t on the v i s c o s i t y at various temperatures. When 1% KC1 i s present, the v i s c o s i t y of HPXan (curve A, Figure 4) i s nearly constant over the entire temperature range between 10-90° C. The v i s c o s i t y of 1% LPXan i n the presence of 1% KC1 decreases steadily with increasing temperature (curve B, Figure 4). Only at 20-25° C are the v i s c o s i t i e s of HPXan and LPXan with 1% KC1 similar. For high temperatures, e.g., 90° C,

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

15.

SAND FORD

Characterization

E T A L .

of Xanthan

Products

199

Shear rate, sec

Figure 3. Viscosity (25°C) of xanthan dispersions vs. shear rate in sec' at various polysaccharide concentrations (w/v). (A) HPXan (4.4% pyruvate); (B) LPXan (2.5% pyruvate). 1

Curve Sample

20

40

60

80 100 80 Temperature, °C

60

40

20

Pyruvate KCI % %

0

Figure 4. Viscosity of 1% dispersions of HPXan (4.4% pyruvate) and LPXan (2.5% pyruvate) at various temperatures with and without added KCI present. On left side of figure, the samples were gradually heated until they reached boiling. Then samples were allowed to cool and viscosity at temperature were remeasured (see right-hand side of figure). Spindle viscometer used.

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

200

EXTRACELLULAR MICROBIAL POLYSACCHARIDES

the v i s c o s i t y of LPXan i s about 1/2 that of HPXan. I f an LPXan dispersion i s f i r s t heated, such as with curve D, and then KCI (II) i s added, the viscosity/temperature curve (E) that results i s very much l i k e that for HPXan (curve A). However, i f KCI (1%) i s present during the heating, no change to HPXan behavior i s seen (curve B). At the 0.5% polysaccharide l e v e l (see Figure 5), the viscosity-at-temperature behavior of both types i s similar to that obtained at the I I l e v e l (Figure 4), but the v i s c o s i t y peaks appear at a lower temperature range (50-60°) and are much smaller. As at the I I polysaccharide l e v e l , heating salt-free dispersions at the 0.51 l e v e l causes LPXan s behavior to become more l i k e HPXan; i . e . , a v i s c o s i t ° 60° and i t s v i s c o s i t y i (II) i s added. 1

At the 0.11 polysaccharide l e v e l (Figure 6), the v i s c o s i t y o f both pyruvate types decreases steadily with increasing temperature. Heating o f the LPXan solutions (no added KCI present) causes subsequent cooling and reheating curves to be higher than i n i t i a l viscosity/temperature curve. Hence, heating changes LPXan to resemble HPXan i n behavior. Effect of Salt on Viscosity. The v i s c o s i t y of HPXan and LPXan dispersions dirTers greatly i n the presence o f s a l t ; however, t h i s difference i s greatly diminished when salt-free LPXan dispersions are heated. In Figure 7, the effect of added KCI on the v i s c o s i t y of I I and 0.51 dispersions of both pyruvate types are compared with and without heating (95°, 3 min). When unheated dispersions are compared, LPXan i s less viscous, p a r t i c u l a r l y at high (1-31) KCI concentrations. At the 0.51 polysaccharide l e v e l , the v i s c o s i t y of unheated LPXan i s nearly unaffected by the addition o f KCI, whereas the v i s c o s i t y of HPXan i s nearly doubled by the addition of as l i t t l e as 0.41 KCI. Heating (95°, 3 min) of a LPXan dispersion causes i t s behavior towards KCI to change to that almost i d e n t i c a l to HPXan provided KCI i s not present during heating but i s present during the v i s c o s i t y measurement. Heating of HPXan under i d e n t i c a l conditions has l i t t l e effect on i t s v i s c o s i t y behavior towards added s a l t . At the 0.1% polysaccharide l e v e l , the effect of heat and s a l t on the v i s c o s i t y of HPXan, LPXan, and a mixture of both pyruvate types i s shown i n Figure 8. Unheated LPXan has s i g n i f i c a n t l y lower v i s c o s i t y than HPXAN (see Figure 8), sometimes 1/3 as much. When heated (95°, 3 min), only LPXan*s v i s c o s i t y i s affected. Heating LPXan causes i t s v i s c o s i t y to increase to or above that o f a HPXan whose v i s c o s i t y i s not affected appreciably (see Figure 8). Also i n Figure 8, the KCl/viscosity curves of a 1:1 mixture o f the HPXan and LPXan product are shown. The v i s c o s i t y at I I KCI o f the

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

Characterization

SAND FORD ET AL.

of Xanthan

Products

Curve Sample

s 15

I·»

D

Pyruvate KCI % _%

^a

A

HPXau

4.4

1

^ Ι ^ Θ ^ - χ - *

C

HPXan

4.4

0

\

a

ο

1.01

Χ χ

·χ

Ε

^0.5 0 § 0 »

20

» 160

1 ε

60

8 Temperature

Figure 5.

I 200

40

Viscosity of 0.5% dispersions of xanthan at various tempera tures. See Figure 4 for experimental details.

Curve Sample ^

J X

* -x

χ

xx

χ

120!

χ *

χ

^"v*

>"* χ'

χ

y *

Pyruvate KCI % %

C

HPXan

4.4

0

0

LPXan

2.5

0

A

HPXan

4.4

1

y n°

y°

* 80

•;-Q-Q-0..0.,

0

20

40

60

80

,..-0-.φ-0-?·'· , 100

0 0

80

60

40 20

Temperature, °C

Figure 6. Viscosity of 0.1% solutions of HPXan (4.4%) pyruvate) and LPXan (2.5% pyruvate) at various temperatures with and with out added KCI present. Ultra-low-adapter of Brookfield LVT vis cometer was heated in an aluminum cylinder; shear rate, 3.0 rpm.

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

EXTRACELLULAR MICROBIAL POLYSACCHARIDES

Curve Sample A HPXan Β HPXan -®

-Δ

-0

C

•

2.0 4,

0

-I

A

44

1.0

Yes

2.5

1.0

Yes

LPXan

2.5

1.0

HPXan HPXan

4.4 4.4

0.5 0.5

LPXan

2.5

0.5

LPXan

D

à

Xan. Pyruvate Cone. % % Heated 10 No 44

Yes No

1.0 2.0 3.0 KCI concentration Lt/w solution), %

Figure 7. Effect of added salt (KCI) on viscosity (25°C, 3.84 sec' ) of heated (95°C, 3 min) and unheated dispersions (1 and 0.5%) of HPXan (4.4% pyruvate) and LPXan (2.5% pyruvate) 1

Sample

3.3 HPXanHPXan (1:1 mixture) lave.) LPXan 2.5

Yes

4.4 HPXan 4.4 HPXan HPXanHPXan 3.3 (1:1 mixture) lave.)

Yes No No

LPXan

0.02 0.05 0 1 0.15 0.2 0.25 0.31.0 KCI concentration (w/w solution), %

Pyruvate Heated %

Yes

2.5

3.0

Figure 8. Effect of added salt on viscosity of heated (95°C, 3 min) and unheated solutions (0.1%) of HPXan (4.4% pyruvate) and LPXan (2.5% pyruvate). A 1:1 solution mixture of both pyruvate types were tested also.

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

15.

SAND FORD ET AL.

Characterization

of Xanthan

Products

203

unheated mixture i s between that of the unheated LPXan and HPXan of the mixture. Heating (95°, 3 min) causes the v i s c o s i t y of the mixture to be equivalent or s l i g h t l y higher than HPXan (heated or not heated) and heated LPXan. In Figure 9, the KCl/viscosity curves before and after heating dispersions of xanthans with intermediate pyruvate values are shown. The product with the highest pyruvate content (3.171) i n t h i s series has the highest v i s c o s i t y and products with lower pyruvate levels have correspondingly lower v i s c o s i t i e s . Heating (95°, 3 min) causes the v i s c o s i t y of a l l three of these intermediate pyruvate xanthans to increase. Effect of gH on Viscosity. In Figure 10, the v i s c o s i t i e s o f 0.5% dispersions oFHPXa d d functio f pH. The v i s c o s i t y obtaine presence of additional s a l t , and f o r the LPXan whether or not the dispersion was heated. Typical U-shaped viscosity/pH curves obtained for HPXan (heated or not) and LPXan (heated) are f l a t tened out by the addition o f 1% KC1. unheated LPXan dispersions gave inverted U-shaped v i s c o s i t y pH curves that were not affected by the addition of KC1. Heating (95° C, 3 min) LPXan dispersions caused t h e i r viscosity/pH behavior to become more l i k e HPXan. I n t r i n s i c Viscosity. Mien measured i n water, the i n t r i n s i c v i s c o s i t y was 102 dl/g f o r HPXan and 70 dl/g f o r LPXan (see Table I I ) . When measured i n ammonium acetate (0.01 M), a solvent previously found suitable f o r molecular weight studies (26), the i n t r i n s i c v i s c o s i t y of HPXan was 43 dl/g while LPXan was"T9 dl/g (see Table I I ) . After heating dispersions of both pyruvate types separately (1%, 95°, 3 min) and d i l u t i n g to proper concentration, the i n t r i n s i c v i s c o s i t y value of HPXan was nearly as before heating, 42 dcl/g, but that f o r the LPXan increased to 39 dcl/g. TABLE I I

No.

I n t r i n s i c Viscosity of HPXan and LPXan. iï^rïïï^ Viscosity!/

Pyruvic Acid Type g/100

Not Heated Solvent 0.01 M Water NH^Ac—^"

Heated^ 0.01 M N^Ac-/

1.

HPXan

4.4

102

43

42

2.

LPXan

2.5

70

29

39

i y dl/g = d e c i l i t e r per gram. •JI Heated = 95° C, 3 min, \% solution i n water. -J, Grams released by hydrolysis per 100 grams xanthan. — NH^Ac = ammonium acetate.

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

EXTRACELLULAR MICROBIAL POLYSACCHARIDES

Pyruvic Curve acid, % Heated ·-· A 3.58 Yes

^

0

3.13

Yes

3.58

No

-A|}A-A-A D

3.17

Yes

+-ο||ο-α-ο I

3.13

No

•Δ|μ-Δ-Δ F 3.17

No

ο...

•-®||o-e-0 c

0. KCI cencentration (w/w solution), %

Figure 9. Xanthan products with intermediate (3.13% to 3.58%>) levels of pyruvate. Viscosity vs. amount of added KCI of heated (95°C, 3 min) and unheated solutions.

Curve \

AA

\i'

\

Pyruvic Sample acid, %

Heated

KCI %

LPXan

2.5

Yes

0

HPXan

4.4

Yes

0·

LPXan

2.5

Yes

1 1 1

l «

HPXan

4.4 Yes or No 0

FΘ 6A

LPXan LPXan

25 2.5

ΘΘ/-Θ-Θ-Θ··Θ-Θ\©

2

4

6

8 PH

J 10

No No

1 0

I L 12 14

Figure 10. Viscosity (3.84 sec ,25°C) vs. pH of dispersions (0.5%) of HPXan (4.4% pyruvate) and LPXan (2.5% pyru vate) 1

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

15.

SAND FORD ET AL.

Characterization

of Xanthan

Products

205

Birefringence. When viewed between crossed p o l a r i z e r s , dispersions or xanthan display birefringence, i . e . , double refraction of l i g h t , even when an external orienting force i s absent or very weak. Several factors such as rate of shear, con centration of polysaccharide, presence of extraneous s a l t , heat, and pH have previously been shown to affect t h i s birefringence (27). When HPXan and LPXan pyruvate xanthans are compared (see Figures 11 and 12), the birefringence [retardation, (Δ) i n nm] o f unheated LPXan i s much lower than that f o r HPXan, p a r t i c u l a r l y at the low rpm's (see Figure 11). After heating (95°, 3 min) and cooling to 25° C, LPXan birefringence i s increased and i t s biréfringent behavior becomes much l i k e that f o r HPXan. LPXan (1%) was heated to various temperatures, cooled to 25° C, and i t s retardation at zero rpm shows that temperatures the LPXan dispersion to display temperature-increased birefringence. Discussion The pyruvic acid content of xanthan i s an indicator of i t s solution properties. A l l xanthans high i n pyruvate (>4.0%) show similar solution properties which are s i g n i f i c a n t l y different from those of xanthans low i n pyruvate (2.5 to 3.0). Plots (see Figures 13 and 14) of v i s c o s i t y vs pyruvic acid content o f various xanthan products indicate that v i s c o s i t y increases cons i s t e n t l y with corresponding increases i n pyruvate content. These data indicate that samples of xanthan with a pyruvate content higher than now normally found might be expected to have higher solution v i s c o s i t i e s . I f every terminal mannose carried a pyruvic acid k e t a l , the pyruvate content would be 8.691 (see Table 1) which i s nearly double that now c a l l e d a "high pyruvate" sample. Likewise, the data i n Figures 13 and 14 show that xanthans with low-pyruvate content would be s i g n i f i c a n t l y less viscous than samples with higher pyruvate content. The temperature and s a l t dependence of v i s c o s i t y i s concent r a t i o n dependent. At high polysaccharide concentrations the rheology of HPXan and LPXan i s s i m i l a r , while at low concentrations they d i f f e r . The molecules of these two pyruvate types evidently interact d i f f e r e n t l y . The anionic carboxyl of the pyruvate, l i k e that of the uronate, influences charge d i s t r i b u t i o n throughout the macromolecule. However, the d i s t r i b u t i o n of pyruvate i n the side chains i s not known. Although a regular d i s t r i b u t i o n i s generally assumed, there i s no evidence to confirm t h i s notion. A l l the pyruvate could be clustered regionally i n each molecule.

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

206

EXTRACELLULAR MICROBIAL POLYSACCHARIDES

Pyruvic Sample acid, % Heated

ΙΟ

HPXan

Ι i

^ΠΓΛΛΙΙ

8

ι

6

g

a-gfl-HPXaii g ^ g ^ e g ; : . ' LLPXan PXan

4.4 4.4 2.5 2.5

No Yes Yes No

ο

I4 m κ 2 0

Figure 11. Retardation (A in nm) vs. rate of shear (rpm) of salt-free aqueous dispersions (1%) of HPXan (4.4% pyruvate) and LPXan (2.5% pyruvate)

10

20

30 40 50 60 70 Temperature, °C

80 90

Figure 12. Retardation (Ain nm) vs. temperature to which LPXan (2.51 % pyruvate) dispersion (1%) was heated to before cooling to 25°C and meas urement of Δ

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

Characterization of Xanthan Products

SANDFORD ET AL.

• S

207

•

2 4

•

X

·

S 20 .2-

I

161

F 12 ΙΟ CM

2Γ

8

©Net heated

Γ

y '

i 4 0

I

1

3.0 4.0 Pyruvic acid, %

2.0

5.0

Figure 13. Viscosity (25°C, 3.84 sec- ) vs. pyruvic acid content of xanthan. Disper sions, 0.5%,1%KCI. 1

Θ Not heated • Heated Θ

2.0

3.0 4.0 Pyruvic acid, %

5.0

Figure 14. Viscosity (25°C, UL adap ter, 3.0 rpm) vs. pyruvic acid content of xanthan. 0.1% solution, 1% KCl.

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

208

EXTRACELLULAR MICROBIAL POLYSACCHARIDES

I t should be noted that the pyruvate content of xanthans i s segregated into two main groups, one at 2.5-3.51 pyruvate and another around 4.61 pyruvate. This grouping i s perhaps s i g n i f i cant i n understanding the biosynthesis and source of pyruvate v a r i a b i l i t y i n xanthans produced by various substrains of Xanthomonas campestris B-1459. Cause of the change i n v i s c o s i t y behavior upon heating aqueous dispersions of low pyruvate xanthan i s not clear. The results could be interpreted as a new physical conformation being formed by the heating process. Alternatively, chemical changes could occur during the heating; e.g., introduction of cross linkages through ketal rearrangement, migration of acetic acid, or freeing of an e s t e r i f i e d carboxyl th spectra of LPXan (and HPXan i d e n t i c a l to spectra taken before heating. These studies also indicate that heating does not remove Q-acetyl groups but they could migrate to other positions i n the molecule. Heating LPXan d i d cause i t s i n t r i n s i c v i s c o s i t y value to increase to nearly that found f o r HPXan, whose value was not affected by heating. These data suggest that heating causes the molecular s i z e , shape, or water-binding capacity of low pyruvate xanthan to become more l i k e that found f o r HPXan. Acknowledgment We thank A. C. Eldridge f o r confirming by gas chromatography the neutral hexose content of several xanthan samples.

Abstract Normal xanthan-producing strains of the bacterium Xanthomonas campestris NRRL B-1459 are characterized by their efficient conversion (~60%) of substrates such as D-glucose into extracellular polysaccharide that gives culture fluids of high viscosity (6,000 to 8,000 cpoise) and pyruvic acid contents of about 4.5%. Various substrains have been found in certain stock cultures that produce xanthan differing i n yield, viscosity and other solution properties, and i n pyruvic acid content. Analysis of xanthan products from these substrains and from commercial sources shows that the pyruvate content can vary at least from 2.5% to 4.8%, while the sugar composition (D-glucose, D-mannose, and D-glucuronic acid) remains constant. TRe precipitation, rehydration, and rheological behavior of all xanthan samples having high (4.0% to 4.8%) pyruvate were similar but significantly different from those samples having low (2.5% to 3.0%) pyruvate which display different properties. At xanthan concentrations of 0.1% to 0.5%, high pyruvate samples are more viscous (sometimes 2 to 3X more), particularly i n the presence of salt, than low

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

15. SANDFORD ET AL.

Characterization of Xanthan Products

209

pyruvate samples. B r i e f heating (95° C, 3 min) of low-pyruvate solutions caused t h e i r solution properties to become more l i k e high-pyruvate when observed i n the presence of s a l t . Other rheological properties o f both pyruvate types are examined. Literature Cited 1. "Xanthan Gum/Keltrol/Kelzan/A Natural Biopolysaccharide for S c i e n t i f i c Water Control," Second E d i t i o n , pp. 1-36, Kelco Company, San Diego, C a l i f o r n i a (1975). 2. Godet, P., Process Biochem., (1973) 8, 33. 3. Jeanes, Α . , J. Polym. Sci., Polym Symp., (1975), 45, 209. 4. Jeanes, Α . , Food Technol., (1974), 28, 34. 5. Jeanes, Α . , P i t t s l e y Polym. Sci., (1961) 6. Sloneker, J. H., and Orentas, D. G . , Nature, (1962), 194, 478. 7. Sloneker, J. H., and Jeanes, Α . , Can. J. Chem., (1962), 40, 2066. 8. Sloneker, J. H., Orentas, D. G . , and Jeanes, Α . , Can. J. Chem., (1964), 42, 1261. 9. Siddiqui, I . R . , Carbohydr. Res., (1967), 4, 284. 10. Jansson, P. -E., Kenne, L., and Lindberg, B., Carbohydr. Res., (1975), 45, 275. 11. Melton, L. D., Mindt, L., Rees, D. Α . , and Sanderson, G. R . , Carbohydr. Res., (1976), 46, 245. 12. Lawson, C. J., and Symes, K. C., t h i s symposium. 13 Moorhouse, R . , Walkinshaw, M. D . , Winter, W. T . , and Arnott, S . , Abstr. Papers Am. Chem. Soc. Meeting, (1976), 171, CELL 97. 14. Sloneker, J. H., and Orentas, D. G . , Can. J. Chem., (1962), 40, 2188. 15. Gorin, P. A . J., Ishikawa, T . , Spencer, J. F. T . , and Sloneker, J. Η . , Can. J. Chem., (1967), 45, 2005. 16. Cadmus, M. C., Rogovin, S. P., Burton, K. A., P i t t s l e y , J. E., Knutson, C. Α . , and Jeanes, Α . , Can. J. M i c r o b i o l . , (1976), 22, 942. 17. Cadmus, M. C., Burton, Κ. Α . , Herman, Α. I., and Rogovin, S. Abstr. Paper Am. Soc. Microbiol. Meeting (1971), 71, A47. 18. Kidby, D . , Sandford, P., and Herman, Α., Appl. Environ. M i c r o b i o l . , in press. 19. Holzworth, G . , Biochemistry, in press. 20. Jeanes, Α . , and P i t t s l e y , J. E., J. Appl. Polym. Sci., (1973), 17, 1621. 21. Duckworth, Μ., and Yaphe, W., Chem. Ind., (1970), 747. 22. McComb, Ε. Α . , and McCready, R. Μ., Anal. Chem., (1957), 29, 819. 23. Knutson, C. Α . , Carbohydr. Res., (1975), 43, 225. 24. Sawardeker, J. S . , and Sloneker, J. H., Anal. Chem., (1965), 37, 945.

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

210

25. 26. 27.

EXTRACELLULAR MICROBIAL

POLYSACCHARIDES

Knutson, C. A., and Jeanes, Α . , Anal. Biochem., (1968), 24, 482. D i n t z i s , F. R . , Babcock, G. Ε., and Tobin, R . , Carbohydr. Res., (1970), 13, 257. P i t t s l e y , J . E.., Sloneker, J. H., and Jeanes, Α., Abstr. Papers Am. Chem. Soc. Meeting (1970), 160, CARB 21.

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

16 Zanflo—A Novel Bacterial Heteropolysaccharide K. S. KANG, G. T. VEEDER, and D. D. RICHEY Kelco, Division of Merck & Co., Inc., 8225 Aero Drive, San Diego, CA 92123

An exocellular bacterial polysaccharide called "xanthan gum" that was originall USDA nearly two decade heteropolysaccharide that is being produced on a large commercial scale. The commercial success of xanthan gum is attributed to its many valuable and often unique properties for industrial applications and to an economic manufacturing process. Among our novel polysaccharides that are produced by many bacterial species that we have isolated in our screening program, a product which is now trade named ZANFLO is especially outstand ing in its fermentation efficiency and product properties. ZANFLO is produced by a bacterium that was isolated from a soil sample taken at Tahiti. The organism is a gram-negative, non -sporeforming rod with a size range of 0.75-1.0 by 1-2μ. The dimensions change during the fermentation. At the beginning of the fermentation they are large rods which quickly change to a coccobacillus 0.75-1.0μ in diameter. It is heavily encapsulated. Some of its biochemical characteristics are shown in Table I. This organism produces a positive lactose reaction within 24 hours. It possesses nitrate reductase, CMCase, urease and lysine decarboxylase. It can utilize citrate as a sole carbon source and will grow in the presence of 8% NaCl. Its optimum growth temperature is 30-33°C. and growth will occur at 45° C. In litmus milk this organism produces an acid curd with peptoniza tion and reduction of the litmus. At the present time we are evaluating these and other significant taxonomic tests to ascer tain the identity of this microorganism. This organism is quite specific about what carbon source it will use for optimum polysaccharide production. It produces an excessive amount of acid with glucose as a carbon source with only minimum polysaccharide synthesis. Even with pH control the conversion efficiency with glucose is still very low. Poly saccharide synthesis is better with sucrose or maltose or mixtures of these than with glucose, but the conversion efficiency is still poor. Improved polysaccharide synthesis is found with 211

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l a c t o s e and hydrolyzed s t a r c h . We t y p i c a l l y hydrolyze our s t a r c h s l u r r i e s with commercially a v a i l a b l e α-amylase p r e p a r a t i o n s . The p r e f e r r e d medium contains phosphate as a b u f f e r i n g agent, ammonium n i t r a t e and a soy p r o t e i n product as n i t r o g e n sources, and magnesium s u l f a t e i n a d d i t i o n t o the carbon source. The data i n F i g u r e 1 shows the r e s u l t s o f a t y p i c a l batch-type fermentation i n a p i l o t p l a n t fermentator. The inoculum s i z e i s t y p i c a l l y 5% with a 3% (as i s ) carbon source c o n c e n t r a t i o n . V i s c o s i t y development s t a r t e d a t approximately 7 hours and reached a maximum o f 4500 cps by 64 hours. Unless otherwise s p e c i f i e d , v i s c o s i t y measurements are made using a Model LVF B r o o k f i e l d viscometer with #4 s p i n d l e a t 60 rpm. The maximum c e l l p o p u l a t i o n o f about 1 χ 1 0 was reached i n 10 hours. By employing an automati system with an oxygen probe, the minimum d i s s o l v e d oxygen concen t r a t i o n was determined t o be 5-10% during the f i r s t 24 hours o f the fermentation. Recovery o f the product i s done by p r e c i p i t a t i o n with such organic s o l v e n t s as acetone, e t h y l a l c o h o l , i s o p r o p y l a l c o h o l , or v a r i o u s isomers o f butanol. A f t e r p r e c i p i t a t i o n , the polymer f i b e r s a r e d r i e d and m i l l e d t o a powder. The s t u d i e s on the chemical components o f t h i s p o l y saccharide were done on m a t e r i a l p u r i f i e d by f i l t r a t i o n o f ZANFLO s o l u t i o n s using diatomaceous earth as f i l t e r a i d followed by repeated r e p r e c i p i t a t i o n s w i t h IPA. The m a t e r i a l was found t o be 97% carbohydrate and 3% p r o t e i n . The p o l y s a c c h a r i d e was hydrolyzed using 2N H2SO4 and heated t o 100° C. f o r 5 hours. The components o f t h i s polymer were i d e n t i f i e d using paper chromatographic techniques. The molar r a t i o was determined using gas l i q u i d chromatography. In Table I I one can see the r e s u l t s o f t h i s chemical component a n a l y s i s . The carbohydrate p o r t i o n was found t o c o n t a i n glucose, g a l a c t o s e , g l u c u r o n i c a c i d , and fucose i n the molar r a t i o o f 3:2:1.5:1. Uronic a c i d accounts f o r approximately 20% o f the p o l y s a c c h a r i d e on a weight b a s i s . I t i s noteworthy t h a t fucose i s not commonly found as a s t r u c t u r a l c o n s t i t u e n t o f e x o c e l l u l a r b a c t e r i a l heteropolysaccharides. We are not y e t c e r t a i n as t o the r o l e o f p r o t e i n i n the p u r i f i e d p o l y s a c c h a r i d e . The completion o f our s t r u c t u r a l work w i l l provide the answers t o these questions. ZANFLO i s a h i g h - v i s c o s i t y polysaccharide, as shown i n the v i s c o s i t y c o n c e n t r a t i o n curve i n F i g u r e 2. I t i s c o n s i d e r a b l y higher than t h a t o f xanthan gum, a well-known b a c t e r i a l heterοp o l y s a c c h a r i d e widely used i n i n d u s t r y . T h i s d i f f e r e n c e becomes more outstanding a t higher c o n c e n t r a t i o n s . At a 1.5% gum con c e n t r a t i o n , the xanthan gum had a v i s c o s i t y of 2500 cps, while ZANFLO had a v i s c o s i t y o f 5000 cps. The r e s u l t s i n F i g u r e 3 show the e f f e c t o f heat on the ZANFLO p o l y s a c c h a r i d e . ZANFLO's v i s c o s i t y , l i k e t h a t o f many m i c r o b i a l p o l y s a c c h a r i d e s , i s d e f i n i t e l y a f f e c t e d by heat. As 1 0

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

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φ

Novel

Beer

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213

Viscosity

φ—Ο

ι

ι

ι

ι

1 0

20

Î0

40

Fermentation

Time

1 r

,0

(hours)

Figure 1. Fermentation parameters of the Zanflo fermentation

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

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EXTRACELLULAR MICROBIAL POLYSACCHARIDES

Table I. NITRATE

Biochemical Characte +

REDUCTASE

CELLULASE

+

(Cx)

UREASE

+

AMYLASE

+

H S

+

2

PRODUCTION

INDOLE

+

VOGES-PROSKAUER METHYL RED LYSINE DECARBOXYLAS GELATIN

LIQUEFACTIO

ACID AND GAS FROM CARBOHYDRATES GLUCOSE

+

LACTOSE

+

SUCROSE

+

MALTOSE

+

CELLOBIOSE

+

MANNITOL

+

INOSITOL

+

ADONITOL DULCITOL

Table II. Carbohydrate Composition of Zanflo URONIC

ACID

19%

GLUCOSE

39%

GALACTOSE

29%

FUCOSE

13%

100% ACETYL

4.5%

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

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—ι

Novel

Bacterial

J

Ο.25

215

Ι

1.00

2.00

POLYSACCHARIDE CONCENTRATION

20

Heteropolysaccharide

60 TEMPERATURE (°C)

80

(%)

90

Figure 2.

Figure

3.

Viscosity vs. concentration

Effect of temperature Zanfio viscosity

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you can see, the r e l a t i o n s h i p between v i s c o s i t y and temperature i s q u i t e l i n e a r . From t h i s graph one can c a l c u l a t e a decrease i n v i s c o s i t y of 25 cps/°C. as the temperature i s i n c r e a s e d . The ZANFLO c o n c e n t r a t i o n i s 1%. The v i s c o s i t y decrease i s temperature r e v e r s i b l e . The r e s u l t s i n F i g u r e 4 show the e f f e c t of pH on ZANFLO i n comparison to that of xanthan gum. The v i s c o s i t y of ZANFLO remains s t a b l e from a pH of 5 to 10 but decreases on e i t h e r side of t h i s range. One of the most s t r i k i n g p r o p e r t i e s of t h i s p o l y s a c c h a r i d e i s i t s c o m p a t i b i l i t y with c a t i o n i c dyes. Anionic gums, such as xanthan gum and many u r o n i c a c i d - c o n t a i n i n g p o l y s a c c h a r i d e s , r e a c t s t r o n g l y with c a t i o n i c dyes, such as methylene blue c h l o r i d e , to form a f i b r o u industrial applications t a i n s a s u b s t a n t i a l amount of u r o n i c a c i d , does not p r e c i p i t a t e w i t h such c a t i o n i c dyes at any pH. I t was noted, nonetheless, that ZANFLO l o s t a l l or most of i t s v i s c o s i t y during the t e s t and f u r t h e r experimentation showed the r e a c t i o n with methylene blue to be i n f l u e n c e d by s a l t concent r a t i o n and pH. The r e s u l t s i n Table I I I demonstrate the e f f e c t of pH on the v i s c o s i t y of the ZANFLO-methylene blue complex using a c e t i c a c i d to a d j u s t the pH. The v i s c o s i t y from n e u t r a l i t y to at l e a s t 4.5 i s as low as water v i s c o s i t y . By the time a pH of 3.9 i s reached, the v i s c o s i t y s t a r t s to increase and reaches a maximum of 110 cps at a pH of 3.1. We a l s o n o t i c e d at t h i s time that the v i s c o s i t y would decrease to that of water again i f the pH were adjusted upward slowly with NaOH. I f one continues to add NaOH, the v i s c o s i t y w i l l begin to i n c r e a s e at a pH of 4.4-4.6 and i t can no longer be brought down to t h a t of water again. T h i s e f f e c t i s caused by the concentrat i o n of Na now present i n the s o l u t i o n . The r e s u l t s i n F i g u r e 5 show the e f f e c t of monovalent and d i v a l e n t c a t i o n s on a s i m i l a r system. Here, v a r i o u s concentrat i o n s of NaCl or MgCl2 were added to n e u t r a l s o l u t i o n s of ZANFLO c o n t a i n i n g MBC and having the v i s c o s i t y of water. The r e s u l t s show that a s t o i c h i o m e t r i c r e l a t i o n s h i p e x i s t s between Mg and Na i n r e s t o r i n g l o s t v i s c o s i t y to the s o l u t i o n . I t appears evident that an increase i n e l e c t r o l y t e c o n c e n t r a t i o n would increase competition with MB f o r the r e a c t i v e s i t e of polymer and t h e r e f o r e increase the v i s c o s i t y . The r o l e of pH i n t h i s phenomenon appears to be centered around the pKa of the uronic a c i d p o r t i o n of the polymer. Glucuronic a c i d has a pKa of 3.2. At pH values below t h i s , the number of r e a c t i v e s i t e s of the polymer from MB would decrease, and, hence, i n c r e a s e i n v i s c o s i t y . The mechanism involved i n c o m p a t i b i l i t y i s not understood at present. We speculate that perhaps the p r o t e i n or peptide moiety may p l a y an important r o l e i n masking the a n i o n i c groups of the gum or s t a b i l i z i n g the gum under these c o n d i t i o n s . +

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

KANG ET AL.

Novel

Bacterial

Heteropolysaccharide

#

2,000 [-

\

/ 1,000 μ

ο.

-Ο-Ο

• s-io O

XANTHAN GUM

2

4

6

8

10

12

PH Figure 4.

Effect of pH on Zanflo viscosity

Table III. Effect of pH on the Viscosity of a Zanflo-Methylene Blue Chloride Complex in Solution PU 7.5

VISCOSITY: W0

CPS

(NO

MBC)

7.5

0 CPS (0.2%

5.8

0

CPS

4.2

0

CPS

3.9

25

CPS

3.1

110

CPS

MBC)

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

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EXTRACELLULAR MICROBIAL POLYSACCHARIDES

CATION CONCENTRATION (M) Figure 5.

Effect of monovalent and divalent cations on restoring viscosity to Zanflo-MBC solutions

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

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Another p o s s i b i l i t y i s that the secondary o r t e r t i a r y s t r u c t u r e o f t h i s polysaccharide, which would give i t a d i s t i n c t i v e conformation i n s o l u t i o n , p r o t e c t s i t against the a c t i o n of c a t i o n i c agents such as methylene blue c h l o r i d e . In summary, we have i s o l a t e d a bacterium from the s o i l which produces l a r g e amounts o f a novel i n d u s t r i a l heteropolysaccharide. Because o f i t s unusual r h e o l o g i c a l p r o p e r t i e s and c o m p a t i b i l i t y with b a s i c dyes, ZANFLO has already e s t a b l i s h e d e x c e l l e n t a p p l i c a t i o n s i n p a i n t and shows e x c e l l e n t p o t e n t i a l i n other a p p l i c a t i o n areas.

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

17 P S - 7 — A N e w Bacterial Heteropolysaccharide

K. S. KANG and W. H. McNEELY Kelco, Division of Merck & Co., Inc., 8225 Aero Drive, San Diego, CA 92123

Dextran p r o d u c t i o n by f e r m e n t a t i o n o r by c e l l f r e e s y n t h e s i s has l o n polysaccharide fermentatio r e l a t i v e l y new f i e l d . The P e o r i a L a b o r a t o r y o f t h e USDA, which c a r r i e d o u t t h e i n i t i a l m i c r o b i a l r e s e a r c h on xanthan gum, a l s o has done development work on p o l y s a c c h a r i d e p r o d u c t i o n by v a r i o u s A r t h r o b a c t e r and yeast s t r a i n s (2). Dextran, xanthan gum, p o l y t r a n , and ZANFLO r e p r e s e n t m i c r o b i a l p o l y s a c c h a r i d e s which a r e now commercially a v a i l a b l e . The most n o t a b l e f e r m e n t a t i o n p o l y s a c c h a r i d e w i t h t h e g r e a t e s t commercial s u c c e s s a t t h i s time i s xanthan gum. T h i s paper i s i n t e n d e d t o r e p o r t on t h e v a r i o u s properties i n relation to possible industrial applic a t i o n s o f a n o v e l p o l y s a c c h a r i d e , PS-7. The m i c r o b i o l o g i c a l and f e r m e n t a t i o n a l a s p e c t s o f t h i s p o l y s a c c h a r i d e are described only b r i e f l y . A preliminary report o f PS-7 as a p o t e n t i a l f o o d a d d i t i v e has been made (3). PS-7 i s produced by a s o i l b a c t e r i u m which was i s o l a t e d from a l o c a l s o i l sample. T h i s organism was i d e n t i f i e d as a s t r a i n o f A z o t o b a c t e r i n d i c u s on t h e b a s i s o f growth c h a r a c t e r i s t i c s , b i o c h e m i c a l and morphological properties. PS-7 i s an e x t r a c e l l u l a r p o l y s a c c h a r i d e produced by means o f an a e r o b i c , submerged f e r m e n t a t i o n . The t y p i c a l f e r m e n t a t i o n medium i s i l l u s t r a t e d i n T a b l e I . The c o n c e n t r a t i o n o f p o t a s s i u m phosphate may be reduced to as low as 0.01% i f t h e pH has been m a i n t a i n e d d u r i n g the f e r m e n t a t i o n , o r d i n a r i l y by u s i n g KOH. By employing an a u t o m a t i c a g i t a t i o n - c o n t r o l l i n g system w i t h an oxygen probe, t h e minimum d i s s o l v e d oxygen c o n c e n t r a t i o n was determined t o be 5-10% d u r i n g the f i r s t 20-25 hours o f t h e f e r m e n t a t i o n . During t h i s f e r m e n t a t i o n time w i t h c o n s t a n t a e r a t i o n r a t e o f 0.25 220

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l i t e r p e r l i t e r o f f e r m e n t a t i o n medium p e r minute, the t i p speed f o r a g i t a t i o n v a r i e d between 60 and 110 meters per minute t o m a i n t a i n the D.O. o f 10%. The v i s c o s i t y o f the f e r m e n t a t i o n l i q u o r s t e a d i l y i n c r e a s e d , and, a t 24 h o u r s , the v i s c o s i t y was a p p r o x i m a t e l y 2000 c p s , as measured by a B r o o k f i e l d v i s c o m e t e r at a r o t a t i o n speed o f 60 rpm and ambient temperature. PS-7 may be r e c o v e r e d from the f e r m e n t a t i o n l i q u o r by a l c o h o l s such as methanol, e t h a n o l and i s o p r o p a n o l , etc., o r by lower a l k y l ketones such as a c e t o n e . The p r e f e r r e d s o l v e n t f o r r e c o v e r y purposes i s IPA. After p r e c i p i t a t i o n , the polymer f i b e r s a r e d r i e d and m i l l e d t o o b t a i n a p a l e , c r e a m - c o l o r e d powder. The c h e m i c a l componen tography and g a s - l i q u i out and the r e s u l t s a r e shown i n T a b l e I I . The r e s u l t s i n d i c a t e t h a t PS-7 c o n s i s t s o f g l u c o s e , rhamnose and a u r o n i c a c i d i n an approximate r a t i o o f 6.6:1.5:1.0. I t has an a c e t y l c o n t e n t o f about 8.0-10.0%. Neither the u r o n i c a c i d nor the l i n k a g e s p r e s e n t i n t h i s polymer have been e l u c i d a t e d a t t h i s t i m e . PS-7 has an u n u s u a l l y h i g h v i s c o s i t y , as demon s t r a t e d i n F i g u r e 1. The v i s c o s i t y o f PS-7 i s much g r e a t e r than t h a t o f xanthan gum, w i t h the d i f f e r e n c e becoming s i g n i f i c a n t a t c o n c e n t r a t i o n s as low as 0.15%. T h i s p o i n t i s f u r t h e r i l l u s t r a t e d i n F i g u r e 2. The f e r m e n t a t i o n l i q u o r i n the f l a s k becomes so v i s c o u s at the end o f the f e r m e n t a t i o n time t h a t t h e r e i s l i t t l e f l o w , even i f t h e f l a s k i s h e l d u p s i d e down. F i g u r e 3 i l l u s t r a t e s another important p r o p e r t y of any commercial p o l y s a c c h a r i d e , and t h a t i s i t s v i s c o s i t y response t o temperature. T h i s d a t a shows t h a t PS-7 i s s i m i l a r t o xanthan gum i n t h a t i t s v i s c p s i t y i s s t a b l e over a wide temperature range. The v i s c o s i t y response o f PS-7 t o changes i n pH i s shown i n F i g u r e 4. The v i s c o s i t y o f PS-7 i s almost as s t a b l e as xanthan gum, a l t h o u g h the v i s c o s i t y s t a r t s t o decrease below pH 3 and beyond pH 12. Another i m p o r t a n t c h a r a c t e r i s t i c f o r some a p p l i c a t i o n s i s p s e u d o p l a s t i c i t y . P s e u d o p l a s t i c i t y i s indicated when the v i s c o s i t y d e c r e a s e s as the shear r a t e i s i n creased. T h i s p r o p e r t y o f PS-7 i s d e p i c t e d i n Figure 5. T h i s v i s c o g r a m was o b t a i n e d by u s i n g the Fann v i s c o meter Model 35. I t s h o u l d be noted t h a t t h e c o n c e n t r a t i o n o f PS-7 i s o n l y h a l f the c o n c e n t r a t i o n o f xanthan gum o r a q u a r t e r o f o t h e r polymers such as CMC, HEC, and guar. There i s a remarkable d e c r e a s e i n the v i s c o s i t y of PS-7 as i t i s s h e a r e d , t h e magnitude w e l l e x c e e d i n g t h a t o f xanthan gum. F i g u r e 6 compares v i s c o s i t y and p s e u d o p l a s t i c i t y o f PS-7 t o xanthan gum a t the same

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

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EXTRACELLULAR MICROBIAL POLYSACCHARIDES Table I

TYPICAL FERMENTATION MEDIUM THE INGREDIENTS

K2HPO4 MGSOZI

CARBOHYDRATE COMPOSITION

5.0 GRAMS

7 H2SO4

0.1

GRAMS

NH14NO3

0.9

GRAMS

PR0M0S0Y

0.5

GRAMS

GLUCOSE

CHEMICAL COMPONENTS OF PS-7

CONCENTRATION

30.0

URONIC ACID

11%

GLUCOSE

73

GRAMS RHAMN0SE

TAP

WATER

TO 1 LITER

16 100%

ACETYL

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

KANG

AND

New Bacterial

M C N E E L Y

H eteropoly saccharide

Figure i

2,000

S ι,οοο h

TEMPERATURE C O Figure 3.

The effect of heat on the viscosity of PS-7 and xanthan gum solutions

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

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EXTRACELLULAR MICROBIAL POLYSACCHARIDES

XANTHA I

ι

I

ι

I

ι

0

2

H

6

8

10

ι 12

PH Figure 4.

The effect of pH on PS-7 and xanthan gum solutions

S h e a r Rate, r p m

Xanthan G u m 1.0lb/bbl

1

10

100

S h e a r Rate, s e c

1000

- 1

Figure 5

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

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Heteropolysaccharide

225

polymer c o n c e n t r a t i o n . Here, the d i f f e r e n c e s i n b o t h v i s c o s i t y and p s e u d o p l a s t i c i t y between PS-7 and xanthan gum a r e more o u t s t a n d i n g t h a n i n F i g u r e 5. PS-7 i s f u l l y s o l u b l e hot o r c o l d i n d i s t i l l e d water, t a p water, b r i n e water o r sea w a t e r . T h i s f a c t i s i l l u s t r a t e d i n T a b l e I I I . The sea water has a s a l t c o n c e n t r a t i o n o f a p p r o x i m a t e l y 3.5%, and the Permian B r i n e water o f West Texas has a s a l t c o n t e n t o f approxi m a t e l y 26%. The l o w e s t v i s c o s i t y was o b t a i n e d i n d i s t i l l e d water. P o l y s a c c h a r i d e PS-7 i s a l s o c o m p a t i b l e w i t h a wide v a r i e t y of s a l t s . Examples o f t h i s c o m p a t i b i l i t y a r e shown i n T a b l e IV. These c o n c e n t r a t i o n s a r e not n e c e s s a r i l y the l i m i t s t a b i l i t y of these s o l u t i o n hours s t i r r i n g and 24 hours s t a n d i n g , l o o k i n g f o r p r e c i p i t a t i o n , g e l a t i o n o r changes i n f l o w p r o p e r t i e s . A l l s o l u t i o n s l i s t e d i n t h i s s l i d e were s t a b l e . The s t a b i l i t y o f PS-7 was a l s o s t u d i e d o v e r a one-month p e r i o d i n many o f t h e s e s a l t s , and t h i s d a t a i s shown i n T a b l e V. A d i s t i l l e d water c o n t r o l was carried. As a p r e s e r v a t i v e , formaldehyde was added t o the s o l u t i o n s a t the c o n c e n t r a t i o n o f 200 ppm. The d a t a i n d i c a t e s t h a t PS-7 was s t a b l e i n a l l c a s e s . P o l y s a c c h a r i d e PS-7 i s i n c o m p a t i b l e w i t h c a t i o n i c o r p o l y v a l e n t i o n s a t h i g h pH. This incompatiblity r e s u l t s i n a g e l . S o l u t i o n s o f PS-7 a l s o e x h i b i t some tendency t o g e l i n the p r e s e n c e o f h i g h concentrations o f monovalent s a l t s above a pH o f 10. While t h i s g e l a t i o n i s c o n s i d e r e d an i n c o m p a t i b i l i t y , i t can a l s o be a d e s i r a b l e e f f e c t , as w i l l be shown l a t e r . Solut i o n s o f PS-7 show l i m i t e d s t o r a g e s t a b i l i t y under c o n d i t i o n s o f s t r o n g a c i d i t y or a l k a l i n i t y . The p r o p e r t i e s i l l u s t r a t e d i n t h e p r e v i o u s s l i d e s demonstrate many c h a r a c t e r i s t i c s o f PS-7 which make i t a h i g h l y u s e f u l agent i n o i l w e l l d r i l l i n g muds. D r i l l i n g muds are g e n e r a l l y aqueous f l u i d s which cont a i n s u b s t a n t i a l q u a n t i t i e s o f c l a y s and o t h e r c o l l o i d a l materials. An optimum d r i l l i n g f l u i d would be one which, f i r s t l y , i s f l e x i b l e i n i t s v i s c o s i t y c h a r a c t e r i s t i c s so as t o p r o v i d e s u s p e n s i o n o f s o l i d s w i t h i n the f l u i d , and, s e c o n d l y , would l u b r i c a t e the d r i l l b i t . The h i g h v i s c o s i t y , p s e u d o p l a s t i c i t y and r e l a t i v e i n s e n s i t i v i t y o f the v i s c o s i t y t o temperature i n d i c a t e t h a t the use o f PS-7 i n a d r i l l i n g mud would come c l o s e t o the optimum c h a r a c t e r i s t i c s d i s c u s s e d e a r l i e r . A v e r y s i m p l e , easy and r a p i d way t o t e s t the s u s p e n d i n g a b i l i t y o f a f l u i d was d e v e l o p e d i n our l a b o r a t o r y (4) . The method employs a s t a n d a r d American P e t r o l e u m I n s t i t u t e s a n d - c o n t e n t t u b e . The tube i s

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

EXTRACELLULAR MICROBIAL POLYSACCHARIDES

Table I I I

VISCOSITY OF PS-7 IN VARIOUS WATERS OF A 0.5% CONCENTRATION WATER

VISCOSITY

DISTILLED

690 CPS

TAP WATER

820 CPS

SEA WATER

860 CPS

PERMIAN BRINE

720 CPS

(CPS)

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

17.

KANG

New Bacterial

A N D M C N E E L Y

Heteropolysaccharide

Table IV Final Salt Concn.

Salt

(40%) (25%) (30%) (65%) (22%) (32%) (55%) (36%) (44%) (30%) (7%) (25%)

Aluminum Nitrate Aluminum Sulfate Ammonium Chloride Ammonium Nitrate Ammonium Sulfate Calcium Chloride Calcium Nitrate Magnesium Chloride Magnesium Nitrate Magnesium Sulfate Potassium Ferricyanide Potassium Ferrocyanide Sodium Chlorid Sodium Dichromat Sodium Nitrat Sodium Phosphate (dibasic) Sodium Sulfate Sodium Sulfite Sodium Thiosnlfate

(40%) (10%) (1 5%) (20%) (40%) (40%) (37%)

Table V

S0II1TI0N S T A B I L I T Y OF P S - 7

1%

PS-7

SOLUTION WITH

VISCOSITY ( C P S ) INITIAL AGED 1 MO,

D I S T I L L E D WATER (CONTROL)

1700

1900

SODIUM CHLORIDE ( 1 5 % )

3150

3450

CALCIUM CHLORIDE ( 1 5 % )

3050

3250

ALUMINUM SULFATE ( 1 5 % )

3000

3100

ZINC SULFATE ( 1 5 % )

3100

3200

AMMONIUM CHLORIDE ( 2 3 % )

2850

3100

CUPRIC CHLORIDE ( 1 3 % )

2950

2850

FERROUS SULFATE ( 1 3 % )

3100

2650

MONOSODIUM PHOSPHATE ( 1 3 % )

3050

3550

ZINC CHLORIDE ( 1 3 % )

3000

3350

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

227

228

EXTRACELLULAR MICROBIAL POLYSACCHARIDES

100

200

300

400

500

600

Settling Time, sec Figure 7

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

700

17.

KANG

AND

M C N E E L Y

New Bacterial

Heteropolysaccharide

229

f i l l e d w i t h t h e f l u i d t o be t e s t e d . Then,1 gram o f 20-35 mesh sand i s p l a c e d i n t o t h e t u b e . The tube i s shaken v i g o r o u s l y t o t h o r o u g h l y d i s p e r s e t h e sand, p l a c e d u p r i g h t , and t i m i n g i s begun. The time t h a t i t t a k e s f o r t h e sand t o r e a c h s e l e c t e d g r a d u a t e d marks a t t h e bottom o f t h e tube i s r e c o r d e d . Figure 7 i s a plot of the s e t t l i n g rates of p a r t i c u l a t e matter i n the d i f f e r e n t f l u i d s . The d a t a i n d i c a t e t h a t PS-7 was by f a r t h e most e f f i c i e n t i n suspending p a r t i c l e s . Guar gum was i n t e r m e d i a t e , and the c e l l u l o s i c s the l e a s t e f f e c t i v e . Figure 8 i s a plot of e f f e c t i v e s e t t l i n g rate vs. concentration f o r the f l u i d s . Here, a g a i n , PS-7 was t h e most e f f e c t i v e h a l f the concentratio parable suspending e f f e c t . D r i l l i n g f l u i d s a r e t y p i c a l l y p r e p a r e d w i t h whate v e r water i s a v a i l a b l e nearby. The good s o l u b i l i t y o f PS-7 makes i t s u i t a b l e t o t h i s a p p l i c a t i o n — e i t h e r the s e a water f o r o f f - s h o r e w e l l s o r f r e s h o r b r i n e water from water w e l l s a t t h e d r i l l i n g s i t e , w h i c h e v e r may be a v a i l a b l e . I n some d r i l l i n g f l u i d s , i t i s des i r a b l e to g e l the f l u i d . P o l y s a c c h a r i d e PS-7 i s a l s o u s e f u l i n t h i s r e s p e c t , as i n c r e a s i n g t h e pH t o 11 causes a d r i l l i n g f l u i d c o n t a i n i n g PS-7 and chromium to g e l . The v i s c o s i t y o f t h e d r i l l i n g f l u i d c a n be a d j u s t e d t o t h e d e s i r e d l e v e l by s i m p l y a l t e r i n g t h e pH and t h e c o n c e n t r a t i o n o f t h e c r o s s - l i n k i n g a g e n t . B e s i d e s t h e o i l w e l l d r i l l i n g a p p l i c a t i o n s , we have e s t a b l i s h e d t h e p o t e n t i a l use o f PS-7 i n o t h e r a p p l i c a t i o n s such as d r i p l e s s water-base l a t e x p a i n t , w a t e r f l o o d i n g systems f o r secondary o i l r e c o v e r y , w a l l j o i n t cement a d h e s i v e s and t e x t i l e p r i n t i n g .

Abstract PS-7 is ananionic heteropolysaccharide produced by a strain of Azotobacter indicus in an aerobic fermentation. PS-7 is composed of glucose, rhamnose, and uronic acid in an approximate weight ratio of 6.6:1.5:1. The polysaccharide has an acetyl content of about 9%. Solutions of PS-7 are characterized by high viscosity and a high degree of pseudoplasticity. The polysaccharide has excellent solubility in sea water and even in brine containing 25% salt. PS-7 exhibits excellent temperature and pH stability and is compatible with a variety of salts. In the presence of Cr , PS-7 gum can be cross-linked at pH 9.0-9.5. These properties indicate that PS-7 w i l l find wide u t i l i t y in a +++

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

230

EXTRACELLULAR MICROBIAL POLYSACCHARIDES

variety of applications. Literature Cited (1)

Jeans, Allene, "Dextrans and Pullulans," Extracellular Microbial Polysaccharides of Practical Importance, ACS Symposium series (1976).

(2)

McNeely, William H. and Kang, Kenneth S., "Xanthan and Some Other Biosynthetic Gums," Industrial Gums (2nd Ed.), p. 473, Academic Press, New York (1973).

(3)

Kang, Kenneth S d Kovacs Peter "New Micro bial Polysaccharide IVth International Congress of Food S c i . and Technol., Madrid, Spain (1974).

(4)

Carico, Robert D . , "New Field Test Improves Fluid-Suspension Measurements," O i l and Gas Journal, (1976) 74, (27), p. 81.

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

18 Applications of Xanthan G u m in Foods and Related Products THOMAS R. ANDREW Kelco, Division of Merck & Co., Inc., 8225 Aero Drive, San Diego, CA 92123

Xanthan gum was approved for use as a food additive on March 19 Administration in accordanc Since then it has been used in a wide variety of foods for a number of important reasons including emulsion stabilization, temperature s t a b i l i t y , com p a t i b i l i t y with food ingredients, and its unique pseudoplastic rheological properties. It has been proposed that xanthan gum, which is produced by the microorganism Xanthomonas campestris, is in fact a survival device for the organism generating it and it has, through millions of years of evolution, been perfected for this purpose (1). Thus, i t is not surprising that a substance generated for the protection of a microorganism should possess such unusual properties and be so resistant to thermal, chemical, and biological degradation. A great deal of progress has been made toward understanding the chemistry of xanthan gum. Figure 1 shows the structure of xanthan gum as i n i t i a l l y postulated. It was shown that i t consisted of a sixteen-residue repeating unit composed of D-glucose, D-mannose, and D-glucuronic acid as shown (2, 3). In a paper published in 1975 Jansson and co-workers proposed the somewhat more simplified structure shown in Figure 2. This structure is composed of a back bone of 1 - 4 linked β-D-glucose units with side chains which consist of two mannose and (4) a glucuronic acid unit on every other glucose unit. Every other side chain carries a pyruvic acid group. In 1972 Rees proposed a double helix solution conformation (Figure 3) for xanthan gum which went a long way toward explaining the yield point phenomenon and the flat temperature-viscosity curve, unique among polysaccharides (5,6). A further extension of Rees's study (Figure 4) provided an 231

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

232

EXTRACELLULAR MICROBIAL POLYSACCHARIDES

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

18.

ANDREW

Xanthan

Gum

in Foods and

Related

Products

233

e x c e l l e n t e x p l a n a t i o n o f why xanthan gum r e a c t s t o form e l a s t i c g e l s w i t h l o c u s t bean gum but not guar gum. The u n i f o r m l y d i s t r i b u t e d g a l a c t o s e s i d e c h a i n s a l o n g t h e mannose backbone o f guar gum p r e v e n t the c l o s e a s s o c i a t i o n o f the m o l e c u l e w i t h the xanthan gum h e l i x w h i l e the e x i s t e n c e o f "smooth" zones ( F i g u r e 5) i n the l o c u s t bean gum m o l e c u l e a l l o w s a s s o c i a t i o n and, t h e r e f o r e , g e l a t i o n ( 7 ) . As i s u s u a l l y t h e case w i t h the use o f gums, t h e o r y and p r a c t i c e have run on almost p a r a l l e l paths b u t the t w a i n have n o t y e t met. Theory h e l p s us u n d e r s t a n d t h e r e s u l t s b u t has not l e d us t o them. When xanthan gum e n t e r e d the food m a r k e t p l a c e i n 1969, i t s b a s i c p h y s i c a l p r o p e r t i e s were a c t i v e l y promoted, f o l l o w e t i o n s , a l l o f whic p r e v i o u s systems s t a b i l i z e d w i t h o t h e r gums. S a l a d d r e s s i n g w i t h e m u l s i o n s t a b i l i t y extended t o a y e a r o r more and s a l a d d r e s s i n g s t h a t c o u l d be r e t o r t e d o r r e p e a t e d l y f r o z e n were d e v e l o p e d . An i n s t a n t pudding which was almost the same as the cooked starch v e r s i o n was f o r m u l a t e d , and i n d u s t r y d e v e l o p e d a number o f o t h e r improved p r o d u c t s . P a s t e u r i z e d P r o c e s s e d Cheese

Spread

More r e c e n t work has been completed which demonstrates the u t i l i t y o f xanthan gum i n p a s t e u r i z e d p r o c e s s cheese s p r e a d ( 8 ) . U s i n g a s t a n d a r d f o r m u l a f o r p a s t e u r i z e d p r o c e s s cheese, samples were p r e p a r e d under d u p l i c a t e c o n d i t i o n s i n a cooker commonly used f o r t h i s purpose. As T a b l e I i n d i c a t e s , a number o f combinations o f xanthan gum, l o c u s t bean gum, and guar gum were e v a l u a t e d t o determine t h e optimum r a t i o f o r good meltdown, f i r m n e s s , s l i c e a b i l i t y , and f l a v o r r e l e a s e . I t i s i n t e r e s t i n g t o note t h a t o n l y one c o m b i n a t i o n g i v e s good r e s u l t s i n e v e r y c a t e g o r y , T r i a l No. 17A. These r e s u l t s n o t o n l y demonstrate the u t i l i t y o f xanthan gum b u t a l s o the n e c e s s i t y f o r u s i n g b l e n d s a t times t o o b t a i n f u n c t i o n a l advantages n o t o b t a i n a b l e w i t h s i n g l e t h i c k e n e r s . Each gum cont r i b u t e s a d e s i r a b l e c h a r a c t e r i s t i c t o the f i n a l p r o d u c t , b u t t h e s y n e r g i s t i c b l e n d i s b e t t e r than t h e sum o f i t s p a r t s . C o t t a g e Cheese D r e s s i n g Table I I again i l l u s t r a t e s the f u n c t i o n a l s u p e r i o r i t y o f the s y n e r g i s t i c b l e n d . C o t t a g e cheese

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

EXTRACELLULAR MICROBIAL POLYSACCHARIDES

GREGATE DOUBLE HELIX AddAcG RANDOM COIL r o nking to Predominant in the sol; Provide cross-linking onsolidastse-lit he gel can exist as "connectingjunctions in the gel. c structure thus acting as lengths" in gel structur and impart elasticity whe they do so. Sol ;j=i Incipient g e l ^ Clear elastic gel ^

Figure 3.

Stiff gel ^

Turbid rigid gel

Phase separation syneresed gel

States of polysaccharide molecules and their role in gel prop erties

Figure 4. Schematic of galactomannan conformation. Each line represents a sugar unit consisting of the backbone composed of β-Ό-mannopyranose units and the side chains composed of a-O-gahctopyranose units.

Figure 5. Possible model for the inter action between xan than gum and locust bean galactomannan, resulting in gel for mation

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

18.

ANDREW

Xanthan Gum in Foods and Related Products

235

cream d r e s s i n g , which i s n o r m a l l y about 10 p e r c e n t b u t t e r f a t w i t h s a l t added, i s f o r m u l a t e d t o p o s s e s s enough v i s c o s i t y t o make i t c r e a m i e r and t o g i v e good c l i n g t o t h e c u r d . I f the v i s c o s i t y o f the cream i s t o o h i g h , t h e r e s u l t i n g c o t t a g e cheese w i l l be t o o " d r y " and t h e e a t i n g q u a l i t i e s w i l l s u f f e r . A n o t h e r p o t e n t i a l problem a r i s e s because o f t h e i n h e r e n t gummy m o u t h f e e l e x h i b i t e d by many gums a t a c o n c e n t r a t i o n o f 0.2 t o 0.3 p e r c e n t , t h e normal use l e v e l o f gums i n c o t t a g e cheese cream. As T a b l e I I i l l u s t r a t e s , a b l e n d o f xanthan gum, l o c u s t bean gum, and guar gum c a n be used t o d e v e l o p t h e v i s c o s i t y n e c e s s a r y f o r good c l i n g , b u t t h e a c t i v e gum conc e n t r a t i o n i s low enough so t h a t m o u t h f e e l and t e x t u r e do n o t s u f f e homogeneous and u n i f o r m l not separate w i t h time. L i q u i d C a t t l e Feed

Supplements

L i q u i d f e e d supplements, a l t h o u g h n o t f o o d i n t h e sense t h a t t h e y a r e consumed d i r e c t l y by humans, a r e n o n e t h e l e s s an i m p o r t a n t f a c t o r i n t h e f o o d supply. L i q u i d f e e d supplements a r e b a s i c a l l y one o r more n u t r i e n t m a t e r i a l s s u p p l i e d i n a l i q u i d v e h i c l e such as water o r m o l a s s e s . The g r e a t m a j o r i t y a r e added t o t h e d r y f e e d o f f e e d l o t c a t t l e and a r e s h i p p e d and s t o r e d i n l a r g e t a n k s where, because o f the formation o f p r e c i p i t a t e s o r f l o c c u l a n t s o r t h e addition o f insoluble material, maintaining uniformity is difficult. I t i s e s p e c i a l l y important t o maintain u n i f o r m i t y because v i t a m i n s and t r a c e m i n e r a l s which a r e added t o t h e r a t i o n t e n d t o adsorb on t h e s o l i d f l o c c u l a n t s t h a t form and w i l l n o t s t a y u n i f o r m l y d i s t r i b u t e d u n l e s s s e d i m e n t a t i o n i s p r e v e n t e d by t h e use o f a s u s p e n d i n g agent o r c o n t i n u o u s a g i t a t i o n . F i g u r e 6 i s a t y p i c a l l i q u i d f e e d supplement formul a t i o n o f the high-molasses, phosphoric-acid type. Note t h e s i m i l a r i t y t o f e r t i l i z e r . Figure 7 i l l u s t r a t e s t h e f u n c t i o n a l i t y o f xanthan gum as a s u s p e n d i n g agent a t room t e m p e r a t u r e , and F i g u r e 8 i l l u s t r a t e s i t s s t a b i l i t y a t 95°F (10). These f i g u r e s i l l u s t r a t e two p o i n t s : (1) xanthan gum i s an e x c e l l e n t s u s p e n d i n g agent because i t p o s s e s s e s a y i e l d p o i n t , and (2) s u s p e n d i n g q u a l i t i e s o f xanthan gum a r e n o t s i g n i f i c a n t l y d i m i n i s h e d by e l e v a t e d t e m p e r a t u r e s as o c c u r s w i t h l i q u i d f e e d supplements i n warm weather. I n a d d i t i o n , because o f i t s p s e u d o p l a s t i c i t y , xanthan gum f a c i l i t a t e s pumping.

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

EXTRACELLULAR MICROBIAL

236

Table

I

POLYSACCHARIDES

F O R M U L A S AND RESULTS

OF EXPERIMENTAL PASTEURIZED PROCESSED CHEESE SPREADS % GUM CONTENT TRIAL NUMBER

ORGANOLEPTIC EVALUATIONS XANTHAN GUM

GUAR GUM

LBG

BODY RESILIENCY

SLICING PROPERTIES

MOUTH FEEL

FLAVOR RELEASE

SANDWICH MELT

1A

0.2

Fair

Tacky

Lumpy

Fair

Excellent

2A

0.5

Fair

Tacky

Lumpy

Fair

Good

3A

0.8

Fair

Tacky

Lumpy

Fair

Good

4A

— —

Good

Good

Lumpy

Fair

Good

Excellent

Excellent

Lumpy

Fair

Excellent

Excellent

Excellent

Lumpy

Fair

Excellent

5A 6A

0.2 0.5 0.8

— —

—

7A

0.2

Poor

Tacky

Smooth

Excellent

Good

8A

0.5

Poor

Tacky

Smooth

Excellent

Excellent

9A

0.8

Fair

Tacky

Smooth

Excellent

Excellent

— — — — —

0.14

Fair

Tacky

Lumpy

Fair

Good

10A

0.06

11A

0.05

12A

0.15

13A

0.25

14A

0.24

0.56

Good

Tacky

Smooth

Good

Excellent

15A

0.06

0.04

0.10

Good

Tacky

Smooth

Good

Good

16A

0.15

0.10

0.25

Good

Good

Smooth

Good

Excellent

17A

0.24

0.16

0.40

Excellent

Excellent

Smooth

Excellent

Excellent

18A*

0.15

0.10

0.25

Good

Good

Smooth

Good

Good

0.4 0.3 0.2

'Trial 1SA contained 0.2 percent pimento solids or 12.50 pounds of drained pimentos per batch.

T^.Ul^

'Cible

Τ Τ

11

Dressing Viscosities (cps) (| itial/24hr.) n

Percent Stabilizer:

0.05

0.10

0.15

Xanthan gum/ galactomannan blend Xanthan gum Guar gum Blend 1 Blend 2 Blend 3 Blend 4 Locust bean gum

27/41 10/10 6/4 7/8 6/5 4/12 7/13 11/16

118/115 40/60 15/31 9/21 8/11 7/19 10/24 23/35

260/245 107/133 33/60 16/42 13/19 18/41 21/49 52/69

0.20

0.30

Not evaluated 373/480 160/250 45/71 27/58 62/105 66/135 175/95

155/225 51/86 23/47 18/35 36/67 51/95 105/130

Cane Molasses, 79.5 Brix

67.5

Urea Liquor, 50%

20.9

Salt

5.0

Trace Minerals

0.2

Phosphoric Acid, 75%

6.4 100.0

%

Figure 6. Liquid supplement for mulation high molasses, range type

Protein Equivalent

32

Phosphorus

1.5

Solids, Calculated

60

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

18.

ANDREW

Xanthan Gum in Foods and Related Products

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

237

238

EXTRACELLULAR

Calf Milk

MICROBIAL

POLYSACCHARIDES

Replacers

A r e l a t e d a p p l i c a t i o n i s t h e use o f xanthan gum f o r s t a b i l i z i n g c a l f m i l k r e p l a c e r s . C a l f milk r e p l a c e r s may c o n s i s t o f d r i e d whey and h e a t p r o c e s s e d soy beans o r s i n g l e - c e l l p r o t e i n . A dry mix i s added t o water, s t i r r e d t o d i s p e r s e t h e s o l i d s , and f e d t o c a l v e s . I f t h e f e e d i s n o t s t a b i l i z e d , t h e i n s o l u b l e s o l i d s w i l l q u i c k l y s i n k and t h e s h o r t term u n i f o r m i t y which i s r e q u i r e d w i l l be l o s t . As l i t t l e as 0.032 p e r c e n t xanthan gum can p r o v i d e enough s t a b i l i t y t o m a i n t a i n u n i f o r m i t y , as F i g u r e s 9, 10, and 11 show ( 1 1 ) . Thermal P r o c e s s i n g Recent work by Cheng and Kovacs has e x p l o r e d t h e r h e o l o g i c a l p r o p e r t i e s o f xanthan gum under h i g h temperature and moderate shear found i n most a g i t a t e d commercial c a n n i n g systems (12). A Fann 50C v i s cometer, an i n s t r u m e n t which p e r m i t s t h e measurement o f v i s c o s i t y a t shear r a t e s from 1.7 - 1075 sec"" , temperatures t o 500°F, and p r e s s u r e s t o 1000 p s i , was used. F i g u r e 12 d e p i c t s t h e v i s c o s i t y drop t h a t xanthan gum undergoes a t two a r b i t r a r i l y s e l e c t e d shear r a t e s , 170 sec"" and 511 s e c " , w i t h and w i t h o u t NaCl. The well-known " f l a t " v i s c o s i t y v e r s u s temperature c u r v e i s seen from ambient temperature t o about 190°F w i t h a l l s o l u t i o n s . The s t a b i l i t y t o t h e r m a l d e g r a d a t i o n imparted by an e l e c t r o l y t e can a l s o be seen. Furthermore, F i g u r e 9 i l l u s t r a t e s that the e l e c t r o l y t e - s t a b i l i z e d solut i o n s l o s e 98 p e r c e n t o f t h e i r v i s c o s i t y a t r e t o r t temperature (250°F) and r e c o v e r about 80 p e r c e n t o f t h e i r o r i g i n a l v i s c o s i t y on c o o l i n g . Obviously, a t h i c k e n e r t h a t i s t h i n a t r e t o r t temperature w i l l f a c i l i t a t e heat t r a n s f e r , thereby shortening the p r o c e s s time. T h i s r e d u c t i o n i n p r o c e s s time i s i m p o r t a n t f o r i n c r e a s i n g p r o d u c t i v i t y and i n t h e t h e r m a l p r o c e s s i n g o f foods which a r e a d v e r s e l y a f f e c t e d by " o v e r c o o k i n g " . 1

1

Other

1

Advances

Xanthan gum has a l s o been used t o s t a b i l i z e f r o z e n d e s s e r t s and t o s t a b i l i z e t o o t h p a s t e , where i t s r h e o l o g i c a l p r o p e r t i e s can be used t o f o r m u l a t e a p r o d u c t t h a t t h i n s when squeezed from t h e tube but has t h e o r i g i n a l c o n s i s t e n c y on t h e b r u s h . A s y n e r g i s m w i t h d e x t r i n has a l s o been used i n d e n t u r e

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

ANDREW

Xanthan

Gum in Foods and Related

Products

Figure 9

Figure 10

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

240

EXTRACELLULAR MICROBIAL POLYSACCHARIDES

Figure 11

Figure 12. The effect of retorting at shear rates of 170 sec' and 511 sec' on the viscosities xanthan gum with and without electrolyte 1

1

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

18.

ANDREW

Xanthan

Gum

in Foods

and Related

Products

241

a d h e s i v e s and i n a number o f f o o d s . Xanthan gum a l o n e and i n c o m b i n a t i o n w i t h galactomannans shows e x c e l l e n t promise i n canned, g r a v y - t y p e p e t f o o d s . I t has a l s o been r e p o r t e d t h a t xanthan gum shows good p o t e n t i a l as a g l u t e n s u b s t i t u t e i n b r e a d (13, 1 4 ) . A l t h o u g h t h i s p o t e n t i a l use i s i m p o r t a n t t o t h e segment o f t h e p o p u l a t i o n who a r e a l l e r g i c t o g l u t e n , i t has much b r o a d e r i m p l i c a t i o n s as a p o s s i b l e a d j u n c t t o l o w - q u a l i t y wheat f l o u r o r grains that are not s u i t a b l e f o r baking. We have reviewed today some o f t h e more r e c e n t developments i n xanthan gum usage. I n view o f t h e unique p r o p e r t i e s i t p o s s e s s e s as a r e s u l t o f i t s f u n c t i o n i n n a t u r e we can undoubtedly e x p e c t even w i d e r usage i n t h e

Literature Cited 1. "Xanthan Gum/KELTROL/KELZAN, A Natural Polysac charide for Scientific Water Control," Kelco Co., Second Edition. 2. Sloneker, J . Η., James Α . , Am. J. Chem. (1962) 40, 2066-71. 3. Siddiqni, Carbohyd. Res. (1967)4 (4), 284-91. 4. Jansson, P. Ε . , Keen, L., and Lindberg, Β . , Carbohyd. Res. (1976) 45 (1), 275-82. 5. Rees, D. Α . , Biophysical Society Winter Meeting, London, England (1973). 6. Rees, D. Α . , Biochem. J . (1972) 126, 257-73. 7. Dea, I. C. Μ., McKinnon, A. A. and Reese, D. A. (1972) J . Mol. B i o l . 68 (1), 153-72. 8. Kovacs, P . , and Igoe, R. S. (1976) Food Product Development, in press. 9. Kovacs, P. and Titlow, B. D. (1976) American Dairy Review 38 (4) 34J-34N. 10. Jackman, K. R., Randel, J . H. and Wintersdorff, P. (1976) A Unique Suspending Agent, National Feed Ingredients Association Meeting, Kansas City, Missouri (April 15). 11. Ibid. 12. Kovacs, P. and Cheng, H. (1976) Effects of High Temperatures and Shear Rates on Hydrocolloid Viscosities During Simulated Canning and HTST Processing Conditions. Unpublished report. 13. Kulp, K . , Hepburn, F. Ν . , and Lehmann, T. A. (1974) The Baker's Digest 48 (3), 34-37. 14. Christianson, D. D. Gardner, H. W., Warner, Κ., Boundy, Β. K. and Inglett, G. E. (1974) Food Technology 28 (6), 23-29.

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

19 Application of Xanthan G u m for Enhanced O i l Recovery Ε. I. SANDVIK and J. M. MAERKER Exxon Production Research Co., P.O. Box 2189, Houston, TX 77001

High molecular weight water soluble polymers find applica tion in two different enhanced oil recovery processes. At present, the principal use is for an improved form of waterflooding in which polymers are used to increase the efficiency with which water can contact and displace reservoir oil. However, it is anticipated that polymer requirements for processes of this type will be overshadowed by the quantity needed to provide mobility control for future micellar-polymer projects. The latter processes have potential for producing oil that is unre coverable by polymer augmented waterflooding. In both applica tions--polymer waterflooding and micellar-polymer flooding--the function of polymer is to reduce the mobility of injected water. Mobility is defined as the flow capacity of a rock-fluid system, or the volumetric flow rate per unit area achieved with a given pressure gradient (Figure 1). It is usually expressed as effective rock permeability divided by fluid viscosity, and the common petroleum reservoir engineering units are darcies (or millidarcies) per centipoise. As will be discussed later, poly mer can reduce mobility by decreasing effective rock permeability and by increasing effective fluid viscosity. Effects of mobility and mobility ratio on the efficiency of reservoir displacements may be illustrated with Hele-Shaw models (1) which give a simplified portrayal of areal displacement effi ciency in a reservoir element. These models commonly consist of two square glass plates that are separated and sealed at the edges by a thin spacing gasket and have provisions to inject and withdraw fluid at opposite corners to simulate injection and pro duction wells. Mobilities of displacing and displaced fluids 242

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

19.

SANviK

AND MAERKER

Xanthan

Gum

for

Oil

Recovery

243

may be v a r i e d b y c h a n g i n g f l u i d v i s c o s i t i e s . The e f f e c t o f m o b i l i t y r a t i o on displacement e f f i c i e n c y i s i l l u s t r a t e d by t h e H e l e - S h a w m o d e l r e s u l t s d e p i c t e d i n F i g u r e 2. The m o d e l s a r e i n i t i a l l y o i l f i l l e d , and water i s i n j e c t e d i n t h e lower l e f t corner. When t h e m o b i l i t y o f d i s p l a c i n g w a t e r ( u n s h a d e d ) e q u a l s the m o b i l i t y o f d i s p l a c e d o i l (shaded), about t h r e e - f o u r t h s o f the o i l i s produced before water a r r i v e s a t t h e p r o d u c t i o n w e l l (Figure 2a). A f t e r water breakthrough, production increases i n water content w i t h continued throughput. At a favorable v i s c o s i t y r a t i o o f 0.03, a b o u t n i n e - t e n t h s o f t h e o i l i s p r o d u c e d before water breakthrough ( F i g u r e 2b). An u n f a v o r a b l e v i s c o s i t y r a t i o o f 30 c a u s e s a n o b v i o u s l y u n s t a b l e d i s p l a c e m e n t t o o c c u r , and o n l y a b o u t o n e - t h i r d o f t h e r e s i d e n t o i l i s p r o d u c e d a t w a t e r breakthrough (Figure 2c) These e x p e r i m e n t s i l l u s t r a t as f a v o r a b l e a m o b i l i t y r a t i o a s p o s s i b l e d u r i n g d i s p l a c e m e n t s . The d i s p l a c e m e n t i l l u s t r a t e d i n F i g u r e 2c c a n be made t o l o o k l i k e t h e o n e i n F i g u r e 2a b y i n c r e a s i n g t h e w a t e r - p h a s e v i s c o s i t y t h i r t y f o l d , and t h i s would s u b s t a n t i a l l y improve o i l r e c o v e r y . S i m i l a r r e s u l t s c a n be e x p e c t e d i n a c t u a l r e s e r v o i r s i t u a t i o n s . C o n s e q u e n t l y , m o b i l i t y c o n t r o l polymers a r e used i n r e c o v e r y p r o c e s s e s t o r e d u c e t h e m o b i l i t y o f i n j e c t e d w a t e r and i n c r e a s e process e f f i c i e n c y . Two b a s i c t y p e s o f p o l y m e r s — x a n t h a n gums a n d p a r t i a l l y hydrolyzed p o l y a c r y l a m i d e s — c o n s t i t u t e the large majority of those c u r r e n t l y used i n enhanced r e c o v e r y . At present, polya c r y l a m i d e s s t r o n g l y dominate polymer w a t e r f l o o d i n g a p p l i c a t i o n s , w h i l e x a n t h a n gums p l a y a v e r y m i n o r r o l e . Substantial increases i n x a n t h a n gum u s e c a n be e x p e c t e d , h o w e v e r , a s m i c e l l a r - p o l y m e r p r o c e s s e s a r e f u r t h e r t e s t e d and then a p p l i e d i n l a r g e r - s c a l e applications. T h i s paper i s devoted p r i m a r i l y t o a comparison o f t h e p e r f o r m a n c e f o r t h e s e two p o l y m e r t y p e s i n l a b o r a t o r y e v a l u a t i o n s and a c t u a l r e s e r v o i r use. H o p e f u l l y , t h i s comparison w i l l serve t o p i n p o i n t s p e c i f i c a s s e t s o r l i a b i l i t i e s and p r o v i d e g u i d e l i n e s f o r needed i m p r o v e m e n t s . Mobility

Reduction

As m e n t i o n e d e a r l i e r , d i l u t e p o l y m e r s o l u t i o n s w o r k i n two ways t o r e d u c e w a t e r m o b i l i t y i n p o r o u s m e d i a : 1) b y i n c r e a s i n g v i s c o s i t y a n d 2) b y d e c r e a s i n g p e r m e a b i l i t y . D i f f e r e n t p o l y m e r s depend o n t h e s e two mechanisms i n v a r y i n g d e g r e e s . However, b o t h mechanisms a r e i n f l u e n c e d b y m o l e c u l a r w e i g h t , m o l e c u l a r w e i g h t d i s t r i b u t i o n , s a l i n i t y , f l o w r a t e and p e r m e a b i l i t y . I n t h e c o n c e n t r a t i o n range u s u a l l y c o n s i d e r e d f o r enhanced o i l r e c o v e r y a p p l i c a t i o n s — 200 t o 1500 ppm — a n d i n w a t e r s a l i n i t i e s n o r m a l l y e n c o u n t e r e d i n r e s e r v o i r s , X a n t h a n gums g e n e r a l l y e x h i b i t h i g h e r v i s c o s i t y and a lower s e n s i t i v i t y o f v i s c o s i t y t o s a l i n i t y changes t h a n p a r t i a l l y h y d r o l y z e d p o l y a c r y l a m i d e s . F i g u r e 3 shows v i s c o s i t y - c o n c e n t r a t i o n b e h a v i o r f o r s e v e r a l

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

EXTRACELLULAR MICROBIAL POLYSACCHARIDES

PRESSURE GRADIENT,

= EFFECTIVE ROCK PERMEABILITY EFFECTIVE FLUID VISCOSITY

=

FLOW RATE/UNIT AREA PRESSURE GRADIENT

Figure

A-VISCOSITY RATIO OF 1.

- FAVORABLE VISCOSITY RATIO OF 0.03 (STABLE).

AC-

UNFAVORABLE VISCOSITY RATIO OF 30 (UNSTABLE).

Canadian Journal of Chemical Engineering

Figure 2.

Disphcements

in HeleShaw cosity ratios (1)

model at different vis-

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

19.

SANViK

A N D MAERKER

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Gum

for Oil

Recovery

245

polymers i n two b r i n e s : 1% NaCl and a s y n t h e t i c r e s e r v o i r b r i n e c o n t a i n i n g calcium and magnesium. Each xanthan sample represents a d i f f e r e n t commercial source, and, although they d i f f e r s i g n i f i c a n t l y from each other i n v i s c o s i t y , each e x h i b i t s l i t t l e s a l t s e n s i t i v i t y . The polyacrylamide, on the other hand, gives s i g n i f i c a n t v i s c o s i t y d i f f e r e n c e s f o r the two b r i n e s used. Many i n v e s t i g a t o r s (2-20) have observed p e r m e a b i l i t y reduct i o n with polyacrylamide s o l u t i o n s by f l u s h i n g a polymerflooded sandstone core o r sand pack with b r i n e and comparing the f l u s h e d , b r i n e m o b i l i t y with that o f b r i n e p r i o r to polymer. The r a t i o of i n i t i a l b r i n e m o b i l i t y t o b r i n e m o b i l i t y a f t e r i n j e c t i o n of a polymer bank has been c a l l e d a r e s i d u a l r e s i s t a n c e f a c t o r , but t h i s f a c t o r i s not always a good measure of p e r m e a b i l i t y reduct i o n during polymer flo responsible for permeabilit mer molecules on main-flow-channel w a l l s which reduces c r o s s s e c t i o n a l area a v a i l a b l e f o r flow, and 2) entrapment of polymer molecules i n narrow pore c o n s t r i c t i o n s which p a r t i a l l y s h u t s - o f f a p o r t i o n of the interconnected pore network. Interested readers may gain an a p p r e c i a t i o n f o r each mechanism by comparing the works o f Thomas (19) and Domingues and W i l l h i t e (20). The degree of p e r m e a b i l i t y r e d u c t i o n v a r i e s i n v e r s e l y with o r i g i n a l b r i n e p e r m e a b i l i t y (2-4_, 10, 21). This r e l a t i o n s h i p i s r e a d i l y understandable by r e c o g n i z i n g that an adsorbed polymer molecule of given s i z e w i l l cause a greater percentage r e d u c t i o n of c r o s s s e c t i o n a l area i n a small diameter pore (lower p e r m e a b i l i t y ) than i n a l a r g e r pore (higher p e r m e a b i l i t y ) . Xanthan gum s o l u t i o n s cause very l i t t l e r e d u c t i o n of permea b i l i t y i n porous media (4, 19). As a r e s u l t , m o b i l i t y c o n t r o l design f o r a secondary (polymer waterflood) or t e r t i a r y (micellar-polymer flood) o i l recovery process i s s i m p l i f i e d ( 7 ) , but the very r e a l advantage o f continued i n j e c t i o n a t a reduced m o b i l i t y i s l o s t f o r b r i n e i n j e c t e d behind a xanthan gum polymer bank. F i g u r e 4 compares r e s i s t a n c e f a c t o r s as a f u n c t i o n of throughput i n one-foot Berea sandstone cores f o r a 600-ppm p o l y acrylamide s o l u t i o n and a 750-ppm xanthan gum s o l u t i o n . Under these t e s t c o n d i t i o n s , steady-state m o b i l i t y r e d u c t i o n during polymer flow and the r e s i d u a l r e s i s t a n c e f a c t o r ( p e r m e a b i l i t y reduction) a f t e r b r i n e flow a r e l a r g e r f o r the polyacrylamide, even though i t has l e s s v i s c o s i t y . However, i t must be noted t h a t , because of the p r e v i o u s l y mentioned dependence o f permea b i l i t y r e d u c t i o n by polyacrylamides on i n i t i a l b r i n e permeabili t y , much l e s s m o b i l i t y r e d u c t i o n would be expected i f the polyacrylamide t e s t of F i g u r e 4 had been conducted i n 500-md, r a t h e r than 100-md, sandstone. In the case of xanthan gum, l i t t l e change i n m o b i l i t y r e d u c t i o n would be expected with changes i n i n i t i a l b r i n e p e r m e a b i l i t y .

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

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0

2

4

6

8

10

12

14

16

PORE V O L U M E S INJECTED

Figure 4.

Mobility reductions in Berea sandstone cores

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

18

19.

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Gum for Oil

Recovery

247

Retention

Polymer r e t e n t i o n i n porous r o c k d e l a y s polymer bank a r r i v a l a t a p r o d u c i n g w e l l and i n c r e a s e s t h e q u a n t i t y o f p o l y mer r e q u i r e d t o p r o v i d e m o b i l i t y c o n t r o l t h r o u g h o u t a n o i l r e s e r voir. C o n s e q u e n t l y , t h i s l o s s o f p o l y m e r t o t h e f o r m a t i o n must not be e x c e s s i v e . The two mechanisms m e n t i o n e d a b o v e f o r perme a b i l i t y r e d u c t i o n — c h e m i c a l a d s o r p t i o n and p h y s i c a l e n t r a p m e n t — a r e a l s o two ways i n w h i c h p o l y m e r m o l e c u l e s a r e removed f r o m s o l u t i o n when f l o w i n g t h r o u g h p o r o u s m e d i a . A l t h o u g h p o l y m e r r e t e n t i o n and p e r m e a b i l i t y r e d u c t i o n a r e d e f i n i t e l y i n t e r r e l a t e d , no o n e has y e t d e t e r m i n e d t h e s e p a r a t e c o n t r i b u t i o n s f r o m a d s o r p t i o n and p h y s i c a l entrapment f o r a g i v e n polymer-rock system. S e v e r a l w o r k e r s ( 6 , 10, 19_, 20^,) h a v e p u b l i s h e d d a t a i n d i c a t i n g t h a t p h y s i c a l entrapmen p o l y a c r y l a m i d e s i n water-we P o l y a c r y l a m i d e s c a n e a s i l y l o s e 300 t o 4 0 0 pounds p e r a c r e - f o o t i n c o n s o l i d a t e d sandstone. As w i t h p e r m e a b i l i t y r e d u c t i o n d i s c u s s e d e a r l i e r , t h i s l o s s i s a l s o a n i n v e r s e f u n c t i o n o f perme a b i l i t y ( 2 1 ) . X a n t h a n gums e x h i b i t l e s s r e t e n t i o n — o n t h e o r d e r o f 1 5 0 t o 300 pounds p e r a c r e - f o o t . F u r t h e r w o r k i s n e c e s s a r y t o a s s e s s r e l a t i v e i m p o r t a n c e o f a d s o r p t i o n and e n t r a p m e n t f o r x a n t h a n gum s o l u t i o n s . I n a c c e s s i b l e P o r e Volume I t has b e e n shown ( 1 9 , 22) t h a t m o l e c u l a r s i z e f o r p o l y m e r s o f i n t e r e s t h e r e c a n e x c e e d t h e d i a m e t e r s o f some o f t h e s m a l l e r pores i n n a t u r a l porous media (16). This i m p l i e s that a p o r t i o n of the i n t e r c o n n e c t e d pore volume i s i n a c c e s s i b l e t o polymer molecules. When p o l y m e r s o l u t i o n s f l o w t h r o u g h p o r o u s m e d i a , t h e r e s u l t i s a n a c c e l e r a t i o n o f polymer through l a r g e r pores r e l a t i v e t o s i m u l t a n e o u s l y i n j e c t e d s o l v e n t ( 2 3 ) , i n a manner r e m i n i s c e n t o f g e l permeation chromatography. T h i s e f f e c t i s i l l u s t r a t e d b y F i g u r e 5, w h i c h shows e f f l u e n t c o n c e n t r a t i o n response t o a p u l s e o f p o l y a c r y l a m i d e and a t r a c e r i n j e c t e d s i m u l t a n e o u s l y i n t o a sandstone c o r e t h a t had p r e v i o u s l y been contacted w i t h a h i g h e r polymer c o n c e n t r a t i o n t o s a t i s f y r e t e n tion. The p o l y m e r p u l s e , t h e r e f o r e , i s n o t d e l a y e d b y r e t e n t i o n and b r e a k s t h r o u g h a b o u t 2 2 % o f a p o r e v o l u m e e a r l y b e c a u s e 2 2 % of the pore space i s i n a c c e s s i b l e t o polymer molecules. I n a c c e s s i b l e p o r e v o l u m e does n o t r e q u i r e p o r e c o n s t r i c t i o n s t o o s m a l l f o r p o l y m e r m o l e c u l e s t o p a s s ; Thomas (19) h a s shown that b r i d g i n g o f adsorbed molecules i n c o n s t a n t - r a d i u s c a p i l l a r i e s w i t h diameters l e s s than f o u r times the average molecular d i a m e t e r can l e a d t o r e d i r e c t i o n o f subsequent polymer i n t o l a r g e r flow channels. P o l y m e r may a l s o b e a c c e l e r a t e d r e l a t i v e t o i t s s o l v e n t b y a mechanism t e r m e d h y d r o d y n a m i c c h r o m a t o g r a p h y (24) w h e r e b y t h e mean v e l o c i t y o f a p a r t i c l e i n f l o w i n g f l u i d i s a r e f l e c t i o n o f the pore v e l o c i t y p r o f i l e . Because o f the s i z e

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EXTRACELLULAR MICROBIAL POLYSACCHARIDES

of polymer molecules, t h e i r c e n t e r s a r e excluded from t h e slowest s t r e a m l i n e s c l o s e s t t o p o r e w a l l s , a n d t h e y move f a s t e r t h a n t h e average s o l v e n t flow r a t e . Transient Flow

Behavior

S e v e r a l i n v e s t i g a t o r s ( 9 , 20, 2 5 ) , have observed t h a t , i n porous media, a s t e a d y - s t a t e e q u i l i b r i u m e x i s t s between r e t a i n e d and f l o w i n g p o l y m e r . I n t e r r u p t i o n s o r c h a n g e s i n f l o w r a t e c a n perturb t h i s steady s t a t e , r e s u l t i n g i n t r a n s i e n t s i n both r e t e n t i o n and s o l u t i o n c o n c e n t r a t i o n . Figure 6 i l l u s t r a t e s this b e h a v i o r f o r x a n t h a n gum. E f f l u e n t c o n c e n t r a t i o n a n d m o b i l i t y r e d u c t i o n ( r e s i s t a n c e f a c t o r ) a r e p l o t t e d v e r s u s pore volumes i n j e c t e d f o r a 500-ppm x a n t h a solutio with 2 NaCl i n a 6 - i n c h , 121-md B e r e r u p t e d f o r 16 h o u r s a n pressur drop This r e s u l t e d i n sharp increases i n both e f f l u e n t c o n c e n t r a t i o n and t h e d e g r e e o f m o b i l i t y r e d u c t i o n r e l a t i v e t o p r e v i o u s s t e a d y s t a t e c o n d i t i o n s . T h i s r e s u l t may be e x p l a i n e d w i t h t h e same m e c h a n i s t i c c o n s i d e r a t i o n s t r e a t e d e a r l i e r — t h a t i s , under a p o s i t i v e p r e s s u r e g r a d i e n t p o l y m e r m o l e c u l e s become p a c k e d i n t o p o r e c a v i t i e s t h a t have downstream o u t l e t s so c o n s t r i c t e d t h a t molecules cannot pass through. This contributes t o permeability reduction. C e s s a t i o n o f f l o w e l i m i n a t e s hydrodynamic drag and p e r m i t s t h e m o l e c u l e s t o assume r e l a x e d c o n f i g u r a t i o n s . M o l e c u l a r d i f f u s i o n i s then able t o reduce t h e c o n c e n t r a t i o n gra d i e n t s e x i s t i n g between c a v i t i e s w i t h r e s t r i c t e d f l o w and main channels. When f l o w i s r e s u m e d , t h e i n c r e a s e d c o n c e n t r a t i o n o f f l o w i n g polymer i n c r e a s e s v i s c o s i t y and, hence, a l s o i n c r e a s e s m o b i l i t y r e d u c t i o n . P e r m e a b i l i t y may a l s o i n c r e a s e , b u t e v i d e n t l y t h i s i s overwhelmed b y t h e a t t e n d a n t i n c r e a s e i n v i s c o s ity. S u b s e q u e n t l y , polymer t r a p p i n g r e c u r s and d e c r e a s e s t h e e f f l u e n t c o n c e n t r a t i o n below t h e i n j e c t e d value. This, i n turn, l o w e r s i n s i t u s o l u t i o n v i s c o s i t y a n d m o b i l i t y r e d u c t i o n . When a l l t r a p p i n g s i t e s a r e once a g a i n s a t u r a t e d , t h e system r e t u r n s to i t s i n i t i a l steady s t a t e . P o s i t i o n s Β a n d C i n F i g u r e 6 i n d i c a t e where p r e s s u r e d r o p a c r o s s t h e c o r e was i n c r e a s e d w i t h o u t i n t e r r u p t i n g t h e f l o w . I n these cases a d d i t i o n a l polymer i s immediately r e t a i n e d , l o w e r i n g b o t h e f f l u e n t c o n c e n t r a t i o n a n d t h e amount o f m o b i l i t y r e d u c t i o n . The m i n i m a a n d a s y m p t o t i c a p p r o a c h e s t o s t e a d y s t a t e w i t h c o n t i n u e d i n j e c t i o n occur as b e f o r e , b u t a lower e q u i l i b r i u m m o b i l i t y r e d u c t i o n r e s u l t s f o r each i n c r e a s e i n p r e s s u r e drop. This i s a t t r i b u t e d t o lower polymer s o l u t i o n v i s c o s i t i e s a t h i g h e r shear r a t e s ( p s e u d o p l a s t i c , non-Newtonian b e h a v i o r ) . Here a g a i n , t h e i n c r e m e n t a l r e d u c t i o n o f p e r m e a b i l i t y a s s o c i a t e d w i t h a d d i t i o n a l polymer r e t e n t i o n , w h i c h opposes t h e e f f e c t o f v i s c o s i t y on m o b i l i t y r e d u c t i o n , i s d o m i n a t e d b y t h e v i s c o s i t y contribution to mobility reduction. Comparing t h e b e h a v i o r o u t l i n e d above f o r x a n t h a n s o l u t i o n s

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In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

EXTRACELLULAR MICROBIAL POLYSACCHARIDES

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w i t h s i m i l a r t r a n s i e n t experiments f o r polyacrylamide s o l u t i o n s (20) again shows a b a s i c d i f f e r e n c e i n porous media behavior, which may be a t t r i b u t e d to molecular conformation d i f f e r e n c e s . Resumption of polyacrylamide s o l u t i o n flow a f t e r a short i n t e r r u p t i o n r e s u l t s i n a m o b i l i t y r e d u c t i o n decrease. T h i s occurs, apparently, because the e f f e c t of increased p e r m e a b i l i t y r e s u l t ing from d i s l o d g i n g molecules that had been trapped i n pore cons t r i c t i o n s i s greater than the simultaneous v i s c o s i t y i n c r e a s e a t t r i b u t a b l e to a higher flowing c o n c e n t r a t i o n . Polyacrylamide s o l u t i o n s a l s o show higher steady-state m o b i l i t y r e d u c t i o n s f o l lowing i n c r e a s e s i n flow r a t e , but i t i s not c l e a r whether t h i s e f f e c t i s due mainly to reduced p e r m e a b i l i t i e s from higher r e t e n t i o n l e v e l s or higher e x t e n s i o n a l v i s c o s i t i e s r e s u l t i n g from the v i s c o e l a s t i c nature of polyacrylamide s o l u t i o n s (26) In con t r a s t , xanthan gum s o l u t i o n Mechanical

Degradation

One c r i t i c a l problem a r i s i n g from i n j e c t i o n of polymer s o l u t i o n s i n t o o i l r e s e r v o i r s i s the p o s s i b i l i t y of imposing f l u i d s t r e s s e s l a r g e enough to rupture molecules and reduce molecular weight. Because of the r a d i a l - f l o w nature of i n j e c t i o n w e l l s , f l u i d s e n t e r i n g a formation a t t y p i c a l flow r a t e s are subjected to very high f l u x e s at the sand face. These l a r g e f l u x e s and the converging-diverging nature of flow channels i n porous media cause s e c t i o n s of entangled molecules to be s t r e t c h e d very r a p i d l y , and some molecules rupture before entanglements can rearrange to r e l i e v e the s t r e s s (27). Polyacrylamide s o l u t i o n s are very s u s c e p t i b l e to t h i s mechanical degradation, while xanthan gum s o l u t i o n s are q u i t e r e s i s t a n t (7, 27, 28). F i g u r e s 7 and 8 compare shear v i s c o s i t i e s vs shear r a t e before and a f t e r high-shear flow through bead packs f o r 300-ppm s o l u t i o n s of a polyacrylamide and a xanthan gum, r e s p e c t i v e l y . The p o l y a c r y l amide s o l u t i o n shows an e i g h t f o l d v i s c o s i t y l o s s f o l l o w i n g bead pack flow, whereas the xanthan s o l u t i o n undergoes n e g l i g i b l e v i s c o s i t y l o s s a f t e r experiencing order of magnitude higher shear r a t e s i n the bead pack. I t should be pointed out that v e l o c i t y gradient i n the flow d i r e c t i o n , or s t r e t c h r a t e , appears to be a b e t t e r measure of deformation r a t e than shear r a t e f o r c o r r e l a t i o n of mechanical degradation (27). The maximum i n apparent v i s c o s i t y f o r polyacrylamide i n F i g u r e 7 i s due to i t s v i s c o e l a s t i c c h a r a c t e r , and there i s a strong c o r r e l a t i o n between v i s c o e l a s t i c s t r e s s e s and mechanical degradation (27). Injectivity

Behavior

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In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

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w a t e r a n d x a n t h a n gum s o l u t i o n s , f l o w r a t e s a n d p r e s s u r e s w e r e monitored t o determine i n j e c t i v i t i e s o f thevarious f l u i d s . I n j e c t i o n was t h r o u g h c a s i n g p e r f o r a t e d w i t h 4 s h o t s - p e r - f o o t i n t o e i t h e r o n e o r two s a n d s a t d e p t h s o f 1600 t o 1800 f e e t s u b surface. S o f t e n e d f r e s h w a t e r was u s e d f o r p o l y m e r h y d r a t i o n t o y i e l d a 6000-ppm c o n c e n t r a t e a n d f o r f u r t h e r d i l u t i o n t o 300 ppm p r i o r t o c a r t r i d g e f i l t r a t i o n and i n j e c t i o n . F i g u r e 9 shows i n j e c t i v i t y a s a f u n c t i o n o f i n j e c t e d volume. D u r i n g i n j e c t i o n o f x a n t h a n gum ( p o i n t s " L " a n d "P", F i g u r e 9 ) , s u b s t a n t i a l i n j e c t i v i t y d e c r e a s e s w e r e o b s e r v e d . Upon r e t u r n i n g t o b r i n e i n j e c t i o n ( p o i n t s M and "Q", F i g u r e 9 ) , i n j e c t i v i t y l e v e l s r e m a i n e d s i g n i f i c a n t l y below pre-polymer v a l u e s . T h i s i n d i c a t e d n e a r w e l l b o r e p l u g g i n g , w h i c h c o u l d b e removed b y two w e l l c l e a n u p procedures: e i t h e r a s i m p l e b a c k wash o r a t r e a t m e n t d e s i g n e d t o remove b a c t e r i a a n d a n d R i n F i g u r e 9. C l e a r l i n j e c t i o n o f x a n t h a n gum c a n r e s u l t i n u n d e s i r a b l e p l u g g i n g a n d injectivity loss. I n j e c t i v i t y p r o b l e m s w i t h x a n t h a n may be a t t r i b u t e d t o t h r e e p r i m a r y f a c t o r s : w a t e r q u a l i t y , polymer c o m p o s i t i o n and injection well configuration. I n g e n e r a l , water q u a l i t y r e q u i r e ments a r e more s t r i n g e n t f o r p o l y m e r t h a n f o r p l a i n w a t e r i n j e c tion. I n t h e C o a l i n g u a i n j e c t i o n t e s t , i t was c o n c l u d e d t h a t f i n e l y d i v i d e d s o l i d s i n t h e i n j e c t i o n s o u r c e w a t e r were b e i n g f l o c c u l a t e d by polymer and c o n t r i b u t e d t o p l u g g i n g problems ( 2 8 ) . In a d d i t i o n , any species present i n t h e i n j e c t i o n water that can c r o s s l i n k x a n t h a n , s u c h a s f e r r i c i r o n o r b o r a t e i o n , s h o u l d be avoided. Composition o f t h e polymer as c o m m e r c i a l l y s u p p l i e d i s another major cause o f xanthan i n j e c t i v i t y problems. Again u s i n g t h e C o a l i n g u a t e s t s a s a n e x a m p l e , i t was c o n c l u d e d ( 2 8 ) t h a t t h e xanthan c o n t a i n e d about 11 weight p e r c e n t c e l l u l a r debris. T h i s c e l l u l a r m a t e r i a l and u n h y d r a t e d polymer " g e l s " w e r e b e l i e v e d r e s p o n s i b l e f o r most o f t h e p l u g g i n g . S e v e r a l p u b l i c a t i o n s and p a t e n t s d e a l w i t h xanthan i n j e c t i v i t y problems and methods f o r improvement. T h e s e i n c l u d e t e c h n i q u e s t o f l o c c u l a t e c e l l u l a r d e b r i s onto c l a y o r other s o l i d s that enable e a s i e r removal by f i l t r a t i o n o r s e d i m e n t a t i o n (29, 3 0 ) , p r o c e d u r e s f o r d i a t o m a c e o u s e a r t h f i l t r a t i o n ( 2 8 , 2 9 , 3 0 , 31) a n d methods f o r d i s s o l v i n g p r o t i e n a c e o u s d e b r i s t h r o u g h a l k a l i n e (32) o r e n z y m a t i c (33) a c t i o n . W h i l e a l l o f t h e s e c l a r i f i c a t i o n p r o c e d u r e s p r o b a b l y do i m p r o v e p o l y m e r - s o l u t i o n p r o p e r t i e s , some q u e s t i o n e x i s t s a s t o t h e v a l i d i t y o f procedures used t o e v a l u a t e t h e degree o f i m p r o v e m e n t . B o t h o f t h e t e s t s commonly e m p l o y e d — M i l l i p o r e f i l t e r t e s t s and r o c k i n j e c t i o n t e s t s — a r e capable o f r a n k i n g polymer s o l u t i o n s on a r e l a t i v e b a s i s , b u t t e s t s r e p o r t e d i n t h e l i t e r a t u r e do n o t b e g i n t o a p p r o a c h t h e i n j e c t i o n l e v e l s e x p e r i enced i n a c t u a l w e l l s . Since the p l u g g i n g u s u a l l y observed w i t h x a n t h a n gum s o l u t i o n s i s a n e a r - s u r f a c e o r s a n d f a c e phenomenon, fl

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i t i s r e a s o n a b l e t o s c a l e i n j e c t i o n on t h e b a s i s o f Volume i n j e c t e d p e r u n i t a r e a o f s a n d f a c e , as i s commonly done i n f i l t r a t i o n s t u d i e s . T h i s means t h a t t h e t y p e o f i n j e c t i o n w e l l c o m p l e t i o n i s an i m p o r t a n t f a c t o r , s i n c e c o m p l e t i o n t y p e w i l l d e t e r m i n e sand e x p o s u r e . The f o l l o w i n g t a b l e shows i n j e c t i o n v a l u e s f o r s e v e r a l w e l l c o m p l e t i o n s commonly e m p l o y e d . Injection v a l u e s c a n r a n g e a b o u t t h r e e o r d e r s o f m a g n i t u d e d e p e n d i n g on t h e SCALED INJECTION VALUES FOR VARIOUS WELL COMPLETIONS ASSUMING 10 BBL/DAY-FT INJECTION 2 W e l l Completion 4 Collapsed Perforations/F 2 Perforations/Ft 6" Open H o l e 18" Underrearned and G r a v e l P a c k e d "Typical" Laboratory

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type of w e l l completion. The c o l l a p s e d p e r f o r a t i o n c a s e i s most u n f a v o r a b l e f r o m an i n j e c t i v i t y s t a n d p o i n t ( t h i s i s p r o b a b l y t h e s i t u a t i o n f o r the Coalingua t e s t s described e a r l i e r ) . The most f a v o r a b l e s i t u a t i o n w o u l d be one w h e r e t h e w e l l i s e n l a r g e d t h r o u g h o u t t h e r e s e r v o i r i n t e r v a l and p a c k e d w i t h c o a r s e s a n d o r g r a v e l behind a s c r e e n . A l l of these w e l l completions have s c a l e d i n j e c t i o n v a l u e s s u b s t a n t i a l l y above t h o s e g e n e r a l l y reported for laboratory tests. I n other words, p u b l i s h e d t e s t d a t a a r e s h o r t b y a f a c t o r o f up t o a t h o u s a n d o f s i m u l a t i n g e v e n one d a y ' s i n j e c t i o n a t a r a t e o f 10 b b l / d a y / f t o f i n t e r v a l . I n an e f f o r t t o i n v e s t i g a t e i n j e c t i v i t y more r e a l i s t i c a l l y , we h a v e c o n d u c t e d i n j e c t i o n t e s t s i n B e r e a c o r e s a t h i g h e r throughput l e v e l s . T e s t s were conducted i n o n e - h a l f - i n c h square by o n e - f o o t - l o n g s a n d s t o n e c o r e s f i t t e d w i t h p r e s s u r e t a p s as shown i n F i g u r e 10. F l u i d s w e r e pumped t h r o u g h e a c h c o r e a t c o n s t a n t r a t e , and m o b i l i t i e s , o r f l o w c a p a c i t i e s , o f t h e v a r i o u s s e c t i o n s w e r e d e t e r m i n e d as a f u n c t i o n o f t h r o u g h p u t . W i t h an a p p r o p r i a t e e x p e r i m e n t a l s e t u p , b r i n e c o u l d be i n j e c t e d for sustained periods without plugging (Figure 11). Injection of sheared, but u n f i l t e r e d , polymer s o l u t i o n caused r a p i d p l u g g i n g a s shown i n F i g u r e 12. F i l t r a t i o n of the polymer s o l u t i o n a f t e r s h e a r i m p r o v e d i n j e c t i v i t y ( F i g u r e 13) b u t c e r t a i n l y d i d not e l i m i n a t e plugging tendencies. R e s u l t s of these l a b o r a t o r y t e s t s may be c o m b i n e d w i t h a m o d e l f o r r a d i a l f l o w o f a n o n N e w t o n i a n f l u i d i n p o r o u s m e d i a (28) t o c a l c u l a t e r e s p o n s e o f a hypothetical injection well. F i g u r e 14 shows p r e s s u r e d r o p b e t w e e n t h e w e l l b o r e and a p o i n t 100 f t i n t o t h e f o r m a t i o n p l o t t e d a s a f u n c t i o n o f i n j e c t i o n t i m e a t 10 b b l / d a y / f t . I f a n o n - p l u g g i n g p o l y m e r s o l u t i o n i s i n j e c t e d , t h e r e w i l l be some p r e s s u r e i n c r e a s e due t o m o b i l i t y r e d u c t i o n i n t h e g r o w i n g p o l y mer b a n k . T h i s i s t h e d e s i r e d p r e s s u r e r e s p o n s e . I n j e c t i o n of

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

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a x a n t h a n gum s o l u t i o n s h e a r e d a n d f i l t e r e d t o 5 m i c r o n s w i l l g i v e one o f t h e upper c u r v e s depending on w e l l c o m p l e t i o n . A g a i n , t h i s c a l c u l a t i o n i s based on l a b o r a t o r y d a t a , b u t i t i s reasonably c o n s i s t e n t w i t h f i e l d experience. Clearly, injectionw e l l p l u g g i n g c a n b e e x p e c t e d f o r some f i e l d s i t u a t i o n s , a n d t h e r e i s s u b s t a n t i a l room f o r improvement i n t h e x a n t h a n p o l y m e r s c u r r e n t l y i n use. Compatibility I n j e c t i v i t y p r o b l e m s e x p e r i e n c e d w i t h x a n t h a n gums c a n b e regarded a s one form o f c o m p a t i b i l i t y p r o b l e m — s o m e t h i n g i n t h e p o l y m e r s o l u t i o n i s t o o b i g t o e n t e r t h e f o r m a t i o n . O t h e r comp a t i b i l i t y c o n s i d e r a t i o n whe p o l y m e r d i enhanced recovery processes i n c l u d e s e n s i t i v i t y t o degradatio a c t i o n , a n d , i n some c a s e s , i n t e r a c t i o n b e t w e e n p o l y m e r a n d micellar fluids. N a t i v e f o r m a t i o n b r i n e s v a r y i n s a l i n i t y from p o t a b l e f r e s h water t o g r e a t e r than 20% d i s s o l v e d i n o r g a n i c s o l i d s . F i g u r e 15 shows how i n t r i n s i c v i s c o s i t y o f a p a r t i c u l a r p a r t i a l l y h y d r o l y z e d p o l y a c r y l a m i d e v a r i e s w i t h b r i n e c o n c e n t r a t i o n and composition. I n s o d i u m c h l o r i d e a l o n e , b e h a v i o r i s t y p i c a l o f most "flexible" polyelectrolytes, i.e. intrinsic viscosity varies l i n e a r l y w i t h i o n i c s t r e n g t h t o t h e m i n u s o n e - h a l f power ( 3 4 ) . Greater i n t r i n s i c v i s c o s i t y r e d u c t i o n s a r e observed w i t h c a l c i u m c o n t a i n i n g b r i n e s — p r o b a b l y due t o c r o s s l i n k i n g . I n g e n e r a l , as water s a l i n i t y i n c r e a s e s , polyacrylamide e f f e c t i v e n e s s decreases due t o d e c r e a s e s i n v i s c o s i t y a n d p e r m e a b i l i t y r e d u c t i o n ( 2 , and a n i n c r e a s e i n m e c h a n i c a l d e g r a d a t i o n ( 2 7 ) . T h e r e i s obvious i n c e n t i v e f o r using t h elowest s a l i n i t y i n j e c t i o n water f e a s i b l e f o r any s p e c i f i c a p p l i c a t i o n . X a n t h a n shows l i t t l e change i n f l u i d p r o p e r t i e s w i t h s a l i n i t y ; t h i s a p p a r e n t l y i s due t o t h e more r i g i d n a t u r e o f t h e h y d r a t e d m o l e c u l e . Most c u r r e n t a p p l i c a t i o n s o f polymers a r e i n r e s e r v o i r s h a v i n g t e m p e r a t u r e s o f 160°F (^70°C) o r b e l o w . I n t h i s range, t h e r m a l d e g r a d a t i o n i s n o t c o n s i d e r e d t o be a s e r i o u s p r o b l e m . I t i s a n t i c i p a t e d t h a t f u t u r e a p p l i c a t i o n s w i l l be a t t e m p e r a t u r e s up t o a t l e a s t 250°F (^125°C). A t some p o i n t , t h e r m a l d e g r a d a t i o n w i l l be a concern f o r both p o l y a c r y l a m i d e s and xanthan, b u t p u b l i s h e d work has n o t y e t c l e a r l y d e f i n e d t h i s point. S u b s t a n t i a l c h e m i c a l d e g r a d a t i o n a t r e l a t i v e l y l o w temperat u r e s h a s been observed w i t h p o l y a c r y l a m i d e s under c e r t a i n conditions. I f b o t h d i s s o l v e d oxygen and redox c a t a l y s t s — f o r example, d i s s o l v e d i r o n — a r e p r e s e n t , r a p i d d e g r a d a t i o n occurs (35, 3 6 ) . G e n e r a l l y t h i s i s a v o i d e d i n l a b o r a t o r y work by e x c l u d i n g m e t a l from t h e e x p e r i m e n t a l system. I n f i e l d u s e , oxygen i s scavenged from i n j e c t i o n water b e f o r e polymer a d d i t i o n . F i g u r e 16 shows t h e e f f e c t o f s o d i u m h y d r o s u l f i t e a d d i t i o n o n

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

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In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

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polymer q u a l i t y . Slow b u t s u b s t a n t i a l d e g r a d a t i o n occurs w i t h d i s s o l v e d o x y g e n p r e s e n t ; much l e s s o c c u r s when o x y g e n i s s c a v enged b y h y d r o s u l f i t e . I n t h e c a s e o f x a n t h a n gum, c h e m i c a l d e g r a d a t i o n i s n o t c o n s i d e r e d t o be a p r o b l e m , b u t biodégradation c a n c a u s e s e v e r e v i s c o s i t y l o s s and p l u g g i n g b y m i c r o b i a l s l i m e s . Biodégradation has b e e n e l i m i n a t e d by u s e o f c h l o r o p h e n o l a t e s , f o r m a l d e h y d e , o r a d j u s t m e n t o f i n j e c t i o n w a t e r pH t o a h i g h v a l u e . Another c o m p a t i b i l i t y aspect f o r m o b i l i t y c o n t r o l polymers a r i s e s t h r o u g h i n t e r a c t i o n s b e t w e e n a p o l y m e r d r i v e bank and t h e s u r f a c t a n t s l u g employed i n m i c e l l a r - p o l y m e r p r o c e s s e s . These p r o c e s s e s depend o n t h e a b i l i t y o f i n j e c t e d s u r f a c t a n t s t o l o w e r i n t e r f a c i a l t e n s i o n between t r a p p e d r e s i d u a l o i l and t h e d i s placing fluid. Becaus f a c t a n t b a n k s c a n be u s e d t i o n s — o r microemulsion y hig t i e s ( t e n t o twenty times water v i s c o s i t y ) . I f ordinary brine were used t o d i s p l a c e a m i c r o e m u l s i o n s l u g , b r i n e would f i n g e r i n t o t h e s l u g because o f t h e u n f a v o r a b l e m o b i l i t y r a t i o , d i l u t e the s u r f a c t a n t , and render i t i n e f f e c t i v e . Consequently, i t i s important t o f o l l o w a s u r f a c t a n t slug w i t h a m o b i l i t y b u f f e r . Polymer s o l u t i o n s having m o b i l i t i e s equal t o o r l e s s than t h e s u r f a c t a n t s l u g have been t h e primary c h o i c e f o r t h i s e s s e n t i a l task. I t has been observed t h a t polymer r e t e n t i o n decreases t o n e a r l y i n s i g n i f i c a n t l e v e l s when a p o l y m e r b a n k f o l l o w s a s u r f a c t a n t s l u g ( 3 7 ) . A s a r e s u l t o f t h i s , and b e c a u s e o f p o l y m e r i n a c c e s s i b l e pore volume, polymer molecules from a m o b i l i t y b u f f e r bank c a n invade a s u r f a c t a n t s l u g . Phase b e h a v i o r s t u d i e s h a v e shown ( 3 8 ) t h a t p r e s e n c e o f x a n t h a n gum m o l e c u l e s i n a m i c r o e m u l s i o n c o n s i s t i n g o f b r i n e , I P A and a p e t r o l e u m s u l f o n a t e c a n c a u s e t h e m i x t u r e t o s e p a r a t e i n t o two p h a s e s . This has been c a l l e d s u l f o n a t e - p o l y m e r i n t e r a c t i o n . S u l f o n a t e - p o l y m e r i n t e r a c t i o n c a n be v e r y d e t r i m e n t a l t o o i l r e c o v e r y o p e r a t i o n s by c o n t r i b u t i n g t o s u r f a c t a n t l o s s . F i g u r e 17 shows a p l o t o f p r o d u c e d s u l f o n a t e , p o l y m e r and t r a c e r c o n c e n t r a t i o n s a s f r a c t i o n s o f i n j e c t e d v a l u e s v s pore volumes produced i n an 8-foot-Berea-core t e r t i a r y f l o o d . S u l f o n a t e conc e n t r a t i o n b e g i n s t o d r o p when p o l y m e r f i r s t a p p e a r s i n t h e effluent. The s h a d e d a r e a b e t w e e n t h e s u l f o n a t e and i t s t r a c e r c o n c e n t r a t i o n p r o f i l e s r e p r e s e n t s s u l f o n a t e l o s s due t o t r a p p i n g o f a second phase. S i n c e t h e s u l f o n a t e t r a c e r c o n c e n t r a t i o n does n o t drop u n t i l t h e polymer t r a c e r appears i n t h e e f f l u e n t , p o l y m e r a p p a r e n t l y h a s moved i n t o t h e s u r f a c t a n t s l u g . I n some instances, pressure gradients required to maintain r e a l i s t i c o i l f i e l d f l o w r a t e s become e x c e s s i v e . M i n i m i z i n g t h i s d e t r i m e n t a l phase b e h a v i o r by l o w e r i n g d r i v e water s a l i n i t y and i n c r e a s i n g c o s u r f a c t a n t / s u r f a c t a n t r a t i o ( 3 8 ) may r e d u c e s u l f o n a t e t r a p p i n g from s u l f o n a t e - p o l y m e r i n t e r a c t i o n and improve t e r t i a r y o i l recovery.

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Field Application P r e v i o u s d i s c u s s i o n has been concerned p r i m a r i l y w i t h l a b o r a t o r y measurements o f t h e many a s p e c t s o f m o b i l i t y - c o n t r o l polymer behavior. Because o f the v a r i e t y o f i n t e r a c t i o n s w i t h porous media and t h e complex n a t u r e o f p e t r o l e u m r e s e r v o i r s , t h e r e a l t e s t o f a polymer and a process i s a c t u a l f i e l d performance. F i g u r e 18 i l l u s t r a t e s t y p i c a l s t e p s a n d i n t e r a c t i o n s i n v o l v e d i n e v a l u a t i o n o f a proposed f i e l d p r o j e c t . F o r each polymer under c o n s i d e r a t i o n , l a b o r a t o r y measurements a r e made o f t h e v a r i o u s aspects o f polymer behavior d e s c r i b e d e a r l i e r . T h i s i s done u s i n g r o c k , w a t e r , and o i l from t h e s u b j e c t r e s e r v o i r and s p e c i f i c a l l y i n c l u d e s r e t e n t i o n and m o b i l i t y r e d u c t i o n a s a f u n c t i o n o f polymer c o n c e n t r a t i o n , rock p e r m e a b i l i t y e t c Measurements o r estimates of i n j e c t i v i t desirable. T h i s polyme a v a i l a b l e r e s e r v o i r d e s c r i p t i o n t o c o n s t r u c t a computer model o f the r e s e r v o i r and process. C a l c u l a t i o n s f r o m t h i s m o d e l may be used f o r o p t i m i z a t i o n and p r e d i c t i o n o f polymer f l o o d and w a t e r f l o o d performance. These performance e s t i m a t e s a r e then used t o p r o v i d e an economic e v a l u a t i o n o f a polymer p r o j e c t r e l a t i v e t o a n o r d i n a r y w a t e r f l o o d p r o j e c t . One way o f c o m p a r i n g b e h a v i o r i s by a p l o t o f produced w a t e r - o i l - r a t i o v s cumulative o i l production (Figure 19). E a c h p r o j e c t i s t e r m i n a t e d when t h e w a t e r - o i l - r a t i o becomes h i g h enough t h a t c o n t i n u e d o p e r a t i o n i s no l o n g e r e c o n o m i c ( i n t h i s c a s e , 9 6 % w a t e r c u t , f o r e x a m p l e ) . I t i s i n t e r e s t i n g t o n o t e t h e r e l a t i v e l y s m a l l amount o f a d d i t i o n a l o i l r e c o v e r e d b y t h e p o l y m e r f l o o d when compared t o t h e w a t e r f l o o d base case. E v e n u s i n g p o l y m e r , t h e amount o f o i l l e f t a t p r o j e c t t e r m i n a t i o n c o u l d e a s i l y b e a b o u t t h e same a s the t o t a l polymer f l o o d r e c o v e r y — a b o u t 300 b b l / A c - F t o f r e s e r voir. This remaining o i l i s then a p o t e n t i a l candidate f o r micellar-polymer flooding. The p r e c e e d i n g d i s c u s s i o n g i v e s a v e r y r o u g h e s t i m a t e o f how o n e e v a l u a t e s a p o l y m e r w a t e r f l o o d p r o j e c t . A n o b v i o u s q u e s t i o n a t t h i s p o i n t i s — " w h e r e d o e s x a n t h a n gum f i t i n ? " The a n s w e r i s t h a t x a n t h a n gum h a s p l a y e d a v e r y m i n o r r o l e . The l a r g e m a j o r i t y o f polymer w a t e r f l o o d p r o j e c t s , p a s t and p r e s e n t , a r e u s i n g some f o r m o f p o l y a c r y l a m i d e . T h i s d o e s n o t mean, h o w e v e r , t h a t x a n t h a n gum w i l l c o n t i n u e t o p l a y a m i n o r r o l e i n t h e f u t u r e . Improvements i n t h e c o m m e r c i a l p r o d u c t s h o u l d e n a b l e x a n t h a n gums t o c a p t u r e a g r e a t e r f r a c t i o n o f p o l y m e r w a t e r f l o o d i n g a p p l i c a t i o n s . On t h e o t h e r h a n d , f o r e c a s t s f o r enhanced r e c o v e r y agree t h a t polymer f l o o d i n g i s expected t o be r e l a t i v e l y m i n o r compared t o m i c e l l a r - p o l y m e r f l o o d i n g . F i g u r e 20 shows t h e d i s t r i b u t i o n o f c u r r e n t a n d p l a n n e d m i c e l l a r p o l y m e r p r o j e c t s i n t h e U.S. ( 4 0 ) . I t has n o t been p o s s i b l e t o d e t e r m i n e what p o l y m e r i s b e i n g c o n s i d e r e d o r u s e d f o r e v e r y p r o j e c t , b u t , based on the polymer chosen f o r a s i g n i f i c a n t f r a c t i o n o f t h e s e t e s t s , x a n t h a n gum w i l l b e u s e d i n

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

sANviK

A N D MAERKER

Xanthan

Gum

for Oil

Recovery

1. 1.4 PV PETROLEUM SULFONATE IN 92% 0.23N NaCI 2. 1.5 PV 700 PPM X A N T H A N G U M IN 0.05N NaCt 3. 2.0 PV 0.05N NaCI

AJ.Ch.L Meeting

Figure 17.

8-ft Berea core tertiary

flood—110°F

(38)

POLYMER FLOOD EVALUATION

RESERVOIR DESCRIPTION

LABORATORY DATA: RETENTION RESISTANCE FACTOR INJECTIVITY DEGRADATION

WATERFLOOD PERFORMANCE

POLYMER FLOOD PERFORMANCE

ECONOMIC EVALUATION

Figure 18.

Steps for evaluating a proposed field project

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

262

EXTRACELLULAR MICROBIAL POLYSACCHARIDES

Figure 20.

Micellar/surfactant

projects testing U.S. reservoirs (40)

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

19.

SANviK

A N D MAERKER

Xanthan

Gum

for Oil

Recovery

263

approximately one-third to one-half. I f t h i s r a t i o holds f o r f u t u r e f u l l - s c a l e p r o j e c t s , c o n s u m p t i o n o f x a n t h a n gum i n enhanced r e c o v e r y p r o j e c t s i s l i k e l y t o be s u b s t a n t i a l .

Literature Cited 1. 2. 3.

Mungan, Ν., Can. J . Chem. Eng. 49, 32-37 (1971). Smith, F. W., J. Pet. Tech. (Feb. 1970) 148-156. Jennings, R. R., Rogers, J . Η., and West, T. J., J . Pet. Tech. (March 1971) 391-401; Trans., AIME, 251 (1971). 4. Hirasaki, G. J. and Pope, G. Α., Soc. Pet. Eng. J . (Aug. 1974) 337-346. 5. Mungan, Ν., Smith, F. W., and Thompson, J . L., J. Pet. Tech. (Sept. 1966) 1143-1150 Trans. AIME 237 (1966) 6. Szabo, M. T., Soc Trans., AIME, 259 (1975). 7. H i l l , H. J., Brew, J . R., Claridge, E. L., Hite, J . R., and Pope, G. A . , paper SPE 4748 presented at the SPE-AIME Third Improved Oil Recovery Symposium, Tulsa, Okla. (April 22-24, 1974). 8. Chauveteau, G. and Kohler, Ν., Paper SPE 4745 presented at the SPE-AIME Improved Oil Recovery Symposium, Tulsa (April 22-24, 1974). 9. Maerker, J . M . , J. Pet. Tech. (Nov. 1973) 1307-1308. 10. Gogarty, W. B . , Soc. Pet. Eng. J . (June 1967) 161-173; Trans., AIME, 240 (1967). 11. Burcik, E. J., Prod. Monthly (June 1965) 29. 12. Burcik, E. J . and Walrond, K. W., Prod. Monthly (Sept. 1968) 12-14. 13. Ershaghi, I. and Handy, L. L., paper SPE 3683 presented at 42nd Annual California Regional Mtg. of SPE of AIME, Los Angeles (Nov. 4-5, 1971). 14. Knight, B. L., U.S. Patent 3,724,545. (April 3, 1973). 15. Norton, C. J . and Falk, D. O., U.S. Patent 3,743,018 (July 3, 1973). 16. Thakur, G. C., paper SPE 4956 prepared for the Permian Basin O i l Recovery Conference of the SPE of AIME, Midland, Texas (March 11-12, 1974). 17. Martin, F. D., Paper SPE 5100 presented at the SPE-AIME 49th Annual Fall Meeting, Houston, Texas (Oct. 6-9, 1974). 18. Sparlin, D., Paper SPE 5610 presented at the SPE-AIME 50th Annual Fall Meeting, Dallas (Sept. 28-Oct. 1, 1975). 19. Thomas, C. P . , Soc. Pet. Eng. J . (June 1976) 130-136. 20. Domingues, J . G. and Willhite, G. P., Paper SPE 5835 presented at the SPE-AIME Improved Oil Recovery Symposium, Tulsa (March 22-24, 1976). 21. Vela, S., Peaceman, D. W., and Sandvik, Ε. I., Soc. Pet. Eng. J . (April 1976) 82-96. 22. Lynch, E. J . and MacWilliams, D. C., J . Pet. Tech. (Oct. 1969) 1247-1248.

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

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23.

Dawson, R. and Lantz, R. B . , Soc. Pet. Eng. J. (Oct. 1972) 448-452; Trans., AIME, 253 (1972). 24. Small, H . , J. Colloid and Interface Science (July 1976) 147-161. 25. Rhudy, J . S., Fullinwider, J . Η., and Ver Steeg, D. J., U.S. Patent 3,734,183 (May 22, 1973). 26. Marshall, R. J . and Metzner, A. B . , Ind. and Eng. Chem. Fund., 6 (1967) 393-400. 27. Maerker, J . Μ., Soc. Pet. Eng. J . (Aug. 1975) 311-322; Trans., AIME, 261 (1975). 28. Tinker, G. Ε., Bowman, R. W., and Pope, G. Α., J . Pet. Tech. (May 1976) 586-593. 29. Lipton, D., paper SPE 5099 presented at SPE-AIME 49th Annual Fall Meeting Housto (Oct 6-9 1974) 30. Abode, Μ. Κ., U.S 31. Yost, M. E. and Stokke, O. M., J. Pet. Tech. (Oct. 1975) 1271-1272. 32. Patton, J . T., paper SPE 4670 presented at the SPE-AIME 48th Annual Fall Meeting, Las Vegas, Nev. (Sept. 30-0ct. 3, 1973). 33. Burnett, D. Β., paper SPE 5372 presented at the 45th Annual California Regional Meeting of SPE-AIME, Ventura (April 2-4, 1975). 34. Smidsrod, O. and Haug, Α., Biopolymers, 10, (1971) 1212-1227. 35. Pye, D. J., U.S. Patent 3,343,601 (Sept. 26, 1967). 36. Knight, B. L., J. Pet. Tech. (May 1973) 618-626. 37. Trushenski, S. P . , Dauben, D. L., and Parrish, D. P., Soc. Pet. Eng. J. (Dec. 1974) 633-642; Trans., AIME, 257 (1974). 38. Trushenski, S. P. paper presented at Α.I.Ch.Ε. meeting, Kansas City (April 1976). 39. Healy, R. Ν., Reed, R. L., and Stenmark, D. G., Soc. Pet. Eng. J . (June 1976) 147-160. 40. O i l and Gas J., 74, No. 14 (1976).

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

20 Production, Properties, and Application of Curdlan TOKUYA HARADA Institute of Scientific and Industrial Research, Osaka University, Yamadakami, Suita-shi, Osaka, Japan (565)

Curdlan was produced in high yield by cultures of a newly isolated and improved mutan myxogenes. Curdlan form should be a useful, new polymer not only as a food additive, but also for industrial purposes. I. Findings The history of the discovery of curdlan is interesting. In 1962, Harada and his colleagues made great efforts to obtain microorganisms which could utilize petrochemical materials. They isolated an organism from soil, capable of growing on medium containing 10% ethylene-glycol as the sole carbon source (1) and named it Alcaligenes faecalis var. myxogenes 10C3 (2, 3). They found that this organism produced a new β-glucan which contained about 10% succinic acid and named it succinoglucan (4, 5) . The structure of the polysaccharide moiety of succinoglucan (6, 7) is shown below: -->Glcl-->4Glcl-->3Glcl-->3Glcl-->6Glcl-->4Glcl-->3Glcl-->3Glcl-->4Glcl-->3Gall-->4Glcl--> During investigations on the production of succinoglucan, one day they found that the culture medium did not become viscous and no succinoglucan was formed, but almost all the added glucose was consumed. They thought that some special compound(s), must have been produced instead of succinoglucan in the culture. So they examined the product and found that it was a neutral polysaccharide (8, 9). They named it curdlan in 1966 (10). Curdlan is composed of β-l,3-glucosidic linkages. A mutant strain 10C3K was isolated from the stock culture 10C3, which produced only curdlan. Strain 10C3K is a spontaneous mutant and it has stable ability to produce the exocellular polysaccharide whereas the ability of strain 10C3 is unstable(11). Thus, by chance, they succeeded in obtaining a suitable organism for pro duction of curdlan. Later Takeda Chemical Industries Ltd.isolated 265

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

EXTRACELLULAR MICROBIAL POLYSACCHARIDES

266

a u r a c i l - l e s s mutant o f s t r a i n 10C3K named s t r a i n * 13140 as a b e t t e r gel-forming B-l,3-glucan producer (12). The polymer from the s t r a i n , designated as p o l y s a c c h a r i d e 13140, i s a k i n d o f curdlan i n a broad sense or a curdlan type p o l y s a c c h a r i d e . II.

Mutation

The d e t e c t i o n o f c u r d l a n using A n i l i n e Blue was t e s t e d using s t r a i n 10C3 and i t s mutant s t r a i n s 10C3k and 22 as shown i n Figure 1.(13). The c u l t u r e medium used i n t h i s p l a t e , c o n s i s t e d o f 1% glucose, 0.5% yeast e x t r a c t , 0.005% A n i l i n e Blue and 2% agar. The middle colony i s t h a t o f 10C3. The s u r r o u d i n g - c l e a r zone i s due to the formation of succinoglucan which i s a s o l u b l e , viscous polymer. Succinoglucan doe t s t a i with A n i l i n Blue Curdla can form a complex wit i n t e r a c t i o n o f the polymer y Nakanishi and h i s colleagues to be p r o p o r t i o n a l to t h e i r concent r a t i o n s and degrees o f p o l y m e r i z a t i o n (14). The l e f t colony i s that o f a spontaneous mutant o f the parent s t r a i n which produces only curdlan. The complex o f the polymer with the dye can e a s i l y be s t r i p p e d o f f . The remaining c e l l s do not s t a i n with the dye. The r i g h t colony i s that o f mutant s t r a i n 22, d e r i v e d from 10C3 by treatment with N-methyl-N -nitro-N-nitrosoguanidine . This s t r a i n produces only succinoglucan. Mutation o f s t r a i n 10C3 to s t r a i n s s t a i n i n g with A n i l i n e Blue was a l s o induced by treatment with mutagens such as NTG, and ethylmethane-sulfonate and u l t r a v i o l e t l i g h t , but not by t r e a t ment with Mitomycin C, ethidium bromide or A c r i d i n e Orange which are reagents causing e l i m i n a t i o n of plasmids (Table 1) (11). Experiments on t r a n s f e r of genes concerned with production o f succinoglucan and(or) curdlan between d i f f e r e n t mutant s t r a i n s have not been s u c c e s s f u l . Thus, a plasmid may not be d i r e c t l y i n v o l v e d i n the production o f the p o l y s a c c h a r i d e s . f

III.

Structure

Curdlan i s composed o f 3-1,3-glucosidic linkages ([ot] +18° IN NaOH) (10, 15). S a i t o and h i s colleagues (15) i n d i c a t e d the presence of two i n t e r n a l 6 - l , 6 - g l u c o s i d i c linkages i n o r i g i n a l curdlan (DPn 455) while Ebata (16) detected one p a r t of g e n t i b i o s e to 360 p a r t s of glucose i n the hydrolyzate o f the glucan by the a c t i o n o f exo-B-l,3-glucanase, although Nakanishi and h i s colleagues could not detect any other g l u c o s i d i c linkages besides 6-1,3-glucosidic linkages i n p o l y s a c c h a r i d e 13140 (12). C e l l u l o s e f i b e r , which i s composed o f 6 - l , 4 - g l u c o s i d i c l i n k a g e s , does not s w e l l i n the presence o f water whereas curdlan swells i n water and can form a r e s i l i e n t g e l on heating. T h i s i s an important and i n t e r e s t i n g f a c t . C a l l o s e and pachyman are g-1,3-glucans which are l a r g e l y composed o f g - l , 3 - g l u c o s i d i c l i n k a g e s . C a l l o s e cont a i n s a l i t t l e g l u c u r o n i c a c i d (17). Pachyman has other glucoD

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

20.

Curdlan

HARADA

267

Table 1 E f f e c t s o f Mutagens on Mutation o f S t r a i n 10C3 with A n i l i n e Blue (11) ~~ Mutagen

ConcenGrowth tration inhibition (per ml) (%)

None

R a t

io w

h

to S t r a i n s S t a i n i n g

o f blue c o l o n i e s i

t

e

c

o

l

o

n

i

e

s

( % )

1.0

X

10"

1.4

X

10"

7

f

N-Methyl-N nitro-N-nitrosoguanidine

- 3

30 jig

Ethylmethanesulfonate 5-Bromouracil

25

3 &

2

Q

1.0

X

10"

7

&

Ultraviolet light irradiation

2 0 X

10"

3

s i d i c linkages and does not form a r e s i l i e n t gel on h e a t i n g (15). Cal l o s e has been found i n a v a r i e t y o f l o c a t i o n s i n the tissues" o f h i g h e r p l a n t s , such as i n s i e v e tubes, young t r a c h e i d e s , p o l l e n , root h a i r s , stem h a i r s and root endodermis. No other p o l y saccharides besides curdlan composed e n t i r e l y o f g - l , 3 - g l u c o s i d i c linkages have yet been found. IV.

Production

Now i t has become p o s s i b l e to o b t a i n h e a t - g e l a b l e g-l,3-glucan e a s i l y from glucose and many carbon compounds. The y i e l d o f the polymer from added glucose i s about 50%. About 5 g o f curdlan can be produced from 10 g o f glucose i n 100 ml of simple defined medium, i f the pH i s maintained at n e u t r a l i t y (_9 , 12_, 18) · Curdlan can a l s o be produced using a c e l l suspension i n medium c o n t a i n i n g only glucose and calcium carbonate (19). A p i l o t p l a n t f o r p r o d u c t i o n o f p o l y s a c c h a r i d e 13140 has been accomplished i n Takeda Chemical I n d u s t r i e s L t d . Nakanishi and h i s colleagues examined the occurrence of curdlan type polysaccharides i n microorganisms, u s i n g the A n i l i n e Blue method (Table 2) (13). Four s t r a i n s o f Agrobacterium r a d i o b a c t e r , one strain~o"f Agrobacterium rhizogenes and a s t r a i n o f Agrobacterium sp. were found to produce curdlan type p o l y saccharides with water s o l u b l e β-glucans (11, 13). Spontaneous mutant s t r a i n s which produce p r i n c i p a l l y curdIan-type p o l y saccharides i n high y i e l d were a l s o induced from the r e s p e c t i v e parent s t r a i n s .

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

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Table 2 Curdlan Type Polysaccharides (Curdlan i n a Broad Sense) Alcaligenes faecalis var. myxogenes 10C3K Alcaligenes faecalis var. myxogenes IFO 13140

r

uirttian

Polysaccharide 10C3K

Polysaccharide 13140

Agrobacterium radiobacter IFO IFO IFO IFO

12607 12665 13127 13256

Polysaccharide 12607 Polysaccharide 12665

Agrobacterium rhizogenes IFO 13259 Agrobacterium sp. IFO 13660

Polysaccharide 13259 Polysaccharide 13660

The structure of the polysaccharide moiety of a water soluble polymer from strain A. radiobacter IFO 12665 seems to be like that of succinoglucan because a specific 3-glucanase, succinoglucan depolymerase from Flavobacterium sp. M64 (20), can attack the polymer to release oligosaccharide with similar Rf value to that of the product released from succinoglucan by the enzyme (21). Succinic acid may be not contained i n the polymer. V.

Rheology

Excretion of curdlan as microfibrils from the cells of strain 10C3K, i s seen by electron microscopy (Figure 2). When a 2% suspension of curdlan i s heated, i t becomes clear at about 54°C and gel forms at higher temperature (22). Agar gel i s formed when the sol of agar obtained by heating i t s suspension i s cooled. This i s a difference between curdlan and agar. Figure 3 i s a photograph of the gel of curdlan obtained by heating a 2% suspension at 90°C. The gel of curdlan i s very elastic and r e s i l i e n t and does not break, whereas agar gel breaks when i t i s pressed between the fingers. The gel as seen i n Figure 4 i s easy to make using curdlan but i t i s not easy to make such gels using agar. It has also been found that curdlan forms a gel when an alkaline solution i s dialyzed i n a cellophan bag(S.0kamoto unpublished), when an aqueous solution of 0.2 - 0.63 M dimethylsulfoxide i s cooled (23)or when calcium ions are added to a weakly alkaline solution (H. Kimura, unpublished). Maeda and his colleagues investigated the effect of temperature on gel formation using a curdmeter. They heated 3% sus-

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

HARADA

Curdlan

Journal of General and Applied Microbiology

Figure 1. Photograph of colonies of strains (left to right) 10C3K, 10C3, and 22 grown on glucose-yeast extract medium containing water-soluble aniline blue (0.005% ) (13)

Figure 2.

Curdlan excreted from the cells of 10C3k as microfibrils, negatively stained with uranyl acetate

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

EXTRACELLULAR MICROBIAL POLYSACCHARIDES

Figure 3.

Photograph of curdlan gel. Aqueous suspension (2%) of this polymer was heated at90°C for 10 min.

Figure 4. Photograph of curdlan gel. Aqueous suspension (2%) of this polymer was heated in special vessel at90°C for a few min.

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

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pensions of curdlan f o r 10 min and then measured the strength o f the r e s u l t i n g gels at 30°C (Figure 5). T h i s curve i s c u r i o u s : the g e l strength i s about the same between 60°C and 80°C and then i t increases from 80°C. The g e l strength depends on the temperature but i s independent o f the i n c u b a t i o n time at 70°C (22). Urea breaks hydrogen bonds and i t s e f f e c t on g e l formation was i n v e s t i g a t e d using a Shimadzu Microviscograph (Figure 6). The s t a r t i n g temperature f o r g e l formation decreased with increase i n the concentration o f urea added. I t i s i n t e r e s t i n g that f o r mation o f g e l i n the second stage was not observed with above 5 M urea. Thus, g e l formation i n the f i r s t stage seems t o r e q u i r e the breakage o f hydrogen bonds whereas that i n the second stage does not. I t i s a l s o i n t e r e s t i n g that the v i s c o s i t y increased markedly from 39°C to 20°C with 2 t 8 M whe th temperatur decreased. The formatio formation of hydrogen bonds E t h y l e n e - g l y c o l a c c e l e r a t e s formation o f hydrogen bonds and i t s e f f e c t on g e l formation was examined i n the same way. The r e s u l t s i n Figure 7 show that i t a l s o decreased the s t a r t i n g temperature f o r g e l formation. However, i n the presence o f a high concentration (5 M to 7 M) o f e t h y l e n e - g l y c o l , no g e l was formed. These r e s u l t s suggest that at the s t a r t i n g temperature f o r g e l formation some o r a l l the hydrogen bonds must be broken. The formation o f g e l o f the polymer was i n v e s t i g a t e d using a Rotovisca Viscometer (Haake) by the members o f Takeda Chemical I n d u s t r i e s Ltd.(24). The s p e c i f i c v i s c o s i t i e s were determined continuously as tEe temperature was r a i s e d t o 60°C and then decreased (Figure 8). From 54°C to 60°C s w e l l i n g occurred due to breakage o f hydrogen bonds. On c o o l i n g the g e l t o about 40°C, the v i s c o s i t y r a p i d l y increased and low-set gel was obtained (25). The e f f e c t o f temperature on transmittance was examined under tïïe same conditions (Figure 9). The transmittance increased on h e a t i n g to 60°C and decreased on c o o l i n g from 60°C (25). Figure 10 shows that the s p e c i f i c v i s c o s i t y a l s o i n c r e a s e d to some extent on c o o l i n g from 85°C with formation o f h i g h - s e t g e l (22, 24). As shown i n Figure 11, the transmittance decreased g r a d u a l l y with increase i n temperature from 60°C to 100°C (25). T h i s was probably due to formation o f hydrophobic bonds during formation o f cross l i n k s . Acetone powders were prepared from gels formed by heating at 60°C, 70°C and 90°C. The two formers formed s i m i l a r gels t o that d e r i v e d from the o r i g i n a l polymer, but a powder from g e l heated at 90°C d i d not. T h i s i n d i c a t e s that gels obtained a f t e r the second stage o f g e l formation have a d i f f e r e n t molecular arrangement from that o f the o r i g i n a l polymer. VI.

Conformation

Figure 12 shows some r e s u l t s o f Ogawa and h i s colleagues. They s t u d i e d the conformational behavior o f polysaccharide 13140 i n a l k a l i n e s o l u t i o n by measuring the o p t i c a l r o t a t o r y d i s p e r s i o n ,

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

EXTRACELLULAR MICROBIAL

50

POLYSACCHARIDES

60 70 80 9 0 100 Heating temperature V

Agricultural and Biological Chemistry

Figure 5. Effect of heating temperature on gel strength of curdhn(22)

Temperature

Figure 6. Effects of urea (0-8 M) on gel formation of curdlan (1% ). Shimazu microviscograph type SN1 was used.

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

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Temperature

Figure 7. Effects of ethylene-glycol (0-7 M) on gel formation of

Figure 8. Effect of heating temperature on specific viscosity of polysaccharide 13140(1%)

Temperature

o| 0

, 30

, , 40 50 Temperature (°C)

1 60

Figure 9. Effect of heating temperature transmittance of polysaccharide 13140 (1%)

o

n

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EXTRACELLULAR MICROBIAL POLYSACCHARIDES

>.10

Journal of Food Science

Figure 10. Ε feet of heating temperature on specific viscosity of polysaccharide 13140(1%) (24)

20

40 60 Temperature (°C)

\

/

\ \

ol

(1%)

8

_jι

/

<.—Polysaccharide

"

ιι

40

Potato stach

/

_ —

Figure 11. Effect of heating temperature on transmittance on polysaccharide 13140

80

—

rellnlnse

ιι

60 80 Temperature ( °C )

.

,40

Concentration of NaOH

Ν

Carbohydrate Research

Figure 12. Dependence of optical rotation [a] oo intrinsic viscosity [η] and extinction angle χ of a polysaccharide 13140 solution on concentration of sodium hydroxide. The value of χ is obtained in 0.5 g/100 ml glucan solution at the rate of shear of 6000 sec' and 30°C (26). 3

1

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

1

100

20.

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vicosity and flow birefringence (26). A l l these characters were found to change greatly i n about 0.2JN sodium hydroxide. Thus, they proposed that at low concentrations of sodium hydroxide, this polymer has an ordered conformation, whereas at higher sodium hydroxide concentrations, i t consists of random coils and that the transition of conformation occurs i n about 0.2 N^odium hydroxide. Saito and Sasaki confirmed this proposal, using C NMR as shown in Figure 13 (27). Increase IrT the salt concentration causes a conformational transition of the glucan from random coils to an ordered structure when the concentration of a l k a l i i s below 0,3 Ν (28) . Change i n conformation of curdlan i n a solution of dimethyl-sulfoxide to a r i g i d ordered structure occurred on addition of nonsolvents, such as 2-chloroethanol, dioxane or water (29) Ogawa and his colleagues also showed degree of polymerizatio glucan hydroxid (Figure 14) (30). The optical rotation of the soluble fractions and that of the original glucans are practically constant, whereas that of the insoluble fractions increased with DPn. Thus, they concluded from their results that the glucan takes an ordered form at low concentration of a l k a l i when the DPn of the glucan i s above 25. The content of the ordered form increases with DPn u n t i l i t reaches a maximum value and becomes constant at DPn values of about 200. This may be the lower limit of DPn for gel formation in neutral media. Formation of a complex of curdlan with Congo Red was studied by Ogawa and his colleagues (31) and by Ogawa and Hatano (32) by measuring the circular dichroism spectra i n the visible region. This results suggested that there may be two kinds of binding systems i n alkaline media. The supramolecular structures of glucans with different degrees of polymerization were compared by electron microscopy using heated^and unheated samples (Figure 15) (33). Microfibrils of 100 - 200 A width composed of many elementary f i b r i l s , were observed i n the original and depolymerized glucan (DPn 400 and 260) and the insoluble fraction of higher molecular weight (DPn 140), but no microfibrils were detectable i n the insoluble fraction of low molecular weight (DPn 36) or the soluble fraction (DPn 13). The microfibrils of the original glucan were very much longer than those of the insoluble glucan. Thus, only glucans with higher degrees of polymerization can form a gel when heated, form a complex with Aniline Blue or Congo Red, show higher optical rotations and form microfibrils seen by electron microscopy. No significant difference was observed between heated and unheated preparations. Jeisma and Kreger reported that 6-1,3-glucan from rhizomorph of Armiliana mellea also showed an X-ray fiber pattern and did not form a r e s i l i e n t gel on heat treatment (34J. Three types of water seem to be involved i n the gel; one i s in the structure of elementary f i b r i l s , another between the ele mentary f i b r i l s and the third between the microfibrils. The existence of three types of water i n this gel was proposed by

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276

EXTRACELLULAR MICROBIAL POLYSACCHARIDES

20 PPM

Abstract of the Annual Meeting of Japanese Biochemical Society

Figure 13.

C Ν MR spectra in water suspension and alkaline solution (90° pulse, repetition time 0.6 sec) of polysaccharide 13140 (27) 13

Carbohydrate Research

Figure 14. Dependence of the spe cific rotation of polysaccharide 13140 at 439 nm on the degree of polymeri zation in 0.1 M sodium hydroxide at 30°C. (O) gel-forming β-glucans; (€)) insoluble fractions; (Φ) soluble fractions (30).

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Figure 15. β-Ιβ-Glucan (polysaccharide 131401 microfibrils negatively stained with uranyl acetate, (a, b, c, d) original glucan (DPn 400); (d, e) depolymerized glucan (DPn 260); (f, g) depolymerized glucan (DPn 140). b, d, j, and h were heated at 95°C for 10 min; a, c, e, and g were not (33).

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

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EXTRACELLULAR MICROBIAL POLYSACCHARIDES

Suzuki and Aizawa from NMR analyses (35). The X-ray d i f f r a c t i o n p a t t e r n s o T " t h i s polymer were analyzed by Takeda and h i s colleagues (Figure 16) (36). The presence o f water i n the p r e p a r a t i o n i s r e q u i r e d f o r X-ray a n a l y s i s . I t i s noteworthy that the center of the d i f f r a c t i o n p a t t e r n has a c r o s s l i k e appearance, suggesting that the molecule has a r a t h e r simple h e l i c a l s t r u c t u r e . S a i t o and Sasaki have succeeded t o observe broad carbon-13 resonance peaks with l i n e - w i d t h , C.A. 150 H ( C l - C5) and H (C6) i n g e l o f curdlan (27). The p r o f i l s of carbon-13 N^R i n the g e l o f curdlan ancT i n the s o l u t i o n o f degraded curdlan (DPn 13) were compared. I t i s i n t e r e s t i n g that the carbon-13 peaks o f CI and C3 i n g e l , s h i f t e d downfield. These s h i f t s could be explained by a p r e f e r r e d rotamer p o p u l a t i o n around the 3-1,3-glucosidic l i n k a g e s Thei studie als d th presence o f a s i n g l e h e l i VII.

Application

Table

The p o t e n t i a l uses of curdlan i n food products 3. Table 3 A p p l i c a t i o n i n Food Function

are shown i n

Products Food

Food m a t e r i a l G e l l i n g agent

J e l l y , j e l l y - l i k e food, custard and dry mixes

Slimming a d d i t i v e (non-caloric material)

D i e t e t i c and d i a b e t i c foods

F i l m and f i b e r former

E d i b l e f i l m s and

sherbet,

fibers

Food a d d i t i v e Improvement o f viscoelasticity

Spaghetti and

Binding agent

Hamburgers and starchy

Water-holding

agent

Sausages, ham

noodles jelly

and starchy j e l l y

Masking malodors or aromas

Boiled rice

Retention o f shape

Starchy j e l l y and dry dessert mixes

Thickener and

Salad dressings and

stabilizer

spreads

The polymer seems to be u s e f u l f o r g e l l i n g m a t e r i a l s , f o r j e l l y products and as a food a d d i t i v e f o r improving the q u a l i t y o f

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

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Figure 16. X-ray diffraction analysis of polysaccharide 13140 (wide-angle x-ray diffraction patterns) (36)

Β

PS 13K0(RE5IUENT G a )

C-2

C-6

A- FRACTION I IC-1

C-3

100

no

C-A

90

80

70

60 PPKTMS) 50

Abstract of the Annual Meeting of Japanese Biochemical Society

Figure 17.

C

13

NMR spectra in gel of polysaccharide 13140 and in solution of the degraded polymer (DPn 13) (27)

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

EXTRACELLULAR MICROBIAL POLYSACCHARIDES 12 / Agar

Polysaccharide 13140 -—

ni υ

0

" Gelatin

1 I1 II Iι L· 4 8 12 16 Breaking strength (dynes/cm )10" 2

5

Figure 18. ing strength and elastic modulus of various gels. Samples of gels were sliced into cylindrically shaped pieces 23 mm thick and then measured with Autograph model IM-100 (Shimazu) (24).

Figure 19.

Gel of polysaccharide 13140

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

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various foods. The p o l y m e r c a n b e added d u r i n g t h e p r o d u c t i o n p r o c e s s b e f o r e h e a t i n g e i t h e r as a p o w d e r , o r as a s u s p e n s i o n o r s l u r r y i n w a t e r o r aqueous a l c o h o l . V a r i o u s k i n d s o f t e s t s showed t h a t t h i s p o l y m e r i s s a f e . The g e l o f c u r d l a n h a s p r o p e r t i e s i n t e r m e d i a t e between the b r i t t l e n e s o f a g a r g e l and t h e e l a s t i c i t y o f g e l a t i n ( F i g u r e 18) ( 2 4 ) . T h i s g e l was shown t o a d s o r b t a n n i n (38). I n a d d i t i o n , n u t r i t i o n s t u d i e s on t h i s p o l y m e r showed t h a t i t h a s no c a l o r y v a l u e . Thus i t i s u s e f u l as a n i n g r e d i e n t o f l o w - c a l o r i c foods. F o r e x a m p l e , a g e l s u c h as shown i n F i g u r e 19 c a n be e a s i l y made u s i n g t h i s p o l y m e r . I t seems t o h a v e many p o t e n t i a l i n d u s t r i a l u s e s as a f i l m , f i b e r o r s u p p o r t f o r i m m o b i l i z e d enzymes, as s e e n i n T a b l e 4. Applicatio Application

Function

Characteristic

Film or fiber

Material

Transparent, e d i b l e , i n s o l u b l e i n h o t water and i m p e r m e a b l e t o oxygen

Tobacco

products

Binding agent

C o m b u s t i b l e and i n s o l u b l e

A r t i f i c i a l feeds f o r f i s h e s and s i l k worms

Gelling agent

Good t e x t u r e

Molecular sieve Carrier and s u p p o r t f o r i m m o b i l i z i n g enzymes

F i l m s and f i b e r s o f t h i s p o l y m e r a r e e a s i l y p r e p a r e d and have t h e c h a r a c t e r i s t i c s o f being e d i b l e , i n s o l u b l e i n water, biodegradible and i m p e r m e a b l e t o o x y g e n . Beads a n d f i b e r s o f t h e p o l y m e r a r e good s u p p o r t s f o r i m m o b i l i z e d enzymes ( 3 9 , 4 0 ) . E x t e n s i v e s t u d i e s on v a r i o u s a s p e c t s o f p o l y s a c c h a r i d e p r o d u c t i o n a r e r e q u i r e d . T h e s e may c l a r i f y t h e r o l e o f p o l y s a c c h a r i d e s i n n a t u r e a n d s o i n d i c a t e t h e v a l u e s o f t h e s e compounds f o r human l i f e . E x t e n s i v e s t u d i e s have b e e n made i n many l a b o r a t o r i e s i n J a p a n on b i o l o g i c a l , o r g a n i c and p h y s i c a l c h e m i c a l 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 o f c u r d l a n a n d a l s o on i t s u s e i n f o o d p r o d u c t s and m e d i c i n e s a n d i n i n d u s t r i a l c h e m i s t r y .

Literature Cited 1.

Harada, T. and Yoshimura, T. J . Ferment. Technol. (1964) 42, 615.

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POLYSACCHARIDES

2. Harada, T. and Yoshimura, T. Biochim. Biophys. Acta. (1964) 83, 374. 3. Harada, T., Yoshimura, T., Hidaka, H. and Koreeda, A. Agr. Biol. Chem. (1965) 29, 757. 4. Harada, T. Arch. Biochem. Biophys. (1965) 112, 65. 5. Harada, T. and Yoshimura, T. Agr. Biol. Chem. (1965) 29, 1027. 6. Misaki, Α., Saito, H., Ito, T. and Harada, T. Biochemistry (1969) 8, 4645. 7. Saito, H., Misaki, A. and Harada, T. Agr. Biol. Chem. (1970) 34, 1683. 8. Harada, T., Masada, Μ., Hidaka, H. and Takada, M. J . Ferment. Technol.(1966) 44, 20. 9. Harada, T., Masada, M., Fujimori, K. and Maeda, I. Agr. Biol. Chem. (1966) 30, 196 10. Harada, T., Misaki (1968) 124, 292. 11. Hisamatsu, Μ., Amemura, Α., Harada, T., Nakanishi, I. and Kimura, K. Abstract of the Annual Meeting of Agr. Chem. Soc. Japan (1976) p289. 12. Nakanishi, I., Kanamaru, T., Kimura, K., Matsukura, Α., Asai, M., Suzuki, T. and Yamotodani, S. 284th Meeting of Kansai Branch of Agr. Chem. Soc. Japan, Osaka (1972). 13. Nakanishi, I., Kimura, K., Suzuki, T., Ishikawa, Μ., Banno, I., Sakane, T. and Harada, T. J . Gen. Appl. Microbiol. (1976) 22, 1. 14. Nakanishi, I., Kimura, K., Kusui, S. and Yamazaki, E. Carbohyd. Res. (1974) 32, 47. 15. Saito, H., Misaki, A. and Harada, T. Agr. Biol. Chem. (1968) 32, 1261. 16. Ebata, J . Abstract of 8th International Symposium on Carbohyd. Chem., Kyoto (1976) p112. 17. Aspinall, G.O.and Kessler, G. Chem. Ind. (London) (1957) 1296. 18. Harada, T., Fujimori, K., Hirose, S. and Masada, M. Agr. Biol. Chem. (1966) 30, 764. 19. Harada, T., Fujimori, K. and Masada, M. J. Ferment. Technol. (1967) 45, 145. 20. Amemura, Α., Moori, K. and Harada, T. Biochim. Biophys. Acta. (1974) 334, 398. 21. Harada, T., Yamauchi, Η., Hisamatsu, M., Ott, I., Nakanishi, I. and Kimura, K. Abstract of the Annual Meeting of Agr. Chem. Soc. Japan (1976) p288. 22. Maeda, I., Saito, Η., Masada, Μ., Misaki, A. and Harada, T. Agr. Biol. Chem. (1967)31,1184. 23. Aizawa, M., Takahashi, M. and Suzuki, S. Chem. Letters (1974) 193. 24. Kimura, H., Moritaka, S. andMisaki,M.J . Food Sciences (1973) 38, 668. 25. Konno, Α., Azeti, Y. and Kimura, H. Abstract of the Annual Meeting of Agr. Chem. Soc. Japan (1974) p310.

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

20.

26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40.

HARADA

Curdlan

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Ogawa, Κ., Watanabe, T., Tsurugi, J . and Ono, S. Carbohyd. Res. (1972) 23, 399. Saito, H. and Sasaki, T. Abstact of the Annual Meeting of Japanese BiochemicalSoc.,(1976)p651. Ogawa, K., Tsurugi, J . and Watanabe, T. Chem. Letters (1973) 95. Ogawa, K., Miyagi, M., Fukumoto, T. and Watanabe, T. Chem. Letters (1973) 943. Ogawa, Κ., Tsurugi, J . and Watanabe, T. Carbohyd. Res. (1973) 29, 397. Ogawa, K., Tsurugi, J . and Watanabe, T. Chem. Letters (1972) 689. Ogawa, K. and Hatano, M. 288th Meeting of Kansai Branch of Agr. Chem. Soc. Japan Osaka (1974) Koreeda, Α., Harada Carbohyd. Res. (1974) , Jeisma, J . and Kreger, D. R. Carbohyd. Res. (1975) 43, 200. Suzuki, S. and Aizawa, M. Abstract of 8th International Symposium on Carbohyd. Chem., Kyoto (1976) p76. Takeda, H., Yasuoka, W. Kasai, N. and Harada T. 283rd Meeting of Kansai Branch of Agr. Chem. Soc. Japan (1973). Nakagawa, T., Moritaka, S. and Kimura, H. Abstract of the Annual Meeting of Agr. Chem. Soc. Japan (1973) p196. Nakabayashi, T. Nihon Shokuhin Kogyo Kaishi (1974) 21, 341. Takahashi, K., Yamazaki, Y., Kato, Κ and Takahashi, T. Abstract of the Annual Meeting of Agr. Chem. Soc. Japan (1976) p401. Murooka, Y., Yamada, T. and Harada, T. Annual Meeting of Ferment. Technol. Japan, Osaka (1976).

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

21 Dextrans and Pullulans: Industrially Significant α-D-Glucans ALLENE JEANES Northern Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture, Peoria, IL 61604 This symposium on extracellular microbial polysaccharides of practical importance consideration of the α-D-glucans The significance of dextran in man's practical affairs was apparent before i t s origin, identity, and name were established. Dextrans develop naturally in sucrose-containing solutions that have become inoculated with dextran-producing bacteria from a i r , plants, or s o i l . The resulting transformation of the solutions to syrupy, viscous, or ropey fluids, or even to gelled masses, doubtlessly has plagued man since the inception of accumulating and storing sucrose-containing foods and beverages. As early as 1813 (1,*2*) , r eports described the mysterious thickening or solidification of cane and beet sugar juices, and later impediment of filtration and crystallization was traced to the occurrence of this condition. In 1861, Louis Pasteur (3) initiated systematic scientific progress by explaining that these "viscous fermentations" resulted from microbial action. In 1878, van Tieghem (4) named the causative bacteria Leuconostoc mesenteroides because i t s growth in colorless flocs resembled that of the green algae of the genus Nostoc. In 1880 (5), Scheibler.established this type of product as a glucan having positive optical rotation and named it dextran. Thus, through the importance of the dextran class of α -D-glucans in man's economy, the dextrans were the f i r s t extracellular microbial polysaccharides to come under systematic scientific investigation. Dextrans from several bacterial strains also were the f i r s t extracellular microbial poly saccharides to be produced and used industrially. A comprehensive review (6) was published i n 1966 on dextran production, 1/

^References other than reviews, which c i t e o r i g i n a l research publications not included here, are marked with an asterisk. 284

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21.

JEANES

Dextrans

and

Pullulans

285

structure, properties, uses, and related considerations. The same topics were reviewed from a different viewpoint i n 1973 (7). The biosynthesis and structure of dextrans was reviewed comprehensively i n 1974 as w e l l as the s t r u c t u r a l l y dependent s p e c i f i c interactions with immunoglobulins (antibodies) and globulins such as concanavalin A (8). An extensive bibliography on a l l s c i e n t i f i c aspects of dextran (exclusive of c l i n i c a l research and testing) and dextran derivatives includes information from 1861 through mid-1976 (9). Summarized here i s the current status of dextran as an established product of world commerce and i n r e l a t i o n to s p e c i f i c industries. Interest i n p u l l u l a n and i t s p r a c t i c a l p o t e n t i a l i t i e s have developed since 195 (10) f i r s t characterize product from Aureobasidium (Pullularia) pullulans and named i t accordingly^ The polysaccharide had been isolated previously and p a r t i a l l y characterized i n studies of micro organisms responsible f o r breakdown of forest l i t t e r (11). The slime-forming black yeastlike fungus, A. pullulans, occurs ubiquitously i n organic waste matter which i t decomposes i n s o i l , r i v e r s , paper-mill effluents, and sewage (12). TTie microorganism has adverse economic importance because of i t s costly deterioration of paint, discoloration of lumber, and attack on plants and plant products (13). In none of these natural occurrences, however, does the polysaccharide seem to have a role except as a slimy nuisance. Already of applied p r a c t i c a l l y , however, i s the enzyme pullulanase (pullulan 6-glucanohydrolase EC 3.2.1.41) which was discovered by Bender and Wallenfels i n Aerobacter aerogenes (14) and shown to depolymerize p u l l u l a n to i t s repeating unit maltotriose by s p e c i f i c attack on the i n t e r u n i t α-1,6-linkages. Pullulanase, now obtainable i n p r a c t i c a l amounts from numerous microbial sources, also cleaves α-1,6-linkages i n starch and i s used i n d u s t r i a l l y to release the unit chains i n starch (15,16). Substrates other than p u l l u l a n , however, may be use3~for producing pullulanase. The production, properties, and potential uses of p u l l u l a n have been reviewed (12). Summarized here are the constitutional bases for p r a c t i c a l applications and the uses that have been proposed. Importance of Naturally Occurring Dextrans The p r e d i l e c t i o n of Leuconostocs for sucrose i n nature has a s p e c i f i c basis. Sucrose induces i n these bacteria formation of the dextran-synthesizing enzyme dextransucrase (sucrose: 1,6-a-g-glucan 6-a-glucosyltransferase, E.C. 2.4.1.5). This enzyme accomplishes dextran synthesis by

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transferase action without need for intermediate substrates. Fructose, the byproduct of dextran synthesis, i s metabolized by Leuconostocs which cannot, however, metabolize either sucrose (they have neither invertase nor sucrose phosphorylase) or dextran. Extracellular dextransucrase i s produced abundantly by many s t r a i n s , although the dextransucrase of some strains i s cell-bound. The rate and extent of a c t i v i t y on sucrose that may result i s i l l u s t r a t e d dramatically by an h i s t o r i c report of the fortuitous conversion of 5000 l i t e r s of molasses to a compact gel mass i n 12 hours (4). In 1972, the status of the situation was tKat, "although i t i s d i f f i c u l t to quantify the effects of polysaccharides on the economics of sugar cane processing, i t i s obvious from the volume of recent l i t e r a t u r e that importance i s attached to t h e i r eliminatio dextrans have a major rol and polysaccharides of plant o r i g i n are involved also (17). The long-known adverse effects of dextran continue i n p o l a r i z a t i o n measurements, c l a r i f i c a t i o n and f i l t r a t i o n , and i n reducing the rate and efficiency of c r y s t a l l i z a t i o n . In addition, traces of dextran cause i n f e r i o r c r y s t a l structure by elongating the c axis (18,19). The beet sugar industry i s less affected by dextran contamination. Sucrose i s less exposed to infection during harvesting and f i r s t stages of processing of beets than of cane. Very sensitive biochemical tests have demonstrated the extent of dextran contamination i n commercial sucrose, including that distributed as a standard of highest purity. N e i l l , Hehre, and coworkers (20) demonstrated serological a c t i v i t y indicative of dextran i n both cane and beet sugars from diverse geographical sources and various methods of manufacture. The majority of the cane products showed higher serological a c t i v i t y than did the majority of the beet products. The weakest a c t i v i t i e s were i n several sanples of reagent grade sucrose of German o r i g i n prepared from beet sugar. Gibbons and Fitzgerald (21), u t i l i z i n g the agglutinizing action of dextran on c e l l s oF"Streptococcus mut ans, also demonstrated dextran i n reagent-grade sucrose. Dextranases are being investigated (22) and used (23) for removal of dextran from cane sugar juices as w e l l as from sucrose solutions and wines made hazy by the presence of dextran. Constitutional Basis f o r P r a c t i c a l Importance Dextran. By d e f i n i t i o n , the generic name dextran applies to a large class of α-D-glucans i n which predominance of a-l,6-linkages i s the common feature. One of the simplest dextrans known i s that from Leuconostoc mesenteroides NRRL B-512(F); the structural features are shown i n Figure 1.

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The α-y-glucopyranosidic linkages are 95% 1,6-and 5% 1,3(24). The 1,3-linkages are points of attachment of side drains of which about 85% are 1 or 2 glucose residues i n length (25). The remaining 15% of the side chains may have an average length of 33% glucose residues and may not be uniformly distributed i n the macromolecule (26). This dextran i s readily soluble i n water; certain other dextrans may be insoluble. In dextrans from other strains, the non1,6-linkages may be 1,2-, 1,3-, or 1,4-. Only one type may occur i n a dextran, or there may be two or three. Great d i v e r s i t y i s thus created. Dextran available i n the United States and western Europe i s produced from sucrose by s t r a i n NRRL B-512(F). Dextrans of apparently similar structure, but from different s t r a i n s are produced i n Japan (27) and Russia (28). Dextrans produce and Asia are from selecte (29*) Dextran having t h i s structure (Figure 1) was selected for production because the fraction (R^ 75,000 +_ 25,000) prepared from i t f o r intravenous administration (blood volume expander) was substantially less antigenic as compared with that from dextrans having higher percentages of non1,6-linkages. Dextran from s t r a i n NRRL B-512(F) i s completely metabolized i n man (30) when either ingested or administered parenterally as a fraction of suitable molecular size and size d i s t r i b u t i o n . Deri vat i z a t ion, however, slows or i n h i b i t s metabolism. An additional asset of s t r a i n NRRL B-512(F) i s i t s copious formation of dextransucrase (31). Production of dextran by use o f c e l l - f r e e culture f i l t r a t e s rather than i n growing cultures results i n enhanced y i e l d , quality and ease of p u r i f i c a t i o n o f the product. And furthermore, by suitable adjustment o f conditions, the major product can be synthesized d i r e c t l y within a chosen molecular weight range (32, 9). The native dextran may have weight-average molecular weight ( F y values (33) (light scattering) of 35-50 X 10 . The structural s i m p l i c i t y of this dextran permits graded p a r t i a l depolymerization and separation into fractions of any desired î% and size d i s t r i b u t i o n , which d i f f e r primarily i n molecular weight. The content of branch points remaining, however, would depend on the method of p a r t i a l depolymerization; i t i s decreased by acid hydrolysis (34) but not by use of endo-acting dextranases (1,6-a-g-glucan 6-glucanohydrolase, E.C. 3.2.1.11). Fractions o f lower molecular weight obtained through such enzymolysis r e t a i n the branch points, and t h e i r structural details would be determined by the action pattern of the s p e c i f i c dextranase (35). The series of fractions produced from p a r t i a l depolymerizates i s unique. Selected fractions or derivatives o f them serve pharmaceutical or other purposes having s p e c i f i c requirements f o r molecular

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size i n order to achieve physiological compatibility or other special objectives. Production of such fractions by d i r e c t , controlled enzymatic synthesis i s not known to be i n use. The high proportion of 1,6-linkages i n dextran NRRL B512(F) confers unusual f l e x i b i l i t y on the chain and leaves numerous s i t e s for substitution, essentially a l l of which are i n secondary positions. The r a t i o of r e l a t i v e rate constants established f o r methylation of the hydroxyl groups, C :C :C^:8:1:3.5 (36) indicates also the r e l a t i v e r e a c t i v i t y towards other substituents such as the sulfate (37). The frequent occurrence o f three hydroxyl groups i n consecutive positions i n the glucopyranosidic residues of dextrans would appear to account f o r t h e i r unusual a b i l i t y to complex with large amounts o f metalli calcium. Such complexe importan pharmaceutica preparations and i n certain metallurgical processes. Thus, the charcteristics of dextran from s t r a i n NRRL B512(F) that determine i t s value i n p r a c t i c a l applications, reside i n i t s composition as a soluble α-D-glucan and i n the properties o f i t s primary structure. In contrast, i t has been emphasized i n t h i s symposium that the unique characteristics of the anionic heteropolysaccharide xanthan which are basic to i t s usefulness, result from secondary and t e r t i a r y structural effects (58,39,40). The s p e c i f i c role of ionic charge, which also may Be" i n f l u e n t i a l i n xanthan properties, has not been established but may be inferred from research on ionogenic derivatives o f dextran (41). Pullulan. The generic name pullulan i s applied to any extracellular α-g-glucan elaborated by A. pullulans from a variety of substrates. A commonly observed feature i s the predominant repeat unit maltotriose polymerized l i n e a r l y through 1,6-linkages (Figure 2). Frequently present also are α-maltotetraose units (42,43,44) contained mainly w i t h i n the polymer chain (43J m amounts of 6.61 (43) and 5-7% (44). In products in~which possible heterogeneity was not excluded, traces of other neutral sugars and uronic acids have been reported (12). Products from other strains and from other genera and species have shown variation on the basic pullulan pattern such as the presence of 1,5linked glucosyl residues (45,46). Thus, "there i s , perhaps, no unique structure of pulTATlin (45). The molecular weight of a pullulan product d i f f e r s with the lengtja o f fermentation time (47,48). Molecular weight of 2 X 10 developed ^ n i t i a l l y during limited fermentation decreased to 1.5 X 10 during continued fermentation (47). The s i t e of degradation i s the i n t e r n a l l y located maltotetraose u n i t s ; the degradative enzyme appears to be an "endoamylase" produced during culture growth (45^47). The modified 2

3

H

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p u l l u l a n resulting from "endoamylase" action i s inert to aamylase (43). Such uncontrolled v a r i a t i o n i n molecular weight can be eliminated, however, by choice of s t r a i n and adjustment of the pH and of the phosphate content of the culture medium (49). Pullulan products having molecular weights as high as 2ζΰ X 10 or as low as 5 X 10 may be obtained i n t h i s way. The mechanism of biosynthesis of pullulan discourages consideration of enzymatic synthesis as a means f o r production. Synthesis i s accomplished through mediation of sugar nucleotide/ l i p o i d c a r r i e r intermediates associated with c e l l membrane fractions (50). The pullulan molecule may be considered as a chain of amy lose, the l i n e a r componen bond replaces every t h i r f l e x i b i l i t y , and the interrruption of regularity results i n making pullulan readily soluble, eliminating rétrogradation and improving rather than impairing f i b e r - and film-forming a b i l i t y . The presence of the 1,6-bonds may influence the p o s i t i o n of substituents and properties of derivatives by introducing a different sequence of free hydroxyl groups. The presence of 1,6-linkages, spaced as they are, prevents attack by salivary and i n t e s t i n a l amylases (43). Isoamylase from Pseudomonas sp., which cleaves a-l,6-boncEs i n amylopectin and glycogen, also i s inert on pullulan (51). Dextran and Dextran Derivatives i n Industry Pharmaceutical Industry. Probably the largest outlet for dextran and dextran derivatives i s through the pharmaceutical and fine chemicals industries. The major developments have originated from fundamental research i n Sweden which was i n i t i a t e d about 1944 and has continued consistently (52,53). Research and development have followed, however, i n numerous other countries throughout the world which produce t h e i r own pharmaceutical products from dextran (9,27,28,29*). Two dextran fractions of major significance are used i n suitably prepared solutions f o r parenteral administration ((^,9). The fraction of M 70,000 i s used to restore and maintain blood volume i n treatment of shock, hemorrhage, extensive burns,_and a variety of other physiological conditions. The f r a c t i o n of M 40,000 i s used to improve flow i n c a p i l l a r i e s , treatment of vascular occlusion, a r t i f i c i a l extracorporeal perfusion of organs, and i n a variety of other ways. These and other sharply cut dextran fractions are used for preparation of numerous derivatives such as the sulfates, diethylaminoethyl (DEAE) dextran, and complexes with iron and other metallic elements. These substances serve a variety of purposes (£,54,55). Dextran sulfates have anticoagulant, antilipemic, and antiulcer a c t i v i t y . They w

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are used i n l i q u i d two-phase s e p a r a t i o n and c o n c e n t r a t i o n o f l i v i n g c e l l s such as those o f v i r u s e s , b l o o d , tumors, and other t i s s u e s . DEAE d e x t r a n enhances b i o l o g i c a l e f f e c t s o f macromolecules and v a c c i n e s . A s o l u b l e complex o f dextran and i r o n i s produced w i d e l y i n numerous c o u n t r i e s f o r i n t r a muscular a d m i n i s t r a t i o n t o a l l e v i a t e i r o n - d e f i c i e n c y anemia i n the human and i n domestic animals. The s o l u t i o n contains 5% i r o n and 20% dextran o f M 5,000 (56). The i r o n i s m a i n l y n o n i o n i c (56) and appears t o be B-FeOOH (57). The i n i t i a l patents Ç5ÏÏ) have been emulated e x t e n s i v e l y ( £ ) . A s o l u b l e c a l c i u m complex c o n t a i n i n g 10-121 c a l c i u m i s administered p a r e n t e r a l l y t o a l l e v i a t e hypocalcemia o f c a t t l e d e l i v e r y p a r e s i s (9). Complexes w i t h antimony and a r s e n i c are e f f e c t i v e a g a i n s t t r o p i c a l i n f e c t i o n s (9). C r o s s l i n k e d dextra employed i n p u r i f i c a t i o n , f r a c t i o n a t i o n and i s o l a t i o n o f enzymes, hormones, and o t h e r s e n s i t i v e b i o l o g i c a l substances w i t h o u t m o d i f i c a t i o n o f t h e i r a c t i v i t y . By covalent bonding t o e i t h e r dextran o r c r o s s l i n k e d dextran g e l s , enzymes, immunoglobulins, and antigens are s t a b i l i z e d and supported f o r use i n s p e c i f i c r e a c t i o n s ( 5 9 , 9 ) . Dextranases, prepared by growth o f v a r i o u s molds on d e x t r a n s , are used i n mouthwashes and toothpaste t o e i t h e r d i s p e r s e o r i n h i b i t f o r m a t i o n o f d e n t o - b a c t e r i a l plaques which c o n t a i n dextrans and f o s t e r c a r i o u s d e n t a l l e s i o n s (9,60,61,62). TFoodTndustry. The p o t e n t i a l i t i e s f o r dextran i n the food i n d u s t r y have been reviewed (63^, 64). The o n l y a c t u a l uses known t o the a u t h o r , however, are i n dextran g e l f i l t r a t i o n processes t o concentrate p r o t e i n s o r t o recover p r o t e i n s from l i q u i d wastes and e f f l u e n t streams. From c e r e a l waste streams, 70% recovery o f p r o t e i n has been effected. In the m i l k i n d u s t r y , skim m i l k or cheese whey i s f r a c t i o n a t e d f o r recovery o f undenatured p r o t e i n components o f enhanced q u a l i t y , n u t r i t i v e v a l u e , and a p p l i c a b i l i t y . A p l a n t having c a p a c i t y o f 1 X 10 l b . per day i s i n o p e r a t i o n (65). P r o t e i n i s separated from l a c t o s e and m i n e r a l c o n s t i t u e n t s ancT f r a c t i o n a t e d m a i n l y on the b a s i s o f molecular weight i n t o c a s e i n , β - l a c t o g l o b u l i n , and α - l a c t o g l o b u l i n . βL a c t o g l o b u l i n , which i s h i g h l y s u p e r i o r n u t r i t i o n a l l y t o c a s e i n (66), had r e s t r i c t e d use when p r e v i o u s l y i s o l a t e d as the degraded and denatured l a c t a l b u m i n (67). Other v a l u a b l e products t h a t may be recovered are l a c t o T e r r i n and immuno globulins. By another a p p l i c a t i o n o f g e l f i l t r a t i o n , the p r o t e i n content o f m i l k i s i n c r e a s e d from 3.351 t o 5.35% w i t h o u t i n c r e a s e o f the l a c t o s e and m i n e r a l contents (68). Atomic F u e l and M e t a l l u r g y . G e l p r e c i p i t a t i o n i s a process i n which dextran (or c e r t a i n other p o l y h y d r i c polymers)

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i s used to produce a metal compound i n the form of a gel under conditions where an insoluble precipitate would be expected (69) (Figure 3). The process i s used for purifying, separating, and concentrating metals from solutions of t h e i r salts or mixtures of salts or from c o l l o i d a l dispersions of. aqueous hydrous sols. The f i n a l product may be i n the form of powder, granules, spheres, rods or shaped rods, or ceramic coatings and moulded objects. Products prepared by use of dextran as the g e l l i n g agent are f o r use as nuclear reactor fuels (70,71), catalysts (71,72,73), ceramic coatings (70), refractories and f e r r o - e l e c t r i c materials Ç72,73), and powder f o r alloys (72), pigments (74) and metallurgical processes (70). The g e l l i n g agent an complexes which, when contacte reagent, produce discrete macrocrystalline gel p a r t i c l e s (69,75). The 1,6-linkages i n dextran are believed to confer a special configuration on the three contiguous free -OH groups which i s p e c u l i a r l y favorable to -OH--complex formation with an unusual number of metal ions (6£, 76). The s p e c i f i c properties of dextran metallic ion complexes are u t i l i z e d i n separating f e r r i c iron from mixtures with copper, n i c k e l or cobalt, or n i c k e l from thorium, or zirconium from copper (76). Dextran (or fractions of stated molecular weight) i s u t i l i z e d i n preparing black magnetic i r o n oxide (Fe^O.) from ferrous s a l t (74,77). Cupric ion may be adsorbed from solution on hydrous gels" ÔT f e r r i c oxide, chromium oxide, or thorium phosphate/dextran g e l , and then eluted (ZI). The procedures reviewed here indicate the potential for dextran application i n gel-precipitation processes. Some of the procedures are known to be i n use. Petroleum Production. A pioneering concept advocated for some years was to make dextran a profitable byproduct of the sugar cane industry by using i t i n petroleum d r i l l i n g muds (78). In i n i t i a l laboratory testing f o r water loss i n h i b i t i o n , a modified dextran gave results equivalent or superior to starch and carboxymethyl cellulose (79). The modified dextran (Viscoba), prepared by treatment of dextran with aldehyde before i s o l a t i o n (80), had improved v i s c o s i t y and was resistant to microbial attack. The concept was advanced further when, during 1956 through 1959, a dextran production p i l o t plant was operated i n conjunction with a sugar m i l l i n Cuba (81). The dextran, produced from s t r a i n NRRL B-512(F) by a modified enzymatic procedure, was precipitated once and drum dried. [The fructose byproduct was recovered and uses investigated (81)]. The output (3-6 tons/day) was used i n the United States i n d r i l l i n g muds under a variety of f i e l d conditions. The price at the well s i t e was 46 cents/lb. ; the demand greatly exceeded the supply (82). The dextran

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Figure

1.

Structural features of dextran from Leuconostoc mesenteroide

Figure 2. The characteristic structural features of pullulans: a-maltotriose polymerized through a-lfi-lirikages

Solution

(acidic)

P r e c i p i t a t i n g Reagent

M e t a l l i c salt Gelling agent

NaOH or NH4OH (Dextran)

Solution

solution,

NH 3 g a s , or amines

Gel

Mixed

Non-coalescent,

Reduce

Non-adherring,

alloy

Mixed Gelling

[acidic)

metallic

salts

agent

Gel

Separated

to

Components

powder

Microcrystalline Dry:

to p a r t i c l e s of

desired shape and size Oxides:

oxidize

Oxides,

hydroxides:

gas to m e t a l

Figure 3.

by air reduce by

powders

The gel-precipitation process and some of the products resulting

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functioned better than some ot i t s competitors and as well as any.— The dextran could not be used i n lime base muds; i t precipitated at pH 11.0-11.2 and l o s t i t s water-binding capacity. Under these conditions, however, l i g h t l y hydroxy ethylated dextran retained i t s water-binding capacity (82). At less basic or neutral pH, dextran tolerates calcium ion and magnesium ion w e l l (83). Dextran i s the hydrocolloid i n an "inhibited mud" composition containing 3500 ppm calcium ion that i s used to i n h i b i t shale hydration (84). Like a l l polymers, dextran i s susceptible to free r a d i c a l degradation, and protection i s advised during processing as well as use (85)· Dextran NRRL B-512(F) ha propertie suitabl f o i n viscous water floodin results superior to many otEêr substances examined. The unfavorable results of a f i e l d t r i a l (87) may have related to lack of protection against free-radical degradation. Photographic Industry. Native high molecular weight dextran has been supplied consistently for an undisclosed i n d u s t r i a l use believed to relate to photographic products. Numerous patents have been issued i n the past and continue to be issued on the superior effects achieved from certain dextran derivatives i n X-ray and photographic emulsions (9^54,55). I t seems probable that some of these derivatives are i n use. Pullulan--Proposed Uses Numerous applications o f pullulan and i t s derivatives have been proposed and patented, but apparently are not yet i n use (88,89). The p o s s i b i l i t y of eventual success for most uses i s increased by the claim that, by proper selection of pullulan-producing s t r a i n s , the molecular weight can be controlled and absence of black pigment i n the product can be assured (49). The efficiency of pullulan, even as the crude fermentation liquors, has been demonstrated for flocculation of clay slimes from aqueous solutions resulting from beneficiation of uranium, potash, and other ores (90,91,92). Films formed from pullulan without~plasticizers have excellent physical properties, are water-soluble, impervious to oxygen and suitable f o r coating or packaging foods and pharmaceuticals especially when exclusion o f oxygen i s desirable (88). Fibers from pullulan have a shiny gloss and high t e n s i l e strength which, after stretching, i s described — Death of the key personnel i n an airplane accident terminated this development.

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as comparable to that of nylon (88). The fibers may be admixed with natural fibers i n special papers and other products (93). Pullulan i s suitable for making adhesives and shaped a r t i c l e s by compression molding. In such molded a r t i c l e s pullulan has characteristics similar to polyvinyl alcohol or to styrene (88). I t has desirable properties for use i n noncaloric and other foods; i t i s nontoxic and nondigestible (88). Pullulan i s biodegradable, however, under usual conditions of waste disposal.

Literature Cited 1. 2. 3. 4. 5. 6. 7.

8. 9.

10. 11. 12.

13. 14. 15. 16.

Browne, C. Α., Jr., J . Am. Chem. Soc. (1906) 28, 453469. Hehre, E. J. and N e i l l 147-162. Pasteur, L . , Bull. Soc. Chim. Paris (1861), 30-31. van Tieghem, P . , Ann. S c i . Nat. Bot. Biol. Veg. (1878) 7, 180-203. Scheibler, C., Ver. Rubenzucker-Ind. (1874) 24, 309335. Jeanes, Α., "Dextran," in Encyclopedia of Polymer Science and Technology, Vol. 4, Bikales, Ν. Μ., Ed. Interscience Publishers, N.Y., 1968, pp. 693-711. Murphy, P. T. and Whistler, R. L., "Dextrans," in Industrial Gums: Polysaccharides and their Derivatives, Second Edition, Whistler, R. L. and BeMiller, J . Ν., Eds. Academic Press, N.Y., 1973, pp. 513-542. Sidebotham, R. L., Adv. Carbohydr. Chem. Biochem. (1974) 30, 371-444. Jeanes, Α., "Dextran Bibliography: Extensive Coverage of Research Literature (Exclusive of Clinical) and Patents, 1861-1976." Miscellaneous Publication, Agricultural Research Service, United States Department of Agriculture, in press. Bender, H . , Lehmann, J., and Wallenfels, Κ., Biochim. Biophys. Acta (1959) 36, 309-316. Bernier, Β., Can. J. Microbiol. (1958) 4, 195-204. Zajic, J . E. and LeDuy, Α., "Pullulan," in Encyclopedia of Polymer Science and Technology, Supplement Vol. 2, Bikales, Ν. M . , Ed. Interscience Publishers, N.Y., in press, 1977. Cook, W. Β., Mycopathol. Mycol. Appl. (1959) 12, 1-45. Bender, H. and Wallenfels, Κ., Biochem Ζ.(1961)334, 79-95. Enevoldsen, B. S., J . Inst. Brew., London (1970) 76, 546-552; Brygmesteren (1971) 28, 41-51. Hathaway, R. J. (A. E. Staley Mfg. Co.), U.S. Patent 3,556,942. January 19, 1971.

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17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42.

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Imrie, F. Κ. E. and Tilbury, R. Η., Sugar Technol. Rev. (1972) 1, 291-361. Sutherland, D. N. and Paton, Ν., Int. Sugar J. (1969) 71, 131-135. Leonard, G. J. and Richards, G. Ν., Int. Sugar J . (1969) 71, 263-267. N e i l l , J . M., Sugg, J. Y . , Hehre, E. J., and Jaffe, E . , J . Exp. Med. (1939) 70, 427-442; Am. J. Hyg. (1941) 34, 65-78. Gibbons, R. J. and Fitzgerald, R. J., J. Bacteriol. (1969) 98, 341-346. Richards, G. N. and Streamer, Μ., Carbohydr. Res. (1972) 25, 323-332. Tate and Lyle Ltd. British Patent 1,290,694 September 27 1972. Van Cleve, J . W., , , , , Am. Chem. Soc. (1956) 78, 4435-4438. Larm, O., Lindberg, Β., and Svensson, S., Carbohydr. Res. (1971) 20, 39-48. Walker, G. J. and Pulkownik, Α., Carbohydr. Res. (1973) 29, 1-14. Misaki, Α., Yukawa, S., Asano, T., and Isono, Μ., Ann. Rep. Takeda Res. Lab. (1966) 25, 42-54; Chem. Abstr. (1967) 66, 54,255t. Rosenfel'd, E. L., Biokhimiya (1958) 23, 635-638; Biochem. English Transl. (1958) 23, 597-600. Ewald, R. A. and Crosby, W. Η., Transfusion (1963) 3, 376-386. Jeanes, Α., ACS Symp. Ser. (1975) 22, 336-347. Koepsell, H. J. and Tsuchiya, Η. M . , J. Bacteriol. (1952) 63, 293-295. Tsuchiya, Η. M., Hellman, Ν. N., Koepsell, H. J. and others, J . Am. Chem. Soc. (1955) 77, 2412-2419. Senti, F, R., Hellman, Ν. Ν., Ludwig, Ν. Η., and others, J. Polym. S c i . (1955) 17, 527-546. Lindberg, B. and Svensson, S., Acta Chem. Scand. (1968) 22, 1907-1912. Walker, G. J. and Pulkownik, Α., Carbohydr. Res. (1974) 36, 53-66. Norrman, B . , Acta Chem. Scand. (1968) 22, 1381-1385. Miyaji, H. and Misaki, Α., J. Biochem. (1973) 74, 11311139. Dea, I. D. M. and Morris, E. R., this symposium. Morris, E. R., this symposium. Moorhouse, R., Walkinshaw, M. D., and Arnott, S., this symposium. Pasika, W. Μ., this symposium. Wallenfels, Κ., Keilich, G., Bechtler, G., and Freudenberger, D., Biochem. Z. (1965) 341, 433-450.

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43. Catley, B. J. and Whelan, W. J., Arch. Biochem. Biophys. (1971) 143, 138-142. 44. Taguchi, R., Kikuchi, Y., Sakano, Y., and Kobayashi, T., Agric. Biol. Chem. (1973) 37, 1583-1588. 45. Sowa, W., Blackwood, A. C., and Aams, G. Α., Can. J. Chem. (1963) 41, 2314-2319. 46. Elinov, N. P. and Matveeva, A. K . , Biokhimiya (1972) 37, 255-257; Biochem. English Transl. (1973) 37, (2, Part 1), 207-209. 47. Catley, B. J., FEBS Lett. (1972) 20, 174-176. 48. LeDuy, Α., Marsan, Α. Α., and Coupal, B . , Biotechnol. Bioeng. (1974) 16, 61-76. 49. Kato, K. and Shiosaka M [Hayashibara Biochemical Laboratories, Inc. 1975. 50. Taguchi, R., Sakano, Y., Kikuchi, Y . , and others, Agric. B i o l . Chem. (1973) 37, 1635-1641. 51. Yokobayashi, Κ., Akai, H . , Harada, T. and others, Biochim. Biophys. Acta (1973) 293, 197-202. 52. Groenwall, A. and Ingelman, B . , Acta Physiol. Scand. (1945) 9(1), 1-27. 53. Tiselius, Α., Porath, J., and Albertsson, P. Α., Science (1963) 141, 13-20. 54. Jeanes, Α., J. Polym. S c i . : Polym. Symp. No. 45, IonContaining Polymers (1974), 209-227. 55. Jeanes, A. i n "Polyelectrolytes," Frisch, K. and Klempner, D., Eds. Technomic Publishing Co., Inc., Wesport, Conn., 1977. 56. Cox, J. S. G., King, R. E . , and Reynolds, G. F . , Nature (London) (1965) 207, 1202-1203. 57. Marshall, P. R. and Rutherford, D., J. Colloid Interface S c i . , (1971) 37, 390-402. 58. London, E. and Twigg, G. D. (Benger Laboratories, Ltd.), British Patent. 748,024, April 18, 1956; U.S. Patent 2,820,740, January 21, 1958. 59. Kagedal, L. and Akerstroem, S., Acta Chem. Scand. (1971) 25, 1855-1859. 60. Keyes, P. H., Hicks, M. Α., Goldman, B. M . , and others, J. Am. Dent. Assoc. (1971) 82, 136-141. 61. Miller, G. R. (Colgate-Palmolive Co.), U.S. Patent 3,630,924, December 28, 1971. 62. Woodruff, H. B. and Stoudt, T. H. (Merck and Co., Inc.), U.S. Patent 3,686,393, August 22, 1972. 63. Glicksman, M., "Gum Technology in the Food Industry," Academic Press, New York, 1969, pp. 335-341. 64. Jeanes, Α., Food Technol. (1974) 28(5), 34-40. 65. Davis, J. C., Chem. Eng. (July 1972), 114-115. 66. Forsum, E . , J. Dairy Sci. (1973) 57, 665-670. 67. Wingerd, W. H., J. Dairy Sci. (1971) 54, 1234-1236.

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21.

68. 69. 70. 71. 72. 73. 74. 75. 76. 77. 78. 79. 80. 81. 82. 83. 84. 85. 86. 87. 88.

JEANES

Dextrans

and

Pullulans

297

Samuelsson, E-G., Tibbling, P., and Holm, S., Food Technol. (1967) 21(11), 121-124. Grimes, J. H. and Scott, Κ. Τ. Β., Powder Met. (1968) 11(22), 213-223. Dress, W. and Grimes, J. H. (united Kingdom Atomic Energy Authority), British Patent 1,175,834, December 23, 1969. Grimes, J. H. and Lane, E. S. (United Kingdom Atomic Energy Authority), British Patent 1,231,385, May 12, 1971. Grimes, J. H. and Lane, E. S. (United Kingdom Atomic Energy Authority), British Patent 1,286,257, August 23, 1972. Grimes, J. H. and Dress W (United Kingdom Atomic Energy Authority) 1972. Grimes, J. Η., Scott, Κ. T. B . , and McKenna, N. J. (United Kingdom Atomic Energy Authority), British Patent 1,350,389, April 18, 1974. B a l l , P. W., Grimes, J. Η., and Scott, Κ. T. B. (United Kingdom Atomic Energy Authority), British Patent 1,420,128, January 7, 1976. Scott, Κ. T. B . , Grimes, J. Η., and Ball, P. W. (United Kingdom Atomic Energy Authority), British Patent 1,325,870, August 8, 1973. Scott, Κ. T. B . , Grimes, J. Η., and B a l l , P. W. (United Kingdom Atomic Energy Authority), British Patent 1,346,295, February 6, 1974. Owen, W. L., Sugar (1950) 45(3), 42-43; Sugar (1955) 50(5), 47-48. Owen, W. L., Sugar (1951) 46(7), 28-30; Sugar (1952) 47(7), 50-51. Owen, W. L., U.S. Patent 2,602,082, July 1, 1952. Ruiz, A. R., Sugar J. (1957) 20(3), 50-52. Richey, Harry and Woods, Jack (Cherokee Laboratories, Tulsa, Oklahoma), personal communications. Mueller, E. P . , Z. Angew. Geol. (1963) 9(4), 213-217; Chem Abstr. (1963) 59, 4935a. Monaghan, P. H. and Gidley, J. L . , O i l Gas J. (1959) 57 (16), 100-103. Heyne, B. and Gabert, Α., Bergakademie (1969) 21(5), 285-288; Chem. Abstr. (1969) 71, 62,733r. Sparks, W. J. (Jersey Production Research Co.), U.S. Patent 3,053,765, September 11, 1962. Lindblom, G. P . , Ortloff, G. D., and Patton, J. T. (Jersey Production Research Co.), Canadian Patent 654,809, December 25, 1962. Yuen, S., Process Biochem. (November 1974), 7-9.

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89.

Yuen, S., "Pullulan and Its New Applications," Hayashibara Biochemical Laboratories, Inc., Okayama, Japan, February 1974. 90. Zajic, J . E. (Kerr-McGee Oil Industries, Inc.), U.S. Patent 3,320,136, May 16, 1967. 91. Goren, M. B. (Kerr-McGee O i l Industries, Inc.), U.S. Patent 3,406,114, October 15, 1968. 92. Zajic, J . E. and LeDuy, Α., Appl. Microbiol. (1973) 25, 628-635. 93. Nomura, T. (Sumimoto Chemical Co. Ltd.; Hayashibara Biochemical Laboratories, Inc.), U.S. Patent 3,936,347, February 3, 1976).

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

22 Extracellular Microbial Polysaccharides—A Critical Overview JEREMY WELLS Biochem Design S.p.A., Via A. Bargoni, 78, 00153 Rome, Italy It is the interest of this paper to present a c r i t i c a l overview of commercially significant extracellular microbial polysaccharides within the context of the industrial hydro colloid or gums market Polysaccharide hydrocolloid plant weed have been used successfully for food, petroleum, textile and numerous industrial applications for several years. Polysaccharides are produced extracellularly by many microorganisms now available. Several of these new hydrocolloids produced microbially, have shown themselves to be commercially significant. The reasons for the commercial exploitation of these microbial polysaccharides is because of their unique physical and constant chemical properties, regularity of supply, better functional properties and a lower biological use of oxygen. The commercial usefulness of polysaccharides is based on their ability to alter the rheological properties of water. Present major markets for these polysaccharides exist in the food and the petroleum drilling industries. Large future growth is expected to come from enhanced oil recovery. This paper considers the use of microbial polysaccharides in competition with other water soluble gums in the food industry. It also studies the use of polymers in enhanced oil recovery, when i t compares polysaccharides with polyacrylamides. To provide this overview of the industrial gums markets, it has been necessary to review data recently published or in publication. Particular thanks are given to Tate and Lyle Ltd. for allowing publication of data recently obtained durina a market feasibility study made on their behalf. Particular thanks are given to Dr. C.J.Lawson, without whose cooperation this paper would have been that much more difficult. THE MARKET FOR WATER SOLUBLE GUMS M o s t w a t e r s o l u b l e gums a r e t h e o r e t i c a l l y i n t e r c h a n g e a b l e . I n p r a c t i c e , most gums p o s s e s s u n i q u e c h a r a c t e r i s t i c s w h i c h guarantee t h e i r commercial use. 299

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TABLE I CLASSIFICATION OF NATURAL AND SYNTHETIC WATER-SOLUBLE GUMS

Exemple

Origin

Tree Exudates

Gum A r a b i c K a r a y a Gum Gum T r a g a c a n t h and o t h e r s Guar Gum

others

Seaweed E x t r a c t s

Natural

Natural

Starches

Natural

Products

S t a r c h and Dérivâtes

Cellulose Derivatives

Synthetic

Petrochemical Derivatives

Agar Alginates Carrageenan and others Corn Starch Potato Starch T a p i o c a and o t h e r s Dextrans X a n t h a n Gums Pectin Gelatin Dextrins Starch Acetates Dialdehyde s t a r ches and o t h e r s Carboxymethylcellulose Methylcellulose Hydroxymethy1cell u l o s e and o t h e r Polyvinyl Alcohol Polyacrylic Acid Salts Ethylene Oxide Polymers and others

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

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Microbial

Polysaccharides

301

T a b l e I l i s t s b o t h n a t u r a l and s y n t h e t i c gums i n common u s e a c c o r d i n g t o c l a s s . Gums h a v e d i v e r s e c h e m i c a l c o m p o s i t i o n , o r i g i n and f u n c t i o n a l i t y a n d a r e c l a s s i f i e d a c c o r d i n g t o t h e i r origin. The m a i n c l a s s e s o f n a t u r a l gums a r e t h e f o l l o w i n g : - Natural products - S t a r c h and s t a r c h dérivâtes - Seaweed e x t r a c t

- Tree exudates - Seed e x t r a c t - Cellulose derivatives

In the united States, i t i s reported that while the t o t a l e x p a n s i o n o f gums i s o n l y 1.3% p e r annum, t h e s y n t h e t i c p o l y m e r and m i c r o b i a l l y p r o d u c e d gums a r e i n c r e a s i n g b y o v e r 8% p e r annum. This i n c r e a s e i n consumptio e x p e n s e o f p l a n t gums. T a b l e I I shows t h e c o n s u m p t i o n o f gums i n t h e U n i t e d S t a t e s d u r i n g 1973 a s r e p o r t e d b y R . L . W h i s t l e r . The o v e r a l l u s a g e o f gums h a s b e e n f a i r l y w i d e s p r e a d t h r o u g h o u t t h e i n d u s t r y . O r i g i n a l l y t h e t r e e e x u d a t e s were t h e most w i d e l y u s e d c l a s s o f gum. I n r e c e n t y e a r s t h e s e e x u d a t e s h a v e b e e n r e p l a c e d b y m a n u f a c t u r e d gums i n c l u d i n g x a n t h a n gum. Improved p r o p e r t i e s o v e r t h e s e p l a n t gums b y t h i s r a n g e o f m a n u f a c t u r e d gums, h a v e caused t h e s h i f t . P l a n t gums v a r y i n q u a l i t y and a r e d i s t r i b u t ed i n t h e raw s t a t e , s o r e q u i r e f u r t h e r p r o c e s s i n g i n c l u d i n g

Gum

TABLE I I THE CONSUMPTION OF INDUSTRIAL GUMS I N THE UNITED STATES (1973) (tons) Total Food Industrial Usage Usage Usage

Cornstarch Carboxymethylcellulose Methylcellulose Guar Arabic Pectin L o c u s t bean Alginate Ghatti Carrageenan Xanthan Karaya Tragacanth Agar Furcellaran

223,214 6,696 900 6,696 10,267 3,357 4,017 4,017 4,464 4,017 1,000 446 580 133 89

1,116,071 43,303 23,660 15,625 3,125 0 1,785 4,017 446 89 2,678 3,125 89 178 0

1,339,285 50,000 24,553 22,321 13,392 5,357 5,803 8,034 4,910 4,106 3,678 3,571 669 311 89

R . L . W h i s t l e r 1974

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c r u s h i n g and c l e a n i n g b y t h e end u s e r . W h i l e t h e m a n u f a c t u r e d gums a r e i n t h e i r f i n a l s t a t e , r e a d y f o r d i r e c t u s e . The m a n u f a c t u r e d gums a r e o f t e n t a i l o r made f o r s p e c i f i c a p p l i c a t i o n o r premixed f o r d i r e c t a p p l i c a t i o n . These premixes b e a r a v a r i e t y o f names a n d a r e i n t e n d e d f o r s p e c i f i c p u r p o s e s . S e a s o n a l v a r i a t i o n s i n q u a l i t y , s u p p l y a n d p r i c e have o f t e n f o r c e d p r o c e s s o r s t o change t o m a n u f a c t u r e d gums. However when c o n d i t i o n s r e v e r t , these processors then p r e f e r t o continue t o use t h e r e l i a b l e m a n u f a c t u r e d gums. X a n t h a n f o r example h a s t a k e n much o f t h e gum t r a g a c a n t h m a r k e t i n t h e U.S. I t i s o n l y t h e f o o d , p h a r m a c e u t i c a l and c o s m e t i c i n d u s t r i e s t h a t s t i l l u s e t h e s e p l a n t gums. The p e r c e n t a g e d i s t r i b u t i o n o f t h e m a n u f a c t u r e d gums i n t h e U n i t e d S t a t e s has been r e p o r t e d follows

D e t e r g e n t s and l a u n d r y p r o d u c t s Textiles Adhesives Paper Paint Food P h a r m a c e u t i c a l and c o s m e t i c Other

16 14 12 10 9 8 7 24

The m a i n demands f o r gums s t e p s f r o m t h e i r v a r i o u s f u n c t i o n a l p r o p e r t i e s and c a n be b r o k e n down i n t o t h e f o l l o w i n g : Functionality S t a b i l i z e r , s u s p e n d i n g a g e n t and dispersant Thickener F i l m f o r m i n g agent Water r e t e n t i o n agent Coagulant Colloid Lubricant o r f r i c t i o n reducer Other purposes

Percent

25 23 17 12 7 6 5 5

C o s t e f f e c t i v e n e s s a n d c o s t o f gums w i l l be t h e p u r c h a s e r s most i m p o r t a n t c r i t e r i a i n d e c i d i n g w h i c h gum t o u s e . T a b l e I I I g i v e s a range o f p r i c e s as r e p o r t e d by t h e Chemical Market Reporter. A l t h o u g h gum p r o d u c t i o n i s f a i r l y d i f f i c u l t t o a c c u r a t e l y d e t e r m i n e , T a b l e I V l i s t s some gum p r o d u c t i o n e s t i m a t e s o b t a i n ed f r o m v a r i o u s s o u r c e s . The v a r i o u s a p p l i c a t i o n s o f gums a r e d e t e r m i n e d by t h e i r cost e f f e c t i v e n e s s i nu t i l i z i n g p h y s i c a l properties t o perform specific applications.

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

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Microbial

Polysaccharides

TABLE I I I Price, $ per l b V a r i e t y o f Gum

1971

A g a r USP Gum A r a b i c Gelatine, edible Guar gum, e d i b l e K a r a y a gum L o c u s t b e a n gum Methylcellulose Pectin CMC ( c a r b o x y m e t h y l c e l l u l o s e Gum T r a g a c a n t h

2.4C ^-2.80 0.42 H).60 0.57 f 0 . 5 8 0.38 H).40 0.80 H).90 0.52 f 0 . 5 8 0 .89 2 .40

1975 8. 15 1. 75 1 .64 v2.75 0 .35 τθ.40 0 .90 v0.95 0 .79 K>.98 0. 74 2 22

S o u r c e : I n t e r n a t i o n a l T r a d e C e n t e r , 1972 u p d a t e d f r o m C h e m i c a l Market Reporter.

TABLE I V WORLD PRODUCTION OF SELECTED GUMS Gum

Year

Agar 1 Alginate 1 Arabic 2 Carrageenan 1 Furcellaran 1 L o c u s t Bean 2 Methylcellulose 2 Pectin 2 Carboxymethylcellulose Xanthan

1973 1973 1966 1973 1973 1970 1972 1971 1969 1975

Sources:

2

INDUSTRIAL

Production

(tons)

7,950 17,000 60,000 8,000 1,200 15,000 25,000 9,000 60,000 5,000

1) J . N a y l o r FA0 P r o d u c t i o n , T r a d e and U t i l i z a t i o n o f Seaweed P r o d u c t s (1976). 2) R . L . W h i s t l e r

I n d u s t r i a l Gums ( 1 9 7 3 ) .

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As p r e v i o u s l y m e n t i o n e d the f o l l o w i n g c l a s s e s :

t h e m a i n gums a r e c l a s s i f i e d

into

N a t u r a l P r o d u c t s . The f o u r most i m p o r t a n t h y d r o c o l l o i d s i n t h i s c l a s s a r e g e l a t i n , p e c t i n , d e x t r a n and x a n t h a n . I n t h e food i n d u s t r y over twenty thousand tons o f n a t u r a l p o l y s a c c h a r i d e s w e r e s o l d i n t h e U n i t e d S t a t e s i n 1975. T h i s was b r o k e n down i n t o 16,000 t o n s g e l a t i n e , 2,000 t o n s x a n t h a n and 3,000 tons p e c t i n . G e l a t i n i s p r e f e r r e d i n g e l a t i n d e s s e r t s , meat p r o d u c t s s u c h a s ham and l u n c h e o n meat and d a i r y p r o d u c t s . The p h o t o g r a p h i c and p h a r m a c e u t i c a l i n d u s t r i e s a r e t h e l a r g e s t u s e r s of h i g h grade g e l a t i n e . P e c t i n , because o f i t s g e l forming p r o p e r t i e s w i t h sucrose i s u s e d i n jams a n d c o n f e c t i n e r y in salad dressings, citru ducts as w e l l as o i l d r i l l i n g recovery Seaweed E x t r a c t s . Seaweed e x t r a c t s a r e o b t a i n e d f r o m two groups o f a l g a e , r e d algae which i s t h e source o f carrageenan and a g a r a n d b r o w n a l g a e w h i c h i s t h e s o u r c e o f a l g i n a t e s . The r e c e n t FA0 Seaweed R e s o u r c e s o f t h e W o r l d r e p o r t s l a r g e p o t e n t i a l f o r expansion i n t h i s group. P r e s e n t h a r v e s t s o f r e d and brown a l g a e a r e p u t a t 0.807 a n d 1.315 m i l l i o n t o n s r e s p e c t i v e l y w i t h p o t e n t i a l o u t p u t s l i s t e d a t 2.66 and 14.6 m i l l i o n t o n s of algae. E a c h o f t h e s e gums, h a s u n i q u e p r o p e r t i e s g i v i n g them e x c e l l e n t market p o t e n t i a l . Because o f i t s l o w g e l l i n g and heat r e s i s t a n c e , agar has a wide usage i n f o o d s . A l g i n a t e s a r e t h e most e x t e n s i v e l y u s e d gum o f t h e g r o u p and a r e u s e d i n d a i r y p r o d u c t s , c i t r u s b e v e r a g e s , b a k e r y f i l l i n g s , l i q u i d a n i m a l f e e d s , p h a r m a c e u t i c a l and many i n d u s t r i a l applications. Carrageenan has t h e l a r g e s t usage o f t h e group i n d a i r y , beverages and bakery p r o d u c t s . I t i s h e a v i l y used i n d a i r y products because o f i t s r e a c t i o n w i t h c a s e i n . Starch Derivatives. N a t u r a l starches are normally processed t o g i v e them p r o p e r t i e s f o r s p e c i a l a p p l i c a t i o n s . B e c a u s e of t h e i r s t r o n g adhesives p r o p e r t i e s , they are used i n adhesives and a r e h i g h l y c o m p e t i t i v e w i t h gum a r a b i c . Other uses i n c l u d e c e r a m i c s , f l o c c u l a t i o n , w e l l d r i l l i n g muds a n d p h a r m a c e u t i c a l s . Seed E x t r a c t s . The two m a j o r gums i n t h e g r o u p a r e g u a r and l o c u s t b e a n gum. G u a r i s o b t a i n e d i n I n d i a a n d P a k i s t a n , w h i l e l o c u s t s b e a n i s h a r v e s t e d i n S p a i n and t h e M e d i t e r r a n e a n a r e a . They a r e b o t h u s e d i n t h e d a i r y i n d u s t r y f o r c h e e s e m a k i n g and i c e c r e a n p r o d u c t i o n . G u a r gum i s t h e p r e f e r r e d i c e c r e a m s t a b i l i z e r . L o c u s t b e a n gum i s a v i s c o s i f i e r a n d b i n d e r o f f r e e w a t e r . Cellulose Derivatives. F o o d , drum, c o s m e t i c and d e n t i f r i c e p r o d u c t s a r e t h e f a s t g r o w i n g u s a g e o f CMC ( s o d i u m c a r b o x y m e t h y l c e l l u l o s e ) a n d MC ( m e t h y l c e l l u l o s e ) . MC c o s t s more t h a n CMC a n d h a s a US p r o d u c t i o n o f 30 m i l l i o n pounds a g a i n s t a US p r o d u c t i o n o f 74 m i l l i o n pounds o f CMC.

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

22.

WELLS

Extracellular

Microbial

Polysaccharides

305

Tree Exudates. The t r e e e x u d a t e s , gum a r a b i c , gum t r a g a c a n t h a n d gum K a r a y a h a v e a l l l o s t m a r k e t s h a r e s t o t h e s y n t h e t i c s a n d p r o c e s s e d gums. However, gum K a r a y a b e c a u s e o f i t s l a x a t i v e p r o p e r t i e s m a i n t a i n s a market. Gum t r a g a c a n t h h a s a w i d e r a n g e o f a p p l i c a t i o n s i n f o o d , t e x t i l e s , c o s m e t i c s and c e r a m i c s . However b e c a u s e o f s u p p l y d i f f i c u l t i e s and p r i c e d i f f e r e n t i a l i t h a s been r e p l a c e d t o a l a r g e e x t e n t b y x a n t h a n gum. Gum a r a b i c i s u s e d m o s t l y t o p r e v e n t c r y s t a l l i z a t i o n o f s u g a r s and a s a n e m u l s i f i e r t o k e e p f a t s u n i f o r m l y d i s t r i b u t e d . CMC, PVA and m o d i f i e d s t a r c h e s have t a k e n a l a r g e s h a r e o f t h e gum a r a b i c m a r k e t . MICROBIAL POLYSACCHARIDE At t h i s time o n l y m e r c i a l s i g n i f i c a n c e are i n commercial p r o d u c t i o n , dextran, p o l y t r a n a n d x a n t h a n . F i v e o t h e r s show p r o m i s e i n d e v e l o p m e n t and a r e a v a i t i n g d e c i s i o n s o n p o t e n t i a l c o m m e r c i a l e x p l o i t a t i o n . Dextran. Dextrans a r e p o l y g l u c a n s and have been produced i n t h e U n i t e d S t a t e s , C a n a d a , H o l l a n d a n d Sweden. They c a n be s y n t h e s i s e d f r o m s u c r o s e m i c r o b i a l l y f r o m many s t r a i n s o f c e l l f r e e c u l t u r e f i l t r a t e s o f l e u c o n o s t o c m e s e n t e r i d e s , though d e x t r a n s from o t h e r s t r a i n s w i l l d i f f e r both i n s t r u c t u r e and p r o p e r t i e s . M o l e c u l a r w e i g h t s may r a n g e w i d e l y . U s u a l p r a c t i c e i s t o o b t a i n a h i g h m o l e c u l a r weight m a t e r i a l and degrade i t by h y d r o l y s i s , s i n c e the d e x t r a n s t h a t are used i n the food i n d u s t r y must h a v e m o l e c u l a r w e i g h t s b e l o w 100,000, s i n c e o n l y t h e y a r e i n c l u d e d i n t h e GRAS l i s t o f t h e FDA. D e x t r a n s o l u t i o n s a r e c l o s e l y s i m i l a r t o l o c u s t b e a n gum. X a n t h a n . X a n t h a n gum i s p r o d u c e d f r o m g l u c o s e s o l u t i o n i n g r o w i n g c u l t u r e s o f xanthomonas c a m p e s t r i s . Commercial p r o d u c t i o n has been c a r r i e d o u t i n the U n i t e d S t a t e s s i n c e 1967 b y t h e K e l c o Company, who a r e c u r r e n t l y t h e m a i n m a n u f a c t u r e r a n d who p r o d u c e d a n e s t i m a t e d 5,000 t o n s o f x a n t h a n i n 1975. L o c a l e x p a n s i o n o f t h e San D i e g o p l a n t , t o g e t h e r w i t h a g r a s s r o o t s p l a n t i n Oklahoma f o r 10,000 t o n s a t an e s t i m a t e d c o s t o f 35 m i l l i o n d o l l a r s p l u s p l a n t b y Rhone P o u l e n c o f F r a n c e a n d G e n e r a l M i l l s c o u l d make a v a i l a b l e b e t w e e n 35-37J m i l l i o n pounds x a n t h a n b y 1978. T a t e a n d L y l e L t d . and H e r c u l e s I n c . h a v e announced a j o i n t - v e n t u r e t o enter i n t h i s market. C u r r e n t development s t a t u s o f m i c r o b i a l p o l y s a c c h a r i d e s i s l i s t e d i n A p p e n d i x A. The u n i q u e p h y s i c a l p r o p e r t i e s o f x a n t h a n h a v e f o u n d many i n d u s t r i a l a p p l i c a t i o n s i n such d i v e r s i f i e d i n d u s t r i e s as t e x t i l e p r i n t i n g , d r i l l i n g muds, s u r f a c t a n t f l o o d i n g , r u s t r e m o v e r s , and l i q u i d t y p e o f a n i m a l f e e d s . Most i m p o r t a n t p r e s e n t u s e i s t h e r e c o v e r y o f c r u d e o i l . The f l o w c h a r a c t e r i s t i c s o f x a n t h a n , c o u p l e d w i t h i t s s t a b i l i t y

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

306

EXTRACELLULAR MICROBIAL POLYSACCHARIDES

t o pH, g i v e s i t a t e c h n i c a l a d v a n t a g e o v e r o t h e r p o l y m e r s i n d r i l l i n g muds. T h e r e i s a n e s t i m a t e d w o r l d u s a g e o f 1,800 t o n s in drilling applications. S c l e r o g l u c a n ( P o l y t r a n ) . T h i s p o l y s a c c h a r i d e has been dev e l o p e d by t h e P i l l s b u r y Company a n d m a r k e t e d u n d e r t h e t r a d e name o f P o l y t r a n . P i l l s b u r y c l a i m i m p o r t a n t f l o w c h a r a c t e r i s t i c s o v e r a w i d e r a n g e o f pH a n d t e m p e r a t u r e and s t a b i l i t y i n t h e presence of s a l t s . P o l y t r a n w i l l s t a b i l i z e bentonite c l a y s d u r i n g s t o r a g e , o v e r r a n g e s o f t e m p e r a t u r e a n d pH. I t i s u s e d i n t h e c e r a m i c , d r i l l i n g mud, and i n k s a n d c o a t i n g s i n d u s t r i e s . P r i c e s i n t h e r e g i o n o f 9-10,000 d o l l a r s p e r t o n p u t i t i n comp e t i t i o n w i t h x a n t h a n gum. OTHER MICROBIAL POLYSACCHARIDE Pullulan. This polysaccharid H a y a s h a b a r a Company i J a p a n C o m m e r c i a l i n t e r e s t h a s b e e n shown o n a c c o u n t o f i t s a b i l i t y t o f o r m s t r o n g r e s i l i e n t f i l m s and f i b e r s a n d t h e e a s e i t c a n be m o l d e d i n t o s h a p e s . A t p r e s e n t p u l l u l a n i s o n l y i n t h e p i l o t plant stage. C o n s t r u c t i o n o f a p r o d u c t i o n p l a n t was r e p o r t e d t o h a v e s t a r t e d i n 1975. P a t e n t s c l a i m i n g b o t h f o o d a n d i n d u s t r i a l a p p l i c a t i o n s have been f i l e d . M i c r o b i a l A l g i n a t e . A l g i n i c a c i d a n d a l g i n a t e s a r e most i m p o r t a n t gums w i t h many a p p l i c a t i o n s i n f o o d , t e x t i l e , pharmac e u t i c a l a n d p a p e r i n d u s t r i e s . P r o d u c t s o b t a i n e d f r o m seaweed v a r y i n b o t h , q u a l i t y and s t r u c t u r e . S e v e r a l m i c r o o r g a n i s m s produce m i c r o b i a l a l g i n a t e s v e r y s i m i l a r t o a l g a l a l g i n a t e . The c o m p o s i t i o n o f t h e s e p o l y m e r s f o r m e d i s r e p o r t e d t o b e u n e f f e c t e d by t h e c a r b o h y d r a t e source used, and o f c o n s t a n t quality. Most o f t h e main development i n m i c r o b i a l a l g i n a t e s has b e e n c l a i m e d b y T a t e a n d L y l e . Curdlan. C u r d l a n h a s b e e n d e v e l o p e d b y t h e T a k e d a Company i n Japan from a c h e m i c a l mutant o f A l c a l i g e n e s f a e c a l i s v a r myxogenes IOCS. I t s i n d u s t r i a l d e v e l o p m e n t depends o n t h e g e l s t r e n g t h o f h i g h s e t g e l s n o t r e v e r t i n g g r e a t l y when c o o l e d . Aqueous s u s p e n s i o n s o f t h e p o l y m e r r e m a i n s o f t and r e s i l i e n t when c o o l e d , a f t e r h e a t i n g . A p p l i c a t i o n s a r e i m m o b i l i z e d enzymes a s w e l l a s b e i n g u s e d i n p r e p a r a t i o n o f f i l m s and g e l . E r w i n a ( Z a n f l o ) . E r w i n a was d e v e l o p e d b y t h e K e l c o Company s p e c i a l l y f o r c a r p e t p r i n t i n g a p p l i c a t i o n s , due t o i t s c o m p a t i b i l i t y t o c a t i o n i c dyes. I t i s produced from a s t r a i n o f Erwina t a h i t i c a . I t has been c l a i m e d t h a t t h i s p o l y s a c c h a r i d e possesses p s e u d o p l a s t i c i t y , pH s t a b i l i t y and f r e e z e - t h a w s t a b i l i t y . The e x c e l l e n t r e s i s t a n c e t o enzyme a t t a c k , a n d i t s f l o w and l e v e l l i n g q u a l i t i e s h a v e a l r e a d y made i t f i n d a p p l i c a t i o n in thepaint industry.

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

22.

WELLS

Extracellular

Microbial

Polysaccharides

307

INDUSTRIAL DEVELOPMENT OF MICROBIAL POLYSACCHARIDES. D e x t r a n was f i r s t p r o d u c e d c o m m e r c i a l l y i n Sweden i n t h e e a r l y f o r t i e s , t h e n l a t e r i n E n g l a n d , Canada and t h e U n i t e d States. X a n t h a n was f i r s t p r o d u c e d c o m m e r c i a l l y i n t h e U n i t e d States i nthe early s i x t i e s f o r i n d u s t r i a l applications. I n 1969 FDA c l e a r e d t h e g e n e r a l u s e o f x a n t h a n gum i n f o o d s where t h e s t a n d a r d s o f i d e n t i t y do n o t p r e c l u d e i t s u s e . I n 1973 FDA a l l o w e d u s e s i n p r o c e s s a n d cream c h e e s e s a s a t h i c k e n i n g and s t a b i l i z i n g a g e n t . I n 1974 MID/PID I n s p e c t i o n D i v i s i o n o f USDA i n c l u d e d x a n t h a n gums o n t h e i r a u t h o r i z e d l i s t o f n o n meat i n g r e d i e n t s . One c a n c o n v e n i e n t l y d i v i d e up t h e m a i n m a r k e t d e v e l o p ment o f m i c r o b i a l gums - Food a p p l i c a t i o n - P e t r o l e u m and o i l i n d u s t r y a p p l i c a t i o n s - Other a p p l i c a t i o n s . Food A p p l i c a t i o n s . Over 60% o f m i c r o b i a l p o l y s a c c h a r i d e s s a l e s go t o t h e f o o d i n d u s t r y . I n 1975, K e l c o Company a r e s a i d t o have s o l d o v e r 5 m i l l i o n pounds o f x a n t h a n gums i n t o t h e US f o o d i n d u s t r y . X a n t h a n gums have g a i n e d r a p i d acceptance i n t o t h e U n i t e d S t a t e s f o o d i n d u s t r y a n d a p p l i c a t i o n s a r e now b e i n g d e v e l o p e d i n b o t h E u r o p e a n d J a p a n . Denmark, E n g l a n d , I r e l a n d , H o l l a n d , S p a i n a n d Canada h a v e g i v e n r e g u l a t o r y a p p r o v a l . A p p r o v a l s i n F r a n c e , Sweden a n d B e l g i u m a r e e x p e c t e d b e f o r e t h e end o f 1976. I n i t i a l l y the applications followed i n the United States f o r u s e i n s a l a d d r e s s i n g s , meat a n a l o g s , p e t f o o d , b a k e r y p r o d u c t s , c a r b o n a t e d b e v e r a g e s a n d f r o z e n f o o d s w i l l be s t u d i e d . New d e v e l o p m e n t s b y t h e USDA, announced d u r i n g 1 9 7 5 , f o r p r o d u c i n g m a t r i x t e x t u r e s f o r f o o d s and s n a c k f o o d s c o u l d open up v e r y l a r g e d e v e l o p m e n t s . J o i n t p a t e n t s b e t w e e n K e l c o Company and DCA g i v e p r o m i s e o f c h a n g e s i n t h e b a k e r y i n d u s t r y . I t i s r e p o r t e d t h a t a j o i n t - v e n t u r e i n Japan w i t h t h i s group and N i s s h i n F l o u r c o u l d open p o t e n t i a l m a r k e t s i n d o n u t s , o n i o n r i n g p r o c e s s i n g , s n a c k i t e m s a n d c e r t a i n new b a k e r y p r o d u c t s . I n o r d e r t o s e e how t h e s e m i c r o b i a l p o l y s a c c h a r i d e s f i t i n t o t h e o v e r a l l m a r k e t i t i s n e c e s s a r y t o s t u d y t h e t o t a l US c o n s u m p t i o n o f gums i n 1 9 7 5 , w h i c h i s g i v e n i n T a b l e V I . ( X a n t h a n w i t h US s a l e s o f 2,500 t o n s i s c l a s s i f i e d a s a n a t u r a l product. I n i t i a l l y x a n t h a n gums h a v e o b t a i n e d t h e i r m a i n m a r k e t s by r e p l a c i n g gum t r a g a c a n t h . However t h i s m a r k e t h a s p r a c t i c a l l y d i s a p p e a r e d i n t h e US. Though t h e r e e x i s t s s e v e r a l t h o u s a n d tons p o t e n t i a l elsewhere. The w o r l d m a r k e t f o r a l g i n a t e s i s o v e r 17,000 t o n s w i t h about f i v e thousand tons u t i l i z e d i n t h e food i n d u s t r y . About 2,000 t o n s a r e u s e d i n t h e U n i t e d S t a t e s and n e a r l y 1,500 t o n s i n Europe. L a r g e s t a p p l i c a t i o n s a r e i n d a i r y and bakery p r o d u c t s , where c o n s u m p t i o n i s e x p e c t e d t o expand. T h i s c o u l d

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

EXTRACELLULAR MICROBIAL POLYSACCHARIDES

308

TABLE V I US CONSUMPTION OF HYDROCOLLOIDS IN FOODS IN Thousand tons

Product

1975 Million dollars

Natural products Starch derivatives Seaweed e x t r a c t s Tree exudates Seed e x t r a c t s Cellulose derivatives

20.5 124.2 4.1 1.4 5.9 5.9

113 64 19 7 10 12

TOTAL

162.0

225

Source:

James H i c k e y -

open e x c e l l e n t m a r k e t s t o m i c r o b i a l a l g i n a t e s i f t h e y c o u l d be produced at e q u i v a l e n t p r i c e s t o the a l g a l product. In g e n e r a l the shortages of n a t u r a l l y o c c u r r i n g hydroc o l l o i d s i n 1974 showed t h e v u l n e r a b i l i t y o f t h i s m a r k e t t o l e s s c o s t l y m a n u f a c t u r e d gums. I n g e n e r a l t h e m a i n f o o d p r o c e s s o r s w i l l t e n d t o p r e f e r t o f o r m u l a t e w i t h t h e s e manuf a c t u r e d gums, whose s u p p l y , q u a l i t y and p r i c e a r e n o t s u b j e c t t o v a g a r a n c i e s o f s u p p l y , w e a t h e r , p o l i t i c s and l a b o u r c o s t s . The E u r o p e a n and J a p a n e s e m a r k e t s a r e e x p e c t e d t o o f f e r l a r g e p o t e n t i a l f o r d e v e l o p m e n t . I t must be remembered t h a t o v e r 17,000 t o n s o f x a n t h a n w i l l be a v a i l a b l e p e r y e a r a f t e r 1978 and o v e r h a l f o f t h i s must be a b s o r b e d by t h e F o o d I n d u s t r y . Hence t h e m a i n m a r k e t i n g e f f o r t s o f x a n t h a n must n e c e s s a r i l y move t o E u r o p e . P r o b a b l y a d i f f e r e n t r a n g e o f a p p l i c a t i o n s w i l l e v e n t u a l l y d o m i n a t e o u t s i d e t h e US s i n c e use o f s a l a d d r e s s i n g s , meat a n a l o g s , and c a r b o n a t e d b e v e r a g e s i s n o t so d e v e l o p e d . F o r example f r u i t y o g h o u r t m a r k e t s a r e many t i m e s l a r g e r t h a n i n t h e US. The l a r g e s t E u r o p e a n h y d r o c o l l o i d f o o d u s a g e i s i n t h e m o d i f i e d s t a r c h f i e l d . O v e r 150,000 t o n s u s a g e has b e e n r e p o r t e d t o be u s e d i n t h e E u r o p e a n Community. P r o b a b l y t h e b e s t g r o w t h r a t e comes f r o m c e l l u l o s e dérivâtes. CMC a t 60 c e n t s p e r pound has made g r e a t i n r o a d s i n t o t h e s e e d gum m a r k e t . L a r g e v o l u m e s o f CMC a r e r e p o r t e d t o be g o i n g i n t o i n s t a n t soups and c a k e m i x e s , a m a r k e t t h a t x a n t h a n i s also t r y i n g to penetrate. I n summary t h e f o o d i n g r e d i e n t s m a r k e t i s v e r y c o m p l i c a t e d and o n l y t h e most t e c h n i c a l l y c o m p e t e n t and t e c h n i c a l l y m a r k e t ed o r i e n t e d w i l l s u r v i v e . To s e l l gums i n t o new f o o d p r o d u c t s demands a s o p h i s t i c a t e d t e c h n i c a l i n p u t . I t i s e s s e n t i a l t o u n d e r s t a n d p o t e n t i a l a p p l i c a t i o n . Gums a r e m u l t i f u n c t i o n a l . X a n t h a n added t o r e p l a c e an e m u l s i f i e r , w i l l a l s o i n c r e a s e viscosity. T h i s can c a u s e p r o b l e m s i f t h e o t h e r t h i c k e n e r s a r e not reduced.

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

22.

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309

Petroleum and O i l I n d u s t r y A p p l i c a t i o n s . Polymers a r e f i n d i n g i n c r e a s i n g usage i n the o i l i n d u s t r y and developments a r e f o r e c a s t w h i c h c o u l d open u n l i m i t e d p o t e n t i a l . A t t h i s t i m e the market remains i n e x p l o r a t i o n usage. Market developments h e r e h a s b e e n d i v i d e d i n t o two s e g m e n t s : o i l d r i l l i n g muds a n d enhanced o i l r e c o v e r y . O i l d r i l l i n g muds. The f o u r m a j o r p o l y m e r s i n u s e a t t h i s time are xanthan, p o l y a c r y l a m i d e s , m o d i f i e d s t a r c h e s and c e l l u l o s e d e r i v a t i v e s p a r t i c u l a r l y CMC. D e x t r a n a n d p u l l u l a n a r e a l s o trying to get into this industry. B e c a u s e o f i t s s t a b i l i t y t o pH, h e a t , c a t i o n s a n d d i v a l e n t i o n s coupled t o i t s p s e u d o p l a s t i c behaviour under c o n d i t i o n s o f h i g h s h e a r , x a n t h a n gums a r e t h e t e c h n i c a l l y p r e f e r r e d p o l y m e r for l u b r i c a t i o n o f b e n t o n i t mud d t d r i l l o i l wells D u r i n g 1^75 a b o u t 1,80 ing operations w i t h a p o t e n t i a usag predicte y 1980. However r e c e n t p r i c e i n c r e a s e s h a v e c a u s e d s e v e r a l o f t h e m a j o r s t o s w i t c h some o f t h e u s a g e t o CMC e v e n t h o u g h i t t a k e s a l m o s t d o u b l e t h e amount o f CMC t o a c h i e v e t h e same p e r f o r m a n c e effects. X a n t h a n i s a l s o much u s e d i n s u m u l t a n e o u s w a t e r f l o o d i n g and p u s h i n g t e c h n i q u e s u s e d i n t h e N o r t h Sea, where s e a w a t e r c o n t a i n i n g s m a l l q u a n t i t i e s o f x a n t h a n (100 ppm) a r e p u s h e d into injection wells. However, p o l y a c r y l a m i d e s a r e a l s o b e i n g c o n s i d e r e d f o r t h i s a p p l i c a t i o n due t o l o w e r p r i c e . The d r i l l i n g s e r v i c e i n d u s t r y i s c o n t r o l l e d b y a s m a l l number o f s e r v i c e c o m p a n i e s i n c l u d i n g B a r o i d , M i l c h e m , Imco, D r e s s e r i n t h e US w i t h C r o d a and C e c a f r o m E u r o p e . They r e s e l l x a n t h a n o b t a i n e d f r o m K e l c o , G e n e r a l M i l l s o r Rhone Poulenc. Enhanced o i l r e c o v e r y . The g r e a t e s t f u t u r e p o t e n t i a l f o r p o l y s a c c h a r i d e s , l i e s i n enhanced o i l r e c o v e r y . Great i n t e r e s t and r e s e a r c h e f f o r t i s b e i n g c e n t e r e d on r e c o v e r i n g the l a r g e f r a c t i o n o f o r i g i n a l o i l remaining i n p l a c e i n o i l s t r a t a a f t e r c o n v e n t i o n a l r e c o v e r y methods have been u t i l i z e d . A l a r g e i n c e n t i v e f o r d e v e l o p i n g r e c o v e r y enhancement methods e x i s t s i n t h e U n i t e d S t a t e s a s t h e p e r c e n t a g e o f i m p o r t ed o i l f o r d o m e s t i c p u r p o s e s i s i n c r e a s i n g v e r y r a p i d l y . A c c o r d i n g t o the American Petroleum I n s t i t u t e , o f t h e 440 b i l l i o n b a r r e l s o f o i l d i s c o v e r e d i n t h e U n i t e d S t a t e s b y t h e e n d o f 1974, 295.8 b i l l i o n b a r r e l s w o u l d h a v e t o b e r e c o v e r e d b y e n h a n c e d r e c o v e r y , o r a d v a n c e d enhanced r e c o v e r y techniques. I n 1973 t h e G u l f U n i v e r s i t i e s R e s e a r c h C o n s o r t i u m i n a s t u d y w i t h many o f t h e l a r g e o i l c o m p a n i e s s t a r t e d a s e r i e s o f e n h a n c e d r e c o v e r y . I n 1974 v e r y h i g h l y o p t i m i s t i c p r e d i c t i o n s o n s u r f a c t a n t f l o o d i n g gave r i s e t o e s t i m a t e s o f r e c o v e r i n g 500,000 s t o c k t a n k b a r r e l s (STB) p e r day. However s i n c e

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

310

EXTRACELLULAR MICROBIAL POLYSACCHARIDES

t h a t t i m e , r e d u c e d e s t i m a t e s and l o n g e r r e a l i z a t i o n t i m e s have been p r e d i c t e d . A t t h e e n d o f 1975, t h e c o n s o r t i u m were p r e d i c t i n g a n n u a l p r o d u c t i o n r a t e s f o r 1985 o f enhanced o i l b e t w e e n 300-400 m i l l i o n STB, w h i c h w o u l d c a l l f o r a n a n n u a l p o l y m e r demand o f b e t w e e n 200-250 m i l l i o n pounds b a s e d o n 5 8 % E0R b y s u r f a c t ant f l o o d i n g . However i n 1976, t h e G u l f C o n s o r t i u m h a s how l o w e r e d i t s 1986 p r e d i c t i o n t o a r e a l i s t i c g o a l o f 200,000 STB p e r d a y , w i t h a s t a r t i n g d a t e f o r l a r g e s c a l e d e v e l o p m e n t i n 1979. Table 7 gives t h e polymer requirements f o r both cases s t u d i e d . T h i s s t u d y h a s assumed t h a t t h e m a r k e t i s e q u a l l y s h a r e d between p o l y s a c c h a r i d e s and p o l y a c r y l a m i d e s .

POLYMER DEMAN (Thousands o f pounds p e r d a y ) Year

Case A

1979 1980 1981 1982 1983 1984 1985 1986

7.3 14.6 32.8 51.1 76.7 11.0 120.5 138.5

Source:

Case Β 18.3 40.2 79.0 131.5 175.2 215.2 233.6 248.2

G u l f U n i v e r s i t i e s R e s e a r c h C o n s o r t i u m , M a r c h 1976 1. P o l y m e r s demand assumed t o b e 5 0 % p o l y s a c c h a r i d e s , 50% p o l y a c r y l a m i d e . 2. C a s e A - assumes d e v e l o p m e n t t o 200,000 STB/day by 1986. C a s e Β - Assumes d e v e l o p m e n t t o 500,000 STB/day by 1986.

Hence t h i s d e l p h i t y p e e x e r c i s e p r e d i c t s m a r k e t s o f b e t w e e n t o 140 t h o u s a n d pounds p o l y m e r demand p e r d a y b y 1986 s t a r t a t b e t w e e n 7,300 t o 18,300 pounds d a i l y i n 1979. A s s u m i n g t h i s p o t e n t i a l t o be c o r r e c t , t h e n e x t p r o b l e m t o be r e s o l v e d b y t h o s e d e v e l o p i n g p o l y s a c c h a r i d e p o l y m e r s , w i l l b e t h e p o t e n t i a l s p l i t b e t w e e n p o l y s a c c h a r i d e and p o l y a c r y l a m i d e s . The d i f f i c u l t y i n p r e d i c t i n g f u t u r e t r e n d s ( a c c o r d i n g t o t h e Gulf Consortium) i s the general d i s s a t i s f a c t i o n w i t h the current generation of materials. P o l y a c r y l a m i d e s a r e i n t h e r i g h t p r i c e range b u t a r e d e s c r i b ed a s u n d u l y s h e a r s e n s i t i v e a n d s a l t s e n s i t i v e . T a b l e V I I I shows d e l p h i a n a l y s i s o f v a r i o u s f i g u r e s d i s c u s s e d i n numerous s t u d i e s s i n c e 1974. 250 ing

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

22.

Extracellular

WELLS

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Polysaccharides

311

TABLE V I I I USE OF POLYACRYLAMIDE AND POLYSACCHARIDE FOR EOR Polyacrylamide Viscosity

thickening

S a l i n i t y maximum o f f l u i d Maximum r e s e r v o i r t e m p e r ature Divalent

i o n maximum

Polysaccharide

Ideal

10-15 cp a t 10-15 c p a t 20 c p a t 500 ppm 100 ppm 500 ppm 1500-2000 ppm

10000 ppm

15000 ppm

175-200°F

200-225°F

up t o 250°F

200 ppm

5000 ppm

5000 ppm

Permeability o i l pluggin C o s t t/lb

1.30+0.3

T h i s i s b a s e d o n t h e G u l f C o n s o r t i u m recommend c h a r g e o f 10 pounds o f s u r f a c t a n t s , 3 pounds o f a l c o h o l s a n d a b o u t 1 pound of polymer per b a r r e l . Though t h e u s e o f a l c o h o l i s i n some d o u b t , s i n c e i t may be b e t t e r t o i n c r e a s e t h e s u l p h o n a t e r a t i o a t t h e expense o f t h e alcohol. I t s h o u l d a l s o be p o i n t e d o u t t h a t s u r f a c t a n t f l o o d i n g i s n o t t h e o n l y method o f e n h a n c e d r e c o v e r y . B a s i c r e c o v e r y p r o c e s s e s have b e e n a p p o r t i o n e d a s f o l l o w s : - Surfactant recovery 58% - Thermal recovery 29% - Carbon d i o x i d e processes 8% -Hydrocarbon m i s c i b l e processes 5% A recent development i s s t u d y i n g f e a s i b i l i t y o f d e v e l o p i n g s m a l l s c a l e u n i t s t o produce s u r f a c t a n t charges i n c l u d i n g p o l y s a c c h a r i d e s a t t h e o i l f i e l d s i t e . I t has been n o t e d t h a t EOR h a s s t i l l many t e c h n i c a l p r o b l e m s t o s o l v e b e f o r e i t w i l l be a c o m m e r c i a l r e a l i t y . However t h e i n c r e a s i n g f i n a n c i a l s u p p o r t by Energy R e s e a r c h and Development A d m i n i s t r a t i o n i s most w e l l c o m e a n d i n d i c a t e s p o l i t i c a l b a c k i n g w h i c h i s e s s e n t i a l t o make t h i s d e v e l o p m e n t a r e a l i t y . O t h e r A p p l i c a t i o n s . The o t h e r a p p l i c a t i o n s o f m i c r o b i a l p o l y s a c c h a r i d e s h a v e come f r o m t a k i n g t h e m a r k e t o f t h e n a t u r a l p l a n t gums w i t h more r e l i a b l e o r t a i l o r made p r o d u c t s . Z a n f l o has o b v i o u s l y been developed f o r p a i n t and d y i n g applications. Other p o l y s a c c h a r i d e s have found markets i n t e x t i l e s , c o s m e t i c s , p h a r m a c e u t i c a l s and l i q u i d f e e d s . S e v e r a l p a t e n t s have r e c e n t l y been i s s u e d i n Japan f o r p r o d u c t a p p l i cations i n anticancer preparations. The i n d u s t r i a l u s e s a r e more c o m p l i c a t e d t h a n f o o d u s e s and a r e due t o r h e o l o g i c a l p r o p e r t i e s a n d w i d e r a n g e s o f s t a b i l i t y and c o m p a t a b i l i t y w i t h c o n v e n t i o n a l t i c k e n i n g agents and s u r f a c e a c t i v e a g e n t s .

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

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EXTRACELLULAR MICROBIAL POLYSACCHARIDES

The s y n e r g i s t i c e f f e c t s o f x a n t h a n w i t h l o c u s t bean gums is well exploited. However o n a c c o u n t o f t h e c o m p l e x i t y , most o f t h i s know-how r e m a i n s t h e c o n f i d e n t i a l p r o p e r t y o f t h e p r o c e s s o r and s u p p l i e r o f t h e gum. CONCLUSION T h i s paper has t r i e d t o e s t a b l i s h t h e p l a c e o f m i c r o b i a l l y p r o d u c e d gums i n a n e x p a n d i n g i n d u s t r i a l gum m a r k e t . I t i s c l e a r t h a t x a n t h a n gum p r o d u c t i o n w i l l r e m a i n a t a p l a t e a u u n t i l t h e the new p r o d u c t coming i n t o p r o d u c t i o n i s a b s o r b e d . The w h o l e i n d u s t r y r e q u i r e s a v e r y h i g h l e v e l o f t e c h n i c a l e x p e r t i s e and m a r k e t i n g s k i l l t o d e v e l o p i n d u s t r i a l u s a g e . The f u t u r e l a r g e p o t e n t i a l s i n enhanced o i l r e c o v e r y a r e s t i l l a l o n g way o f f a n d much the o i l p r o d u c e r s a n d cial reality. P r o d u c t i o n problems o f f e r m e n t a t i o n d r y i n g and r e c o n s t i t u t i n g d i l u t e s o l u t i o n must be s o l v e d . On a c c o u n t o f t h e l a r g e d e v e l o p m e n t c o s t s n e c e s s a r y f o r b o t h t e c h n i c a l a n d m a r k e t d e v e l o p m e n t i t must b e c o n c l u d e d t h a t o n l y companies d e v e l o p i n g whole range o f m i c r o b i a l p r o d u c t s w i l l predominate. APPENDIX "A" BIOPOLYMER CAPACITY SUMMARY ( M a i n l y Xanthan) tons/year) Date

Capacity

Company

Affiliates

Location

KELCO

Merck subsidiary

San Diego

KELCO

Merck subsidiary

Oklahoma

10,000

End 1976

BIOSYNTHESEMELLE

Rhone P o u l e n c Melle General M i l l s (France)

2,000

Existing

GENERAL MILLS

Rhone P o u l e n c

Iowa

2,500

Mid

TATE & LYLE

Hercules Inc.

NA°

NA°

NA°

TATE & LYLE

Hercules Inc.

NA°

NA°

NA°

T o t a l known c a p a c i t y b y end 1979

3,500

x

Existing

1977

18,000+ M e t r i c

Tons

E s t i m a t e d " c o n v e n t i o n a l " m a r k e t s b y end 1979 15-16,000 M Tons x B e i n g expanded t o 5 , 0 0 0 t / y r b u t i n c l u d e s d e v e l o p m e n t facilities. ° NA = n o t announced.

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22.

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Microbial

Polysaccharides

313

BIBLIOGRAPHY A r n o l d C.W, " C h e m i c a l C h a l l e n g e s i n t h e Q u e s t f o r E n h a n c e d O i l R e c o v e r y " - A m e r i c a n C h e m i c a l S o c i e t y ( A p r i l 4-9 1976). G o g a r t y W.B. " S t a t u s o f S u r f a c t a n t o r M i c e l l a r Methods S.P.E." N a t i o n a l M e e t i n g , D a l l a s , T e x a s ( S e p t . 28 - O c t . 1 1975). H i c k e y J.R. " T h i c k e n e r s a n d S t a b i l i z e r s f o r F o o d ECMRA M e e t i n g of M a r k e t " - Development A n a l y s t s M e e t i n g , London ( A p r i l 1976). K a n g K . S . a n d K o v a c s P. " I n t . C o n g r e s s o f F o o d S c i e n c e " M a d r i d (1974). J e a n e s A. "Food T e c h n o l o g y " (May 1974) 34-39. K e l c o Company " T e c h n i c a K i m u r a H. " A b s t r a c t 3 2 M i n n e a p o l i s (1972). Lawson J . , S u t h e r l a n d I.W. " P o l y s a c c h a r i d e s f r o m M i c r o o r g a n i s m s i n Economic M i c r o b i o l o g y ; ed. by A.H. Rose - A c a d e m i c P r e s s ( i n p r e s s ) . " M a r k e t i n g o f P r i n c i p l e W a t e r S o l u b l e Gums i n P r o d u c i n g C o u n t r i e s " I n t e r n a t i o n a l T r a d e C e n t e r , Geneva ( 1 9 7 2 ) . N a y l o r J . " P r o d u c t i o n Trade and U t i l i z a t i o n o f Seaveeds and Seaweed P r o d u c t s " FAO, Rome ( 1 9 7 6 ) . " R e p o r t o n C h e m i c a l Demand and S u p p l y S t u d y " . " R e l a t i n g on M i c r o e m u l s i o n F l o o d i n g G u l f U n i v e r s i t i e s Research C o n s o r t i u m " Houston, Texas (March 26, 1976). S h a r p J.M. "The p o t e n t i a l o f E n h a n c e d R e c o v e r y P r o c e s s e s " S.P.E. M e e t i n g D a l l a s , T e x a s ( S e p t . 2 8 - O c t . 1 1975) Umland C.W. " P r e s e n t a t i o n f o r F e d e r a l E n e r g y A d m i n i s t r a t i o n " E n h a n c e d O i l and Gas R e c o v e r y Symposium, W a s h i n g t o n (Dec. 1 9 7 5 ) . W h i s t l e r R.L. " I n d u s t r i a l Gums" 2nd E d i t i o n A c a d e m i c P r e s s , New Y o r k ( 1 9 7 3 ) .

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

INDEX Azotobacter (continued) A vinehndii Acetolysis alginic acid production by 14 for the elucidation of structure 183 exopolysaccharide production by 20,24 of D-mannan 121 in relation to alginate synthesis, oligosaccharides 189 metabolism of 22 of xanthan gum 183,185 Acetyl, monosaccharide content Β pyruvic acid 196 Acid whey, xanthan gum from 27 Backbone conformation angles 96 Acylation mechanisms, exopolyBacteria growing in methanol, saccharide 5 Aerobacter aerogenes 17,28 Bacteria, methane and methanol Agrobacterium utilizing 67 radiobacter 267 Bacterial heteropolysaccharides 211,220 rhizogenes 267 Bacterial polysaccharides 107 species 49 Bacterium, soil 211,220 Alcaligenes faecalis 265,306 Batch Alditol acetates, partially methylated .. 187 culture, production of alginic Aldoses, peracetylated aldononitrile acid in 14 derivatives of 115 fermentation 32-38 Alginate(s) 174 kinetics, nitrogen-limiting 74 apparent viscosity vs. rate of shear nitrogen-limiting 71 plots for Azotobacter 21 Berea sandstone cores 246,253,261 microbial 306 Binding, ion 140 synthesis, metabolism of Bingham body, stress vs. shear Azotobacter vinelandii in rate for a 153 relation to 22 Biological function, technological Alginic acid by Azotobacter relevance of 174 vinehndii, production of 14 Biological utility 177 Alginic acid, structure of 20 Biopolymer capacity summary 312 Ammonia chemical ionization 117 Biopolymer injectivity tests, test-well.. 254 Amorphophallus konjac tubers 180 Birefringence 205 Amylose («(l,4)-D-glucan) 104 Block structure 19 Raman spectrum of V 105 Bragg reflection 92 Anabolic fate of glucose 42 Branching, degree of 118 Antimutagenesis 4 Application C field 260 in food products 278 Calcium ion concentration of the growth medium 19 in industry 281 238 Arthrobacter 220 Calf milk replacers 59 Ash separation vs. time 237 Candida boidinii Atom labeling 92 Carbohydrate C-13 N M R spectra on temperature, dependence of 124 Attractive interaction in the X. campestris helices 99 Carbohydrate composition of Zanflo .. 214 Carbon-limited chemostat 63 Aureobasidium ( Pullularia ) monod growth kinetics in 64 pullulans 285 yield coefficients for 69 Azotobacter 155 alginates 21 K-Carrageenan-milk-sugar system 154,155 indicus 220 Casson equation a

317

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318

EXTRACELLULAR MICROBIAL POLYSACCHARIDES

Cations on viscosity, effect of mono- and divalent 218 Cattle feed supplements, liquid 235 C D , effect of temperature on 83 Cell suspension, polymer formation rate in washed 72 Cellobiose 190 Cells, preservation of 10 Cellulose derivatives 304 Cheese spread, pasteurized processed 233 Chemical shifts for O-phosphonomannans and related materials, P-31 .. 125 Chemostat carbon-limited 63,64,69 nitrogen-limited 68, 69, 72 Chondroitin 4-sulfate 107 Chondroitin 6-sulfate 10 Chromatography gas 187 gas-liquid 114,117 high-pressure 120 C M C (sodium carboxymethylcelluluose) 151 Coefficient, non-growth associated 71 Compatibility 257 Concentration behavior, polymer viscosity246 viscosity vs 215 for xanthan solutions, 147 Cone and plate, flow between 165 Conformation 271 angles, backbone 96 of xanthan gum, molecular 90 Continuous culture, production of alginic acid in 14 Core test, injectivity 254-256 Cores, Berea 253,261 Cottage cheese dressing 233 Cottage cheese whey and whey permeate 27 Counter ion, effect of 140 Culture collections 2 maintenance and productivity 1 production of alginic acid in batch and continuous 14 set whey 32 Curdlan 306 gel 270 as microfibrils 269 production properties and application of 265 -type polysaccharides 268

D Darcy's law Degradation, mechanical

156 250

Derivation, peracetylated aldonitriles ( P A A N ) 115 Dermatan sulfate 107 Deuteromethyl groups 118 Dextran(s) 284,286,305 C-13 N M R spectra of 123 derivated 129 in industry 289 from Leuconostoc mesenteroides .... 292 methylated D-glucose components in hydrolyzates of permethylated 120 naturally occurring 285 polyelectrolyte behavior 132 Diffraction pattern for Xanthomonas campestris and Xanthomonas phaseoli 92

Azotobacter vinehndii at various on production of an exopolysac charide by Pseudomonas species, effect of on production of xanthan by Xanthomonas campestris, effect of Dioctyl phthalate Disaccharide backbone height Disaccharide E , lead tetraacetate oxidation of Dynamic viscosity

20

18

18 165 92 188 148

Ε Edamin medium, hydrolyzedpermeate/ 34 Effluent concentration for xanthan gum solutions 249 Elastic modulus of gels 280 Electron impact-M.S. yields 117 Electroviscous effect 131 E n d uses, rheological properties in .... 151 Enterobacter aerogenes 44 Enterobacteriaceae 43 E O R , polyacrylamide and poly saccharide for 311 Erwina tahitica 306 Erwina (Zanflo) 306 Escherichia coli 2,41 Ethylene glycol on gel formation, effect of 273 Exopolysaccharide acylation mechanisms 51 formation of 44 production by Azotobacter vinehndii 20,24 effect of mutations on 50 by Pseudomonas species 16,18

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

319

INDEX Exopolysaccharide ( continued ) synthesis, effect of growth rate on . synthesis, isoprenoid lipids in Extinction angle, effect of sodium hydroxide on Extracellular polysaccharide of Xanthomonas campestris Extracellular substrates, initial pathways for Extrusion

17 46 274 197 42 49

F Feed supplements, liquid cattle 235 Fermentation batch 32-34 conditions 31 modes 3 process 2 repeated-batch 38 semi-continuous 73, 77 Zanflo 213 Fermentor, rate constants for the semi-continuous operation in 75 Field application 260 Field project, evaluating proposed .... 261 Flavobacterium 268 Flood evaluation, polymer 261 Flooding, micellar-polymer 242 Flow behavior, transient 248 between cone and plate 165 through porous media 156 Food(s) additive 231 applications 307 industry 290 products, application in 278 and related products, xanthan gum in 231 systems 151 U.S. consumption of hydrocolloids in 308 Formation of mixed gels 174 Fragments, isolation of 184 Friction reduction 154,156 Fucose synthesis, control of 45 Fuel, atomic 290

G Galactomannan conformation interaction, xanthan gum and locust bean α-D-Galactopyranose residues Galactose medium, glucoseGel(s) breaking strength vs. elastic modulus of

176,178 178,234 234 178 32,33

280

Gel(s) (continued) curdlan formation effect of ethylene glycol on effect of urea on .... mechanism, mixed precipitation process properties, role of polysaccharide molecules in -sol transition, reversible strength, effect of heating temperature on structure, polysaccharide synergistic xanthan unctuous Genes, stabilized

270 234 273 272 174 292 234 149 272 175 174 152 11

improvement of 9 maintenance of 3 Glucans 119 α-D-Glucans, industrially significant.... 284 /M,3-Glucan microfibrils 277 « ( 1 , 4 ) - D - G l u c a n (amylose) 104 Glucose anabolic fate of 42 components in hydrolyzates of permethylated dextrans, methylated 120 -galactose medium 32,33 /M,3-Glucosidic linkages 265 Gluten substitute 241 Glycosaminoglycans 106 Glycitol from oligosaccharide C , pero-trimethylsilyl ether of derived .. 187 Growth kinetics 59 in carbon-limited chemostat, monod 64 -limiting nutrient on exopolysac charide production by Azoto bacter vinehndii, effect of 24 -limiting substrate 15 medium, calcium ion concentra tion of the 19 rate data, specific 60 on exopolysaccharide synthesis, effect of 17 inhibitory specific 59-60 yield for M . mucosa and other microorganisms 66 Guluronic acid 19 Gum(s) consumption of industrial 301 locust-bean 176,178 market for water soluble 299 natural and synthetic water-soluble 300

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

EXTRACELLULAR MICROBIAL POLYSACCHARIDES

320

Gum(s) (continued) solutions, rheological properties of aqueous 144,146 structure 183 world production of selected industrial 303 xanthan 27 H Hansenula capsuhta Hansenula polymorpha

46 59

Heat on viscosity of PS-7 and xanthan gum solutions, effect of 223 Hele-Shaw models 242,244 Helical models 91,96 Helices, attractive interaction in the X. campestris

Ionization ammonia chemical constants of sodium dextran sulfate methane chemical Ionogenic function, effect of nature of the Ionogenic groups Isolation procedures Isoprenoid lipids i n exopolysaccharide synthesis

117 142 117 137 129 2 46

J

Junction zones

175

Κ

9

Helix formation, double 17 Helix, xanthan 93,94,97 Heteropolysaccharide, PS-7 220 Heteropolysaccharide, Zanflo 211 Hexose substrate, fate of 42 HPXan 199 dispersions 204,206 Hyaluronic acid 107 Hydrocolloids in foods, U.S. consumption of 308 Hydrogen bonds, possible 98 Hydrolysates of permethylated dextrans, methylated D-glucose components in 120 Hydrolysates of permethylated D-mannans, methylated D-mannose components i n 119 Hydrolysis, polymer 114 Hydrosulfite on polymer degrada tion, effect of 258 Hyphomicrobium

61

Hystersis loop treatment

150

I Industry application i n 281 applications, petroleum and oil 309 dextran and dextran derivatives 289 pharmaceutical 289 photographic 293 Injection values for various well completions, scaled 253 Injection well pressure response 256 Injectivity behavior 250 Injectivity core test 254-256 Instability of microbes, inherent 3 Instrument ranges 164 Ion(s) counter 128,140 binding 140 macro 131

in carbon-limited chemostat, monod growth constants for the respiration grow ing of bacteria in methanol, Michaelis-Menten of growth nitrogen-limiting batch respiration of substrate inhibition for M. mucosa

64

63 59 74 61 62

L Lead tetraacetate oxidation of disaccharide Ε 188 Length, calculated 172 Leuconostoc mesenteroides 119,284,292 Limitation, molybdate, oxygen, and phosphate 23 Lineweaver-Burk plot 60,62 «-l,6-Linkages, α-maltotriose poly merized through 292 Lipids, carrier 50,51 Lipids, isoprenoid 46 Liquid supplement formulation 236 Locust bean galactomannan inter action, xanthan gum234 Locust bean gum 176,178 LPXan 199 dispersions 204,206

M Macromolecular structure, effect of .... Maintenance coefficients for M. mucosa and other microorganisms a-Maltotriose polymerized through «-l,6-linkages Mannans, structure of D-Mannan, methylation and acetolysis data for

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

135 66 292 118 121

321

INDEX

D-Mannans, methylated D-mannose components in hydrolysates of permethylated 119 β-D-Mannopyranose residues 178 Mannose containing oligosaccharides, structures of 186 Mannose synthesis, control of 45 D-Mannose components in hydro lysates of permethylated D-mannans, methylated 119 α-Mannosidase on oligosaccharide C, action of 187 Mannuronic acid 19 Market for water soluble gums 299 Medium(a) calcium ion concentration of the growth 1 flow through porous 15 formulation 2 glucose-galactose 32,33 hydrolyzed permeate 30 hydrolyzed permeate/edamin 34 set whey 35-37 Metabolism of Azotobacter vinelandii in relation to alginate synthesis .. 22 Metabolism, intermediary 41 Metallurgy 290 Methane chemical ionization 117 Methane utilizing bacteria 67 Methanol effect of the 61 Michaelis-Menten kinetic constants for the respiration of bacteria growing in 63 utilization rate 63 utilizing bacteria 67 Methylation data for D-mannan 121 Methylomonas mucosa

growth yield and maintenance coefficients for kinetics of substrate inhibition for .. respiration rate data of Methylomonas, polysaccharide forma tion by a Micellar-polymer flooding Micellar/surfactant projects testing U.S. reservoirs Michaelis-Menten kinetic constants for the respiration of bacteria growing in methanol Microbes inherent instability of selection and preservation sources of Microbial alginate exopolysaccharide synthesis polysaccharides of commercial significance

58

66 62 62 58 242 262

63 3 10 1 306 40 305

Microbial (continued) polysaccharides, industrial develop ment of 307 Microfibrils, curdlan as 269 Microfibrils, 0-1,3-glucan 277 Microorganisms, growth yield and maintenance coefficients for 66 M i l k replacers, calf 238 Milk-sugar system, Casson plots for /(-carrageenan155 Mobility definition of 244 reduction(s) 243 in Berea sandstone cores 246 for xanthan gum solutions 249 Modification 49

Molybdate limitation Monod growth kinetics in carbonlimited chemostat Monosaccharide content, pyruvic acid acetyl M.S. yields-electron impact Mutability regions, high Mutagens on mutation of strain, effects of Mutants conditional control in a growing culture, proportion .... Mutation(s) development of a stable limiting the opportunity for on production of exopolysaccharides, effect of sequence of events in

23 64 196 117 3 267 9 9 7 266 4 6 50 5

Ν Natural products 304 Newtonian region 169 Newtonian viscosity 145 Nitrogen-limited chemostat 68 polymer production in 69 yield coefficients i n 72 Nitrogen-limiting batch 71,74 N M R of polysaccharides 114 N M R relaxation 82 Nomenclature 172 Nutrient on exopolysaccharide pro duction by Azotobacter vine landii, effect of growth-limiting .. 24 Ο Oil drilling muds industry applications

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

309 309

322

EXTRACELLULAR MICROBIAL POLYSACCHARIDES

O i l (continued) production response, polymer and waterflood 262 recovery, enhanced 242,309,310 Oligosaccharide ( s ) from acetolysis of D-mannan 121 from acetolysis of xanthan gum 188 C, action of α-mannisodase on 187 C, per-o-trimethylsilyl ether of derived gylcitol from 187 in repeating unit of xanthan gum, acetolysis 189 structures of mannose containing .... 186 xanthan acetolysate neutral 185 Optical activity 82 Ordered state 84 Origin of xanthan solution properties molecular 8 Origin of xanthan synergism, molecular 177 Ostwald, power law of 169 Oxygen limitation 23

Ρ Pachysolen tannophilus .118,120 Pentasaccharide repeating unit 90, 92,183 Peracetylated aldonitriles ( P A A N ) derivation 115 Permeability reduction 245 Permeate cottage cheese whey and whey 27 /edamin medium, hydrolyzed34 medium, hydrolyzed30 Permethylation gas-liquid chroma tography /mass spectrometry 117 Permethylation, polymer 114 Per-o-trimethylsilyl ether of derived glycitol from oligosaccharide C .. 187 Petroleum industry applications 309 Petroleum production 291 H of H P X a n and L P X a n dispersions, viscosity vs 204 on PS-7 and xanthan gum solutions, effect of 224 viscosity vs 194,203 on Zanflo viscosity, effect of 217 Pharmaceutical industry 289 Phosphate limitation 23 O-Phosphonohexosans, extracellular yeast 124 O-Phosphonomannans, P-31 chemical shifts for 125 Photographic industry 293 Physiology of polysaccharide synthesis 14 Plate, flow between cone and 165 P

Polyacrylamide for E O R intrinsic viscosity of partially hydrolyzed partially hydrolyzed viscosity vs. shear rate for Polyampholytes Polyelectrolyte behavior, dextran Polyelectrolytes, polysaccharide Polymer degradation, effect of hydrosulfite on demand for enhanced oil recovery .. flood evaluation flooding, micellarformation rate in washed cell suspension

311 258 243 251 128 132 128

258 310 261 242 72

permethylation 114 production in nitrogen-limited chemostat 69 production rate, specific 68 retention 247 -sulfonate interaction 259 synthesis, direction to 41 viscosity-concentration behavior .... 246 and waterflood oil production response 242,262 Polymerization, specific rotation vs. degree of 276 Polysaccharide ( s ) bacterial 107 biosynthesis of Xanthomonas 54 C N M R spectra of 276,279 of commercial significance, microbial 305 concentration, viscosity vs 195,197 curdlan type 268 effect of heating temperature on transmittance and viscosity of 273,274 for E O R 311 formation by a Methylomonas 58 gel structure 175 industrial development of microbial 307 infrared and Raman spectroscopy of 103 molecules in gel properties, role of .. 234 optical rotation and viscosity variations w. temperature for Xanthomonas 83 polyelectrolytes 128 synthesis, control of 54 synthesis, physiology of 14 of Xanthomonas campestris, struc ture of extracellular 197 x-ray diffraction analysis of 279 Polyion 128 Polytran, scleroglucan 306 1 3

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

323

INDEX Pore volume, inaccessible 247,249 Potassium carboxymethyl dextran 135 Potassium dextran sulfate 132 Power law constants 170 fit of data 171 of Ostwald 169 region 145 Precipitation process, gel 292 Preservation of cells 10 long-term 8 of microbes 10 short-term 8 Pressure response, injection well 256 Productivity, culture 1 Products, natural 304 PS-7 effect of heat on viscosity o a new bacterial heteropolysaccharide 220 effect of p H on 224 viscosity of 222, 226 Pseudomonas C 59 Pseudomonas species 15,289 effect of dilution rate on production of an exopolysaccharide by 18 exopolysaccharide production by .. 16 Pseudoplastic system, viscosity vs. shear rate for 147 Pseudoplasticity (shear thinning ability) 160 Pseudoplasticity, xanthan gum 161 Pullulan(s) 284,288,306 proposed uses 293 Purification procedure 163 Pyruvate, xanthan products with intermediate levels of 204 Pyruvic acid acetyl monosaccharide content 196 content, xanthan products of differing 192 content of xanthan, viscosity vs 207

R Rate constants for the semi-continuous operation in fermentor Repeating unit, pentasaccharide Reservoirs, micellar/surfactant projects testing U.S Respiration of bacteria growing in methanol .... kinetics rate data for M . mucosa rates exopolysaccharide production by Azotobacter vinelandii at various

75 183 262 63 61 62 23

24

Respiratory quotient, maximum 78 Retardation vs. shear rate of H P X a n and L P X a n dispersions 206 Retardation vs. temperature 206 Retention times of peracetylated aldononitrile derivatives of aldoses 115 Retorting on viscosities of xanthan gum, effect of 240 Reynolds number 154 Rheological properties of aqueous gum solutions, effect of salts on the 146 in end uses 151 of gum solutions 144 Rheology 268 of xanthan gum solutions 160,167,168 vs. degree of polymerization, specific effect of sodium hydroxide on optical variations w. temperature for Xanthomonas polysaccharide optical

276 274

83

S Saccharomyces cerevisiae 9 Salmonella mutants 49 Salt on viscosity, effect of 200,202,205 Salts on the rheological properties of aqueous gum solutions, effect of .. 146 Sandstone cores, mobility reductions in Berea 246 Scleroglucan (Polytran) 306 Seaweed extracts 304 Seed extracts 304 Selection limiting the opportunity for 6 of microbes 10 sequence of events in mutation 5 Semi-continuous experiment, final yield data for the .. 78 fermentation 73 operation in the 14 L fermentor, rate constants for the 75 Shear plots for Azotobacter alginates, apparent viscosity vs. rate of .... 21 rate for a Bingham body, stress vs 153 vs. retardation of H P X a n and L P X a n dispersions 206 vs. stress for thixotropic material 150 vs. viscosity 198 for polyacrylamide 251 for pseudoplastic or shear thinning system 147 for xanthan solutions ...147,199,251

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

324

EXTRACELLULAR MICROBIAL POLYSACCHARIDES

Shear (continued) thinning ability ( pseudoplasticity ) 160 thinning system, viscosity vs. shear rate for 147 Sodium alginate 23 carboxymethylcellulose ( C M C ) .... 151 dextran sulfate, ionization constants of 142 hydroxide on optical rotation, effect of 274 Soil bacterium 211,220 Solution(s) properties, molecular interpre tation of 84 properties, molecular origin of xanthan 8 rheological properties of gu rheology of xanthan gum 160 viscosity 81 Spectra C NMR 123,124,276,279 Fourier transform infrared 110, 111 polarized infrared 108 Raman 105 Spectrometry, mass 117 Spectroscopy infrared and Raman 103 mass 114,187 nuclear magnetic resonance 114 C-13 122 P-31 124 Staphyhcoccus aureus 46 Starch derivatives 304 Sterilization 30 Strains, colonies of 269 Strength vs. elastic modulus of gels, breaking 280 Streptococcus mutans 286 Stress response, effect of substitution degree on 153 vs. shear rate for a Bingham body .. 153 vs. shear rate for thixotropic material 150 yield 149,169 Structure(s) aggregation of rigid 175 effect of macromolecular 135 polysaccharide gel 175 of xanthan gum, acetolysis for the elucidation of 183 Substitution, effect of degree of 132 on stress response 153 Substrate(s) concentrations, inhibitory specific growth rate at different initial.. 60 fate of hexose 42 1 3

Substrate ( s ) ( continued ) growth-limiting 15 inhibition for M . mucosa, kinetics of 62 initial pathways for extracellular . . . 42 uptake 40 utilization rate correlation, specific 64 Sugar-sugar linkage type 118 Sugar system, Casson plots for K-carrageenan-milk155 Sulfonate-polymer interaction 259 Supermolecular structure 149 Surfactant projects testing U.S. reservoirs, micellar/ 262 Surfactant slug 259 Symmetry, helical 92 Synergism, molecular origin of xanthan 177 Synergistic xanthan gels 174

Τ Temperature on C D , effect of 83 dependence of carbohydrate C-13 N M R spectra on 124 dependence of viscosity 205 on gel strength, effect of heating . . . 272 retardation vs 206 on transmittance and viscosity of polysaccharide, effect of heating 273,274 vs. viscosity 193,198 for Xanthomonas polysaccharide, optical rotation and viscosity variations with 83 on Zanflo viscosity, effect of 215 Thermal changes 109 Thermal processing 238 Thixotropic material, stress vs. shear rate for 150 Thixotropy 149 Time, ash separation vs 237 Tom's effect 154 Transmitance of polysaccharide, effect of heating temperature on 273,274 Tree exudates 205

U Unctous gels Urea on gel formation, effect of

152 272

V Viscosity (ies) 129 commercial vs. purified 163 vs. concentration 215 -concentration behavior, polymer .... 246 determination, intrinsic 164

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

325

INDEX Viscosity ( ies ) ( continued ) dynamic 148,150 effect of mono- and divalent cations on 218 effect of p H o n 203,217 effect of salt on 200,202 effect of sodium hydroxide on intrinsic 274 effect of temperature on Zanflo 215 functions 130 intrinsic 171,194,203,258 measurement of xanthan 193 Newtonian 145 vs. p H 194 of H P X a n and L P X a n dispersions 204 vs. polysaccharide concentration 195,197 of polysaccharide, effect of heating temperature on 273,27 ofPS-7 222 effect of heat on 223 vs. pyruvic acid content of xanthan 207 vs. rate of shear plots for Azotobacter alginates, apparent.. 21 ratios, displacements in Hele-Shaw model at different 244 reduced 129 vs. shear rate 198 and concentration for xanthan solutions 147 for polyacrylamide 251 for pseudoplastic or shear thinning system 147 for xanthan solutions 199,251 solution 81 steady shear 150 temperature dependence of 193,198,205 xanthan gum 161 effect of retorting on 240 for Xanthomonas polysaccharide .... 83 W

Waterflood oil production response .... 262 Waterflooding, polymer 242 Well completions, scaled injection values for various 253 Whey acid-set 34 composition, acid 28 culture set 32 medium, set 35-37 and whey permeate, cottage cheese 27 xanthan gum from acid 27 X Xanthan 107,305 acetolysate neutral oligosaccharides 185 Fourier transform infrared spectra of 110, 111

Xanthan (continued) gels, synergistic 174 gum(s) 243 acetolysis for the elucidation of structure 183,185 acetolysis of oligosaccharides in repeating unit of 189 from acid whey 27 effect of retorting on viscosities of 240 for enhanced oil recovery 242 in foods and related products 231 injectivity core test 256 -locust bean galactomannan interaction 234 molecular conformation and interactions 90

pseudoplasticity 161 rheology 167,168 solutions effect of heat on viscosity of .... 223 effect of p H on 224 effluent concentration and mobility reduction for 249 rheology of 160 structure 233 viscosities 161 viscosity vs. shear rate for 251 helix, 5/1 93,97 helix, 5/2 94 laboratory purification of 193 native conformation 176 pentasaccharide repeating unit of .... 92 products of differing pyruvic acid content 192 products with intermediate levels of pyruvate 204 solution properties, molecular origin of 81 solutions, viscosity vs. shear rate for 147,199 synergism, molecular origin of 177 viscosity measurement of 193 viscosity vs. pyruvic acid content of 207 by Xanthomonas campestris, effect of dilution rate on production of 18 Xanthomonas campestris 2,15,27,43,90,160, 177,183,192,231 diffraction pattern for 92 effect of dilution rate on production of xanthan by 18 helices, attractive interaction in .. 99 structure of extracellular polysaccharide of 197 jughndis 190

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

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EXTRACELLULAR MICROBIAL POLYSACCHARIDES

Xanthomonas (continued)

phaseoli 91,190 diffraction pattern for 92 polysaccharide, optical rotation and viscosity variation with tem perature for 83 polysaccharides, biosynthesis of 54 X-ray diffraction analysis of poly saccharide 279 Xylose 190

Yield (continued) coefficients in nitrogen-limited chemostat data for the semi-continuous experiment, final stress

Yield coefficients for carbon-limited chemostat

69

78 149,169

Ζ

Zanflo carbohydrate composition of Erwina

Y

72

214 306

fermentation 213 a novel bacterial heteropoly saccharide 211 viscosity, effect of p H on 217 viscosity, effect of temperature on .. 215

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.