Advances in Applied Microbiology, Volume 28

ADVANCES IN

Applied Microbiology VOLUME 28

CONTRIBUTORS TO THIS VOLUME

Sumbo H. Abiose

K. E. Aidoo M . C. Allan P. Brodelius

R. Hendry Rup La1 Torbjorn G. I. Ling Vedpal Singh Malik Bo Mattiasson

K. Mosbach Matts Ramstorp B. J. B. Wood

ADVANCES IN

Applied Microbiology Edited by ALLEN 1. LASKIN Exxon Research and Engineering Company Linden, New Jersey

VOLUME 28

@

1982

ACADEMIC PRESS A Subsidiary of Harcourt Brace Jovanovich, Publishers

New York London Paris San Diego San Francisco SBo Paulo Sydney Tokyo Toronto

COPYRIGHT @ 1982, BY ACADEMIC PRESS,INC. ALL RIGHTS RESERVED. NO PART OF THIS PUBLICATION MAY BE REPRODUCED OR TRANSMITTED IN ANY FORM OR BY ANY MEANS, ELECTRONIC OR MECHANICAL, INCLUDING PHOTOCOPY, RECORDING, OR ANY INFORMATION STORAGE AND RETRIEVAL SYSTEM, WITHOUT PERMISSION IN WRITING FROM THE PUBLISHER.

ACADEMIC PRESS,INC.

111 Fifth Avenue, New York, New York 10003

United Kingdom Edition published b y ACADEMIC PRESS, INC. ( L O N D O N ) LTD. 24/28 Oval Road, London N W l 7DX

LIBRARY OF

CONGRESS CATALOG CARD

NUMBER:5 9 - - 1 3 8 2 3

ISBN 0-12-002628-7 PRINTED IN THE UNITED STATES OF AMERICA

82 83 84 85

9 8 7 6 5 4 3 2 1

CONTENTS LIST OF CONTRIBUTORS ....................................................

ix

Immobilized Plant Cells

I. Introduction

P. BRODELIUSAND K. MOSBACH ......................... .. .. .... ... ... .

11. Immobilized Plant Cells 111. Riosynthetic Capacity of

Permeabilized Plant Cel V. Immobilization of Protoplasts

IV.

References

.......... ...., .., ....., ., .... ....., ., . ., .. .. ... ...

.......... . . . . . . . . . . . . . . . . . . . , . . . . . . . . ... ......................... ...... .. ....... .

1

4 14 22

24 24 25

Genetics and Biochemistry of Secondary Metabolism

VEDPAL SINGH MALIK .............................................

1. Introduction 11. Illegitimate Genome Sequences . . . . . . . . . . . . . . . . . . . 111. Enzymes of Semndary Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

IV. Controlling Effect of the Environment . . . . . . . . . . . V.

V1. VII. VIII. IX.

X.

..........

Genetics of Sermdary Metabo Control of Secondary Metaboli ............... Regulation of Autotoxicity . . . . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . Secondary Metabolism, Sporulation, and Exoenzyme Formation . . . . . . . . . . . Role of Semndary Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Epilogue.. . . . . . . . ............................................ References . . . . . . . . . . . . . . . .

28 31

32 38 39 53

86

94 97 101 101

Partition Affinity Ligand Assay (PALA): Applications in the Analysis of Haptens, Macromolecules, and Cells

€30 I. 11. 111.

IV.

MATTIASSON, MATTS RAMSTOW, AND

TORBJORN G. I. LING Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Phase Partitioning.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Principle of Partition Affinity Ligand Assay (PALA) . . . . . . . . . . . . . . . . . . Phase Systems . . . . . . . , . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V

117 118 118 120

vi

CONTENTS

V. VI . VII . VIII . IX . X. XI . XI1. XI11.

Modifcation of Partition ..................................... Influence of Salt on Partition .................... Modification of Partition by Addition of Charged Polymers . . . . . . . . . . . . . . . . Use of Hydrophobicity in Alteration of Partition .................... Biospecifc Interactions-Afinity Partition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Separator Molecules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

....................... Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Discussion . . . . . . . . . . . ...........

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

121 122 123 124 126 126 127 133 145 146

Accumulation. Metabolism. and Effects of Organophosphorus Insecticides on Microorganisms

RUP LAL I. I1. 111. IV. V. VI .

Introduction .......... ..................... Entry of Organophosphorus Insecticides into Microbial Environments . . . . . . Accumulation . . . . . . . . Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Effects of Organophosphorus Insecticides on Microorganisms . . . . . . . . . . . . . . Summary and Future Prospects ....................................... Appendix ............... References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

149 151 152 153 177 191 193 195

Solid Substrate Fermentations

K . E . AIDOO. R . HENDRY.AND B . J . B . WOOD ........... I. ................................ I1. I11. Design Considerations and Types of Solid-state Fermentors . . . . . . . . . . . . . . . IV . V . Advantages and Disadvantages of Solid-state Fermentation . . . . . . . . . . . . . . . VI . Future Developments of Solid-state Fermentation Systems VII . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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

201 202 224 226 229 231 232 233

Microbiology and Biochemistry of Miso (Soy Paste) Fermentation

SUMBOH . ABIOSE. M . C . ALLAN.AND B . J . B . WOOD I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I1. Fermented Soy Products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

239 241

CONTENTS

. .

111 IV V. VI . VII . VIII .

IX. X. XI.

XI1.

INDEX

vii

Fermented Rice Products ............................................ History of Miso Production ........................................... Types ofMiso ...................................................... Raw Materials for Miso Production .................................... Ratio of Raw Materials ............................................... Treatment of Raw Materials .......................................... Koji ............................................................... Moromi ............................................................ Chemical Composition of Miso ........................................ Future Developments in Miso Production .............................. References .........................................................

242 245 247 248 251 252 253

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

267

CONTENTS OF PREVIOUSVOLUMES...........................................

2.55 257 259

261

271

This Page Intentionally Left Blank

LIST

OF CONTRIBUTORS

Numbers in parentheses indicate the pages on which the authors' contributions begin

SUMBOH. ABIOSE, Department of Applied Microbiology, University of Strathclyde, Glasgow G1 IXW, Scotland (239)

K. E. AIDOO,~Biotechnology Unit, University of Strathclyde, Glasgow G1 IXW, Scotland (201) M. C . ALLAN, Department of Applied Microbiology, University of Strathclyde, Glasgow GI 1 X W, Scotland (239)

P. BRODELIUS, Department of Pure and Applied Biochemistry, Chemical Center, University of Lund, S-22007 Lund 7, Sweden (1) R. HENDRY,Department of Chemical and Process Engineering, University of Strathclyde, Glasgow G1 IXW, Scotland (201) Department of Zoology, University of Delhi, Delhi 110007, India (149)

RUP L A L , 3

C . I. LING, Department of Pure and Applied Biochemistry, Chemical Center, University of Lund, S-22007Lund 7, Sweden (117) VEDPAL SINGH M A L I K , ~The Upjohn Company, Kalamazoo, Michigan 49001 (27) TORBJORN

Bo MATTIASSON, Department of Pure and Applied Biochemistry, Chemical Center, University of Lund, S-22007 Lund 7, Sweden (117) K. MOSBACH, Department of Pure and Applied Biochemistry, Chemical Center, University of Lund, S-22007 Lund 7, Sweden (1) 'Present address: Department of Food Science, University of Ife, Ile-Ife, Nigeria. 2Present address: Department of Biological Sciences, University of Science and Technology, Kumasi, Ghana. 3Present address: Department of Zoology, Sri Venkateswara College, University of Delhi, Dhaula Kuan, New Delhi 110021, India. 4Present address: Philip Morris Research Center, P.O. Box 26583, Richmond, Virginia 23261. ix

X

LIST OF CONTRIBUTORS

MATTS RAMSTORP, Department of Pure and Applied Biochemistry, Chemical Center, University of Lund, S-22007 Lund 7, Sweden (117) B. J. B. WOOD, Departinent of Applied Mimohiology, University of Strutltclyde, Glasgow G1 IXW, Scotland (201, 239)

Immobilized Plant Cells P. BRODELIUSAND K. MOSBACH Depurttneiit of Pure and Applied Biochemistry, Cheiiiicul Center, litiivmsit!l of Lutzrl, L u d , Sweden

I. Introduction . . . . . . . . . . . ........................ A. Immobilized Biocatalysts . . . . B. Plant Cell Cultures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Immobilized Plant Cells . . . . . . . . . A. Iminobilization Techniques . . . . . . . . . . . . . . . . . . . . . . . . . . B. Viability of Immobilized Plant Cells C. Reactors for Imrnobili 111. Biosynthetic Capacity of Immobilized Plant Cells . . . . . . . . . . . A. Biotransformations . . . . . . . . . . . . . . . . . . . . . . . . . . B. Synthesis from Precursors . . . . . . . . . . . . . . . . . . . . . . . . . . . C. De N o w Synthesis., . . . . . . . . Iv. Permeabilized Plant Cells . . . . . . . . . . . . . . . . . . . . . . . V. Immobilization of P VI. Perspectives . . . . . .

1 1 3 4 6 10 12 14 14 18 20 22 24 24 25

I. Introduction A. IMMOBILIZED BIOCATALYSTS There has been considerable interest during the last decade in the immobilization of enzymes and cells (Mosbach, 1976; Brodelius, 1978). Immobilization is usually carried out by one of the following procedures: (1) covalent binding, (2) adsorption, (3) entrapment or microencapsulation, or (4) aggregation of enzymes using bifunctional agents leading to insoluble aggregates (Fig. 1). Naturally occurring polysaccharides such as agarose, alginate, various acrylate polymers, or controlled pore glass are usually used as support material. The support (i.e., carrier or matrix) is generally in the beaded form, although enzymes and/or cells bound to or within membranes are also being applied. Such immobilized preparations have found use in three major areas: (1) for the production of biotechnologically interesting substances, (2) in analysis, and (3) in medicine. The advantages that are gained using immobilized preparations are considerable. For instance, in the medical area, missing enzymes can be held in “extracorporal shunts” while immobilized within small polymer beads. On passage of the blood through such beads, toxic components can be removed (Chang, 1977). The alternative approach of injecting soluble enzymes leads to immunogenic reactions and other complications. In the analytic field, immobilization of enzymes 1 ADVANCES I N APPLIED MICROBIOLOGY, VOLUME 28 Copyright 0 I%?. hy Academic Press, Inc All rights of reproduction In an) form resewed ISBN 0.12-002628-7

P. BRODELIUS AND K. MOSBACH

FIG. 1. Alternative principle methods to immobilize enzymes and other biocatalysts. (a) Covalent coupling; (b) entrapment; (c) adsorption; (d) cross-linking.

directly on or close to the transducer (..a,, an electrode or thermistor) gives rise to quicker and more sensitive analyses (Guilbault, 1976; Mosbach and Ilanielsson, 1981). Finally, for production purposes, inimobilization allows reuse of the biocatalyst, often leads to increased stability, and permits continuous processing. A cost analysis comparing the use of an immobilized cell system producing aspartic acid with that of the conventional batch process using intact cells revealed a cost reduction of 40% due mainly to decreased costs of catalysts and labor (the additional price of the support material does not adversely change the overall balance) (Chibata, 1980). The procedure of choice for the immobilization of cells is usually entrapment, whereby the size of the polymer network surrounding the cells is chosen to allow in and/or out diffusion of substrate and/or product and at the same time keep the cells physically encaged. Apart from early isolated attempts to entrap cells in polyacrylainide (Mosbach and Mosbach, 1966), a major breakthrough first came about in the early seventies. Today, some processes employing immobilized cells are already in commercial use including the formation of L-malic acid and L-aspartic acid (Linko and Larinkari, 1980). The immobilized cells hitherto studied and put to use have been predominantly of microbial origin. Originally, single enzyme activities, such as aspartase for

IMMOBILIZED PLANT CELLS

3

+

the conversion of fumarate NH4+ to asparate, were utilized. Prior to entrapment, the cells are usually treated with organic solvents, metals, or heat to minimize side reactions which usually cause them to become nonviable. However, during a series of studies on steroid transformation it was found that single step steroid transformations, such as 11p-hydroxylation leading from Reichstein’s compound S to cortisol and subsequently to prednisolon by A’, 2-dehydrogenation, could also be carried out using living and growing cells (within the polymer beads) without the formation of disturbing side products (Mosbach and Larsson, 1970). In fact, the use of immobilized Iiving cells is gaining in importance because it is possible to utilize these cells for more complex coenzyme-requiring multistep enzymic reactions, such as those involved in ethanol production from glucose or penicillin biosynthesis. Since cells can grow within these beads, the original biotransformation or de novo synthetic capacity of the immobilized species can often be increased severalfold (Larsson et al., 1976). More recently, even spores have been entrapped and allowed to grow within the beads yielding a uniformly distributed biocatalyst. The technique of immobilization has been refined during the last 10 years so that now any of the members of the sequence enzyme-multienzyme-organelle-cell can be immobilized with retention of activity. B. PLANTCELLCULTURES Before we describe the immobilization of plant cells and the properties of such preparations, we would like to discuss briefly the potential applications of plant cell cultures. A large number of natural products isolated from higher plants are today utilized in the food, cosmetic, and pharmaceutical industries. For instance, about 25% of all prescribed drugs contain compounds isolated from higher plants; the most common of these substances are listed in Table 1. As can be seen, most of them are various kinds of alkaloids. In recent years the supply of some of these plants has become difficult to maintain, and it is likely that the number of such species will increase in the future. Therefore, there has been a search for alternative ways to obtain these valuable substances, and plant tissue cultures appear to be the most, if not the only, promising alternative. A large number of different substances have been obtained in culture, but low productivity is a recurring problem that can, however, be overcome by selection of high-producing cell lines. It was not until very recently that such selection techniques were developed, and the number of high-producing cell lines reported is therefore rather limited. In Table I1 some examples of cultures producing the compound of interest in equd or higher amounts than the parent plant are listed. Many of the listed com-

4

P. BRODELIUS AND K . MOSBACH

TABLE I THEMOST COMMON AND ESSENTIAL DRUG COMPOUNDS DERIVED FROM HIGHER PLANTSO 1. 2. 3. 4. 5. 6.

7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.

Steroids (from diosgenin) Codeine Atropine Reserpine Hyocyaniine Digoxin Scopolaminc Digitoxin Pilocarpine Quinidine Colcinine Emctine Morphine Quinine Sennosides Tuhocurarine Vincristine

"Zenk (1978).

pounds are of commercial interest. Plant tissue cultures thus have a potential for the production of secondary plant products, notably high-cost substances such as drug and fragrant compounds. Biotransforination is another area where plant tissue cultures have been considered as a potential catalyst. The most studied system, i.e., the stereospecific hydroxylatiori of digitoxin to digoxin, has reached a state close to commercialization (Reinhard, 1979). Another biotransformation that may prove possible with plant cell cultures is the conversion of thebain to codein, a very important drug, even though no reports have been published yet on this subject. The potential use of plant tissue cultures in the synthesis and transformation of natural compounds has been more extensively discussed in a number of recent reviews (Zenk, 1978; Kurz and Constabel, 1979; Reinhard and Alfermann, 1980; Staba, 1980).

II. Immobilized Plant Cells The area of immobilized cells was recently extended with the report on the immobilization of plant cells grown in suspension (Brodelius et al., 1979a). These investigations were initiated to study plant cell behavior in a milieu simulating that prevailing in normal tissues, as well as to establish possible advantages of using immobilized plant cells for the production or biotrans-

CELL CULTURES

Prnd1ic:t

W’HICH

PRODUCE

PRODUCTS IN

Culturtt condition

Plant sprtrirs

Ginsengoside Anthraquinorits Rtismalinic acid

Pflntrx ginseng .t!irinrln citrifoliu C h h s hiumei

C S

Shikoriin

I,itho.rpmwum i,t-ythrorhi;oii L’wwiu turn Dioscoreu deltoides Stephania cepharantha Coffen arabica Gathnranthu.s roseua d ndrographis puniculnte Cathuranthus roseus Cutharanthus roseus Macieayn microcarpa Ammi t-isnuga Nicotiatia tahocrrm Micotiarin tnhncum

c c

Antlimr~uiiionrs Diosgenin Biscoclaurine Caffein .4jmalicine

Paniculide B Serpentine Serpentine Protopine Visnagin Clutathione Ul)iquinonc-10

“ C , Calliis trulture; S, cell suspension

(iiiltiiw

TABLE I1 AMOUNTS EQUALTO

S

S C C S

C S C C C S

S

(from Z e r k 1978).

OR

Cell culture (% dry weipli~!

LARGERTHAN

Yit:ld (gm/litrr)

27

-

18 15

2.5 3.6

12 6 2 2.3 1.6 1.0 0.9

-

0.8 0.5 0.4 0.31 -

O.O.%

-

THE

PARENT PLANTa

Plant (% dry wciglit)

6 8 5

1.5 0. fi

8 10

n

2,

-

0.8 1.6 0.3 0 0.5 0.5 0.32 0.1 0.1 0.008

0.26

0.16 -

-

0.22 0.045

parent plant

4.5 2.2

-

Kitio ccll ciiltiirc/

1 3 1 3 m

1.6 1 1.25 3 10 12

6

P. BRODELIUS AND K. MOSBACH

ii

.

U

I product

callus culture

suspa~Slarculture

Immobilized @ant cells

'd

immobilized protoplasts with cell walls

I /

process lor BIodKtbn or trmslormation 01 natural products

immobilized prolopl.sIs

FIG.2 . Alternative approaches to prepare and immobilize plant cells. (From Brodelius et a l . , 1980.)

formation of valuable plant cell products. As to the latter, special emphasis was laid on the question of whether plant cells in the immobilized state show higher production of secondary metabolites as well as changes in the excretion pattern, i.e., release of product into the medium. The combination of the plant cell culture technique with that of the immobilization technique is schematically illustrated in Fig. 2. As can be seen, after the establishment of a culture the cells can be immobilized and used in a process. Alternatively, protoplasts can be made and subsequently immobilized and utilized in a similar way.

A. IMMOBILIZATION TECHNIQUES In our initial studies on the immobilization of whole plant cells (Brodelius et al., 1979a,b), we chose to entrap the cells in Caz+-alginate (Kierstan and Bucke, 1977) since in previous studies on the immobilization of microbial cells, we found this method to be simple, mild, and giving a high yield of active biocatalyst. Alginate is a polysaccharide composed of guluronic acid and mannuronic acid and thus containing one carboxyl group on each sugar monomer. In the presence of Ca2+or other multivalent ions, alginate forms a gel through the formation of ionic bridges. This gel can be solubilized by addition of a Ca2+complexing agent such as phosphate ion, citrate, or EDTA. The possibility to reverse the immobilization, i. e., to obtain the immobilized biomaterial back

7

IMMOBILIZED PLANT CELLS

in free solution andlor suspension, was an additional major advantage of

this immobilization technique in our initial studies. The immobilized plant cells could thus be released and studied in various ways. Furthermore, it is relatively simple to immobilize cells under sterile conditions, which is essential when working with slowly growing plant cells. The immobilization in alginate is simply to carry out as mentioned previously, and large quantities of immobilized cells are readily prepared. Filtered cells [up to 50% cells (wet weight) can easily be entrapped] are added to the alginate solution (2-5% wlw of the sodium salt), and the suspension is dripped into a medium containing at least 50 inM CaC12.The beads formed are left for 30-60 minutes in this medium and then washed and transferred to the appropriate medium, which should contain at least 5 mM CaCI, to stabilize the beads. Small batches are easily prepared by the utilization of sterile plastic syringes with or without a needle. If needles are used in order to obtain small beads, care must be taken to avoid clogging of the needle by larger cell aggregates. If possible, the cell suspension can be filtered through a nylon net of appropriate mesh to avoid problems of this kind.

FIG. 3. Device for large-scale preparation of alginateentrapped plant cells. (A) Medium container; (B) lid; (C) air outlet; (D) reservoir for alginate-cell suspension; (E) six nipples (4 = 1 mm) for bead formation; (F) sterile air inlet.

C

.

8

P. BRODELIUS AND K . MOSBACH

For larger batches of alginate-entrapped cells, we have designed a special device for the immobilization procedure. This device is shown in Fig. 3. The alginate suspension, contained in a reservoir (D), is forced through six nipples [inner diameter (4)= 1 mm] (E) by a light pressure ofsterile air (F).Up to 300 gin of beads can be made within a few minutes, but there should be no problem in scaling up. The whole device is autoclaved with the Ca‘+-containing medium in the medium container (A), and the cells and alginate are mixed and subsequently poured into the reservoir (D). Beads of uniform size are formed (Fig. 4). Also, in this case it is advantageous to remove larger cell aggregates from the suspension before immobilization. Recently, we investigated some other gel-forming polymers for the entrapment of whole plant cells (Brodelius and Nilsson, 1980). Some of the polymers tested form a gel on cooling from elevated temperatures (40-50°C). Beads can be molded by pouring a warm suspension of the polymer and the plant cells into a form. We use two teflon plates, one of which is tightly covered with holes (4 = 1-2 mm), for the molding. The plates, shown in Fig. 5 , are held together by clamps. After the gel has solidified, the plates are taken apart and the “cylindrical beads” (Fig. 6) are taken out, washed, and placed in an appropriate medium. This molding technique was originally used for the immobilization of animal cells (Nilsson and Mosbach, 1980).It can be pointed out that there are no major problems to working under sterile conditions with this technique, which, however, is less suitable for the preparation of large quantities of immobilized cells. The method has been used for the immobilization of plant cells in agarose (2.5% wlw final concentration), agar (2%wlw final concentration), and K-carrageenan (1.5%wlw final concentration). Carrageenan is a polysulfonated polysaccharide that forms a relatively strong gel in the presence of potassium ions. After molding, the carrageenan beads are, therefore, placed in a medium containing 0.3 M KCI for 30-60 minutes to improve the stability of the gel. Beads of carrageenan can also be made by dripping the warm suspension of carrageenan and cells into a medium containing 0.3 M KCI. The beads formed are, however, not of uniform size and they are also irregular. The molding form has also been used to immobilize cells in polyacrylamide and gelatin (10%w/w final concentration) cross-linked with glutaraldehyde. Finally, we have immobilized plant cells in copolymers of alginate or agarose and gelatin. After bead formation, treatment with glutaraldehyde was carried out to cross-link the gelatin and thereby make the beads more stable, As can be seen in Table 111, however, the glutaraldehyde treatment adversely affected cell respiration and cell growth; consequently, these immobilization methods have been abandoned.

IMMOBILIZED PLANT CELLS

9

FIG. 4. Alginate beads containing cells of Catharanthus ruseus made with the device schematically shown in Fig. 3.

So fidr we have tested a few methods for the immobilization of plant cells, but there are still many other methods which deserve to be investigated. The most convenient technique of those we have tested, especially for the preparation of large quantities of immobilized cells, is entrapment in Ca2+-

10

P. BRODELIUS AND K. MOSBACH

FIG.5. Teflon plates for molding of “cylindrical beads.” (A) Molding plate; (B) support plate; (C) clamps for holding plates together; (D) piston for removal of heads from the molding plate.

alginate. Agarose and agar have the advantage that no additions to the medium are required as with alginate (Ca”) and carrageenan (K+).

B. VIABILITYOF IMMOBILIZEDPLANTCELLS The most interesting property of plant cells from a biotechnological point of view is their ability to carry out specific biosynthetic reactions. This aspect will be discussed in the subsequent section, and in this section more basic characteristics related to cell viability will be discussed. The viability of immobilized plant cells can be tested in various ways. By exposing the cells to a plasmolyzing agent, e.g., glycerol, some information on the integrity of the plasma membrane can be obtained. Naturally, an intact plasma membrane is essential for a retained viability of the cells. This test is carried out in the following way. The bead containing the plant cells is cut into thin slices which are studied under the microscope after addition of the plasmolyzing agent. The various preparations of immobilized plant cells were tested in this way, and the results are listed in Table 111. As can be seen, all the immobilized cells, except for those entrapped in polyacrylamide, were plasmolyzed and thus appeared to have intact plasma membranes.

IMMOBILIZED PLANT CELLS

11

FIG. 6. Agarose beads containing cells of Catharunthus roseus made by molding with the plates shown in Fig. 5 .

Another requirement for viability of immobilized cells is the ability to respire. Respiration is conveniently measured with an oxygen electrode. With a preparation of immobilized cells there can, however, occur diffusion barriers that may hinder quantitative measurement of the respiration. To minimize such diffusion limitations, the beads should preferably be cut into small pieces before the measurement. As can be seen in Table 111, entrap-

12

P. BRODELIUS AND K . MOSBACH

TABLE 111 COMPAHISON OF VARIOUS PREPARATIONS OF IMMOBILIZED Catharanthus roseus CELLS" ~~

Preparation

~

~~

~~~~

~~~

Plasmolysis

Respiration

Cell growth

+ + + + + + +

+ + + +

+ + + +

Alginate Agarose Agar Carrageenan Alginate gelatin Agarose gelatin Gelatin Polyacrylainide

+ +

-

-

-

-

-

-

-

-

-

"Brodelius and Nilsson (1980).

inent in alginate, agarose, agar, or carrageenan did not affect cell respiration. The cells within the beads treated with glutaraldehyde had, however, lost their capacity for respiration, and thus these immobilization techniques cannot be employed if viable cells are desired. The ultimate criteria of cell viability are cell growth and cell division. The data in Table 111demonstrate the growth of cells that retained their respiratory ability. After incubation in a complete medium (7-12 days), the number ofcells had increased to the extent that the beads started to burst. To avoid this occurrence in biosynthetic studies, a growth limiting medium can be used. In conclusion, plant cells immobilized by entrapment in alginate, agarose, agar, or carrageenan are fully viable. We have even reestablished suspension cultures from cells that have been immobilized for some time ( 2 3 weeks).

c. REACTORSFOR IMMOBILIZED PLANT CELLS Plant cells are grown efficiently in suspension in a batch culture. To obtain larger quantities it is, however, necessary to employ some kind of fermentor. The utilization of ferinentors used for cultivation of microorganisms can rarely be used directly for plant cells. Often the fermentor must be modified to suit plant cell cultivation (Martin, 1980). One of the main problems of such cultivation is the sensitivity of plant cells to shear forces. In an immobilized state, these problems will be reduced or eliminated by the protection of the embedded cells by the polymeric matrix. No investigations have yet been carried out with immobilized plant cells in fermentors. Immobilized plant cells have been studied in batch reactors (Brodelius et al., 1979a; Alfermann et al., 1980) and in column reactors of continuous type (Veliky and Jones, 1980; Brodelius and Nussbaum, unpublished results) and

13

IMMOBILIZED PLANT CELLS

hole for sampling

LCI aqueous phase containing precursors chloroform for extraction products of

rmp I

column containing immobilized plant cells

FIG.7 . Schematic diagram ofa recirculated column reactor with continuous extraction. (From Brodelius et a l . , 1980.)

FIG. 8. Schematic diagram of a column bioreactor for immobilized plant cells. (A) Column with immobilized cells; (B) air inlet; (C) air outlet; (D) airlift pump; (E) medium reservoir; (F) sampling outlet. (From Veliky and Jones, 1980.)

14

P. BRODELIUS AND K. MOSBACH

recirculating type (Brodelius et al., 1979a, 1980). In the latter reactor, a substrate solution is recirculated through a column containing the immobilized cells, and lipophilic compounds produced by the cells can be continuously extracted with methylene chloride or chloroform (Fig. 7). A column bioreactor for immobilized plant cells (Fig. 8) has been designed weliky and Jones, 1980). The aeration of the reactor was carried out in two different ways, and it was found that a straight upward aeration was more efficient than aeration through an airlift pump system.

Ill. Biosynthetic Capacity of Immobilized Plant Cells From a biotechnological point of view, the ability of plant cells to transform and synthesize very complex compounds of commercial interest is most important. We will divide the biosynthetic reactions into three categories, i. e., simple biotransformations, synthesis from added precursors, and de nova synthesis.

A. BIOTRANSFORMATIONS A great number of transformations have been carried out with plant cells in culture (Reinhard and Alfermann, 1980). The transformations studied are mostly stereospecific and involve the addition of a chemical group, such as in hydroxylation, acetylation, or glucosylation, or the removal of a chemical group, such as in demethylation. In our first studies on immobilized plant cells we wanted to investigate whether immobilized cells retained their transformation capacity. We chose the 126-hydroxylation of the steroid digitoxin to digoxin by cells of Digitalis lanatu as a representative example of biotransformations (Fig. 9, line A). The alginate-entrapped DigitaZis cells transformed digitoxin added to the medium to digoxin at approximately the same rate as freely suspended cells of the same strain (Brodelius et al., 1979a). The immobilization does not obviously affect the hydroxylating capacity of these cells to any great extent. As can be seen in Fig. 10, the hydroxylation continued for at least 33 days. With no digitoxin added to the medium, no digoxin was found, proving that de n o w synthesis of digoxin did not occur. It can be pointed out that the cell strain used in this experiment had a comparably low hydroxylation capacity. Recently, Reinhard and co-workers described suspension cultures of D. lanata with high 120-hydroxylating capacity (Alfermann et al., 1977). The cell culture specifically hydroxylates 0-methyldigitoxin to 0-methyldigoxin (Fig. 9, line B). Alfermann et al. (1980) have studied the hydroxylation of P-methyldigitoxin by alginate-entrapped Digitalis cells. The hydroxylation was carried out using batch procedure with the addition of substrate every

0

R

sf<

A. Digitoxin D-D-DB. p-Methyldigitoxin H:,C-D-D-DD-D-& C. Digitoxin D. Digitoxigenin HE. Gitoxigenin Ha

R .*

HHHHHO-

R Digtafis lanata Digiralis lanata

?@?!~s??!?~

Daucus carold Daucus carota

..'i

D-D-DDigoxin H:$G-LD-D,+Methyldigoxin G-D-D-DPurpureaglycoside A 56-Hydroxydigitoxigenin H5/3-Hydroxygitoxigenin H-

R,

RI

R,

HHHHHO-

HO-

HHHHOHO-

HOHHH-

D. digitoxose; G. glucose. Frc:. 9. Biotransforinatiori d glycosidris (.4-C) and aglyconcs (D-E) cirrric:d out with immcjbilized plant cell.;.

References Brodelius et a/. (1979) Alfermann etal. (1980) ALfermann etal. (1980) Jones and Veliky (1980) Veliky and Jones (1980)

16

P. BRODELIUS AND K. MOSBACH

incubation time

(days)

FIG.10. Biotianaformation of digitoxin to digoxin by immobilized cells of DigitaEis Zanata as a function of incubation time. (-0) With addition of digitoxin, (0-0) without addition of digitoxin. (Froin Brodelius et d.,1979a.)

second day for 61 days, when the experiment was stopped because the beads started to disintegrate. It is not stated whether this disintegration of the beads is due to mechanical forces or to an increased number of cells. The result of the experiment is shown in Fig. 11, and it can be seen that with 1120 mg/liter methyldigitoxin added, a total of 443.5mg/liter methyldigoxin can be isolated along with 531.5 mg/liter unreacted substrate which can be reused. Based on the actual consumption of substrate, a yield of 76% can be calculated. Furthermore, it was concluded that the immobilized cells expressed approximately half the hydroxylating activity of freely suspended cells. This decrease in activity was, however, well compensated for by the constant hydroxylating activity over an extended period of time (60 days). When these immobilized cells of D. Zanata were incubated in a medium containing digitoxin instead of P-methyldigitoxin, most of the substrate was glucosylated to purpureaglycoside A (see Fig. 9, line C), which is an undesired product. Freely suspended cells also carry out this reaction. Small quantities of digoxin and deacetyllanatoside C (the 12P-hydroxyl derivative of purpureaglycoside A) were also found in the medium. The 5P-hydroxylation of the aglycones digitoxigenin and gitoxigenin (see Fig. 9, lines D and E) has been studied with alginate-entrapped cells of Daucus carnta (Jones and Veliky, 1980: Veliky and Jones, 1980). Disintegration of the beads was prevented by limiting the cell growth by decreasing the concentration of the nitrogen source, i.e., nitrate, and simultaneously replacing the phosphate of the medium by 2-(N-morpholino)ethanesulfonic

17

IMMOBILIZED PLANT CELLS

?l E"

1.0

Y

c

.-a c

FIG.11. Biotransformation of p-methyl-digitoxin to p-methyldigoxin by alginate entrapped cells of Digitalis Ianta. (A-A) P-methyldigitoxin added to the medium, (m-). 0-methyldigitoxin untransformed and ( L O ) p-methyldigoxin found in the medium. (From Alferniann et al., 1980.)

.8-

Q)

0

C

0

0 Q)

e

.6-

J

u)

0

0

I

0

I

20

f

I

40

incubation time

I

60 (days)

acid. In this medium the Daucus cells remained viable (respired) over a period of more than 20 days. Immobilized cells of Cannabis sativa also remained viable in this medium over an extended period of time. The biotransforination was carried out using a batch procedure in which the immobilized cells (60 beads containing 6-7 mg dry weight of cells) were placed in 25 ml of the modified medium and incubated for four days at 26"C. At this point, the medium was replaced by 25 ml of fresh medium containing the substrate (10 mg/liter of digitoxigenin). After 48 hours on a shaker, the beads and the medium were separated, extracted, and analyzed for digitoxigenin and 5P-hydroxydigitoxigenin (periplogenin). The substrate was hydroxylated at a rate of approximately 70% of that with freely suspended cells. The decreased activity was ascribed to diffusion barriers of the substrate. The yield was relatively high (up to 70%).No change in hydroxylation was observed over a pH range of 5.2-6.7 and a temperature range of 2237°C. The transformation rate was linear with a substrate concentration of up to 20 mgAiter digitoxigenin at an immobilized cell concentration of 7 mg dry weight per incubation sample (25 ml). The transformation of gitoxigenin (Fig. 9, line E) was carried out in the column bioreactor described previously (Fig. 8). Beads containing the D. carota cells were filled in the reactor under sterile conditions, and the sub-

18

P. BRODELIUS AND K. MOSBACH

strate solution (5 mg/liter gitoxigenin) was passed through the reactor. The mechanical stability of the alginate beads as well as the performance of the hioreactor were good for at least 30 days. It was shown that in a column with straight upward aeration, the average conversion rate was 73%,while in the column with the airlift pump the average rate was only 43%during the period from day 6 to 26. Furthermore, the bioconversions were as high or even higher in this bioreactor than in batch fermentations. In order to maintain the alginate beads intact, a relatively high Ca2+ concentration (20 mM), which inhibited growth to some extent and affected bioconversion in both free suspension and immobilized state, was used. Finally, it was concluded that the immobilized cells maintained their 5P-hydroxylating activity for a longer period of time than freely suspended cells.

B. SYNTHESISFROM PRECURSORS Synthesis from precursors can be referred to as a multistep biotransformation and could thus be included in the preceding section. We would, however, like to discuss these kinds of reactions separately because of their higher complexity. The feeding of precursors to suspension cultures of plant cells is a widely useful technique for the investigation of biosynthetic pathways. It should also be possible to use this approach biotechnologically for the synthesis of complex compounds. Knowledge of the biosynthetic pathway is required, however, but in principle it should be possible to convert inexpensive precursors to more valuable chemicals with the aid of cultured plant cells. The yield of a particular product may be increased considerably by the feeding of appropriate precursors to the cells as compared to de novo synthesis (Brodelius and Nussbaum, unpublished results). Furthermore, this approach may be used to produce compounds normally not found in the intact plant or in the cultured cells, by feeding the cells analogs of the natural precursors. From biosynthetic studies it is well known that suspension cultures of Catharanthus roseus can produce the indole alkaloid ajmalicine from the distant precursors tryptamine and secologanin (Fig. 12) (Stockigt et al., 1976; Kreuger and Carew, 1978). We have immobilized cells of C. ruseus and investigated if such cells have a preserved ability to carry out the synthesis of ajmalicine from the precursors. Radiolabeled tryptamine was used to monitor the reaction. A column reactor with recirculation of the medium containing the precursors was used, and lipophilic compounds in the medium were continuously extracted with chloroform (Fig. 7). The added tryptamine was rapidly metabolized, and lipophilic compounds were produced as shown in Fig. 13. Ajmalicine constituted approximately one-third

19

IMMOBILIZED PLANT CELLS

strictosidine

cathenamine

secologanin

NADP' 19-H

20-H

ajmalicine

P

P

tetrahydroalstonine

p

P

FIG. 12. Biosynthetic sequence for the formation of cathenamine and ajmalicine isomers from distant precursors. (From Brodelius et a l . , 1980.)

I

00

30 60 reaction time (h)

90

FIG. 13. Synthesis of indole alkaloids of the ajmalicine group by immobilized cells of Catharanthus roseus in a recirculated reactor (as shown in Fig. 5) according to the reaction scheme shown in Fig. 12. ( 0 4 )Unreacted tryptamine, ( H A )metabolized tryptamine in the form of chloroform-extractable compounds, and (A-A) ajmalicine isomers in the chloroform phase. (From Brodelius et a l . , 1974.)

P. BRODELIUS AND K. MOSBACH

20

of the radioactivity found in the organic phase, which corresponds to a yield of about 10%.This yield could probably be increased by adding the precursors not in eyuimolar amounts but rather in a five-fold excess of secologanin according to optimization experiments carried out with cell-free extracts (Stockigt, 1979). Obviously, the immobilized cells can condense the two precursors to strictosidine (Stiickigt and Zenk, 1977), which then is transferred in a number of steps to ajmalicine. Figure 12 shows that the last step in this sequence, i. e., reduction of cathenamine to ajmalicine, requires reduced coenzyme which is efficiently supplied by the cells. Furthermore, the product is normally stored in the vacuoles of the cells, but in this experiment the product is released and continuously extracted. Traces of chloroform in the aqueous phase alter the permeability of the plasma membrane of the cells within a couple of hours (no plasmolysis) (P. Brodelius, unpublished results), but the synthesis of ajmalicine is continuous for at least 60 hours. Even though the cells are made permeable, the “cell-machinery’’ is working for an extended period of time. This synthesis has been a model system for the investigation of the biosynthetic capacity of C . roseus cells immobilized by entrapment in various gels. In these studies a batch procedure without continous extraction of products was employed, and the results are summarized in Table IV. As can be seen, the activity of the immobilized cells varied between 82 and 176%of that for freely suspended cells for the various preparations. All four immobilization techniques give relatively high activities, and they should therefore prove useful for the immobilization of plant cells.

C . De Novo SYNTHESIS The de n o w synthesis of complex secondary products from a simple carbon source is the most important biutechnological feature of plant cell culT A B L E IV SYNTHESISOF AJMALICINE ISOMERS FROM PRECURSORS BY FREEA N D IMMOBILIZED CELLSOF Cutharanthus roseus“

Cell preparation ~~

Percentage of incorporation

Relative incorporation

7.9 7.5 9.0 13.9 6.5

100 95 114 176 82

~

Free cells Agar Agarosr Algindte Carrageenan ~

~

~~

“Brodeliua and Nilwin (1980)

IMMOBILIZED PLANT CELLS

21

tures. The development of plant cell systems of this kind has been hampered by the fact that plant cells in culture often synthesize secondary products in low yields, and sometimes the compounds found in the parent plant are not found at all in the cultured cells. However, by selecting plant cell lines through cloning techniques, high-producing cultures might be obtained, which of course would reduce these problems considerably. Utilizing plating techniques, Zenk and co-workers have established highproducing cell lines (Zenk et al., 1975, 1977). For instance, cultures of Morindn citrifolia with high anthraquinone-producing capacity have been established (Zenk et al., 1975). To investigate whether such highly producing cells preserve their synthesizing capacity after immobilization, Mminda cells were entrapped in alginate beads (Brodelius et al., 1979a, 1980). Using batch procedure in a growth-limiting medium (no hormones added) it was found that the immobilized cells even had an increased synthesis of anthraquinones as compared to freely suspended cells under the same conditions (Fig. 14). The observed increased biosynthesis by the immobilized cells is of fundamental importance, and if it is of a general character, the im-

FIG. 14. Kinetics of the de novo synthesis of anthraquinones by Mwinda oitrijlolia cells. (0---0) Free cells and (O+) alginate entrapped cells. (From Brodelius et a l . , 1980.)

Incubation t i m e ( d a y s 1

22

P. BRODELIUS AND K. MOSBACH

mobilization of plant cells may be of great importance for the production of natural compounds. An increased synthesis by immobilized cells has also been observed in other systems under growth-limiting conditions (Brodelius et al., 1979b; L. Linse and P. Brodelius, unpublished results). An increased synthesis of ajmalicine by immobilized cells of C. roseus with sucrose as the sole carbon source has been observed (L. Linse and P. Brodelius, unpublished results). The reason for this increased synthesis is not fully understood. Whether this effect is due to immobilization as such or to changed microenvironmental conditions prevailing within the beads is at present under investigation (Linse et ul., 1981).

IV. Permeabilized Plant Cells In many cases the product of interest is stored within the plant cells and only very small quantities of the product may be released into the medium. One of the inherent advantages of an immobilized biocatalyst is the possible utilization in a continuous process. For such a process, however, it is essential that the product of interest is released into the medium, and therefore we have initiated studies on the permeabilization of plant cells using irnmobilized preparations (Felix et al., 1981). Permeabilization of plant cells can be carried out with organic solvents, such as ether, toluene or dimethylsulfoxide, with proteins such as cytochrome C or protamine, or with other lipophilic compounds such as nystatin or lysolecithin. Enzyme activity in uiuo requiring charged substrates or coenzymes, which normally cannot penetrate the plasma membrane, can readily be assayed after permeabilization of the cells. Five enzymes of the primary metabolism, i.e., hexokinase (ATP), glucose-6-phosphate dehydrogenase (NADP+), isocitrate dehydrogenase (NADP+), malic enzyme (NADP+), and citrate synthase (AcCoA), which require coenzymes as indicated, have all been measured within permeabilized cells of the plant Catharantus roseus. It was found that the enzymes within the immobilized permeabilized cells are considerably more stable than the corresponding enzymes in freely suspended cells. For instance, the activity of the two sequential enzymes hexokinase and glucose-6-phosphate dehydrogenase within freely suspended cells after 1day was only 10%of the initial activity. For immobilized cells, on the other hand, 20% of the initial activity remained after 7 days. A model system, i.e., the transformation of cathenamine to ajmalicine requiring NADPH (see Fig. 12), has been studied utilizing permeabilized plant cells of Cutharanthus roseus entrapped in agarose. The most efficient transformation was observed when relatively high concentrations of NADPH (1 mM) were added together with cathenamine to the immobilized cells

23

IMMOBILIZED PLANT CELLS

z 0

loo

1

v)

K

W

5

0 0

50

a9

0

0

1

2

3

INCUBATION TIME

4 (h)

FIG. 15. Biotransformation of cathenamine to ajmalicine isomers by agarose entrapped cells of Catharanthus roseus as a function of' incubation time. (W-W) Cathenainirie NADPH, (A-A) cathenamine NADP+ isocitric acid, ( 0 2 )cathenamine isocitric acid, and (V-'I) cathenamine. (From Felix et al., 1981.)

+

+

+

SECONDARY

+

NADP*

METABOLISM

ERMEABILIZED LASMA MEMBRANE

FIG. 16. Schematic illustration of a perineabilized plant cell with the coupling of an enzynic of the primary metaholism (E,) with an cnzyme of the secondary metabolism (Es), S,, Priinary substrate; P,, primary product; S,, secondary substrate; P,. secondary product. Note rrlrase of' ,,'l which norrnally may be stored within the cell. In the model system studied (see text): E,, isocitrate dehydrogenase; E,, cathenamine reductase; S,, isocitric acid; P,, a-ketoglutarate; S,, cathenamine; P,, ajinalicine isomers.

24

P. BRODELIUS AND K . MOSBACH

(Fig. 15). A somewhat lower transformation rate was observed on replacement of the reduced coenzyme with NADP+ and isocitric acid (the substrates for the above mentioned isocitrate dehydrogenase). Omitting the addition of coenzyme completely, some ajmalicine was still continuously formed on addition of isocitrate, possibly due to reduction and/or recycling of endogeneous NAL)P+. In the latter two cases, the enzyme of the primary metabolism, which is present in relatively large amounts, was utilized to regenerate and recycle the coenzyme required in the transformation step of the aforementioned secondary metabolites as schematically illustrated in Fig. 16. In contrast, cathenamine alone was converted only in the initial phase to a small degree. With the recycling system, a relatively low concentration of the expensive coenzyme is needed and, therefore, might make large-scale transformations of an externally added substrate with such permeabilized immobilized plant cells economically feasible. Such an attractive system would be the aforementioned transformation of digitoxin to digoxin. This example resembles multienzyine systems, using isolated enzymes coimmobilized on a solid support, that have been extensively studied in this laboratory (Srere et al., 1973; Mosbach and Mattiasson, 1978).

V. Immobilization of Protoplasts Protoplasts are obtained after the removal of the cell wall of plant cells. Since cells of this kind are held together only by the plasma membrane, they are very fragile and therefore difficult to handle. On the other hand, such cells might show lesser dif€usion restrictions. Protoplasts can, however, be stabilized by immobilization and stay intact over an extended period of tirne [up to two weeks (P. Brodelius, unpublished results)]. When immobilized protoplasts are placed in an appropriate medium, they can resynthesize a cell wall within several days (Brodelius ct a l . , unpublished results). Scheurich et al. (1980) have studied alginate-entrapped protoplasts of Vicia faba. They found that no morphological change or alteration of the membrane permeability could be observed after 14 days in an immobilized state. Furthermore, the immobilized protoplasts shrank reversibly when the osmolarity of the medium was increased. However, when the osmolarity was reduced, the cells did not swell, as in free suspension, but a cell turgor pressure was built up. It was suggested that the mechanical support of the cross-linked alginate was responsible for this effect.

VI. Perspectives It is difficult at this point to speculate about the future use of immobilized plant cells because this development is only of very recent date. More research is needed to evaluate fully the potential of the technique. However,

1MMOBILIZED PLANT CELLS

25

a comparison to the situation prevailing in the microbial field may be of interest. Originally, after the introduction of the immobilization technique, great excitement was felt, and expectations ran high. As time passed only, a limited number of processes employing immobilized systems had been put to use, and general disappointment was felt. However, one major reason for this relatively slow development was the fact that in each case the immobilization process had to compete with already existing and optimized processes. Now we see a regular and steady growth of the number of processes utilizing immobilized systems. Of particular interest is the tendency to change from traditional fermentation processes to immobilized systems. The situation is different with regard to plant cell cultures to date, as there is no commercial process using plant cell suspension cultures, and thus an immobilized process would not need proof superior to an already existing process. As most researchers in the field are optimistic about the prospects of using plant cell suspension cultures for large-scale production in industry, it may well be that the first operating process will be based on immobilized plant cells. Such a development will necessarily influence the reactor design, and the special airlift fermentor type found so useful for plant suspension cultures may have to be modified or substituted with reactors of the packed-bed type. In summary, we feel optimistic about the future of immobilized plant cell systems because of the inherent advantages of immobilized systems including the ease of obtaining high cell densities, minimizing waste problems (plant cell material), and because of the prospects of increasing secondary metabolite formation and permeability discussed in this chapter. A(;KNOwLk;I>C:>f k v r s

The authors thank Professor M. H. Zenk for providing a suspension culture of Cutharmthus roseus. This work has been supported in part by the National Swedish Board for Technical Development (80-3619).

HEFEHESCES Alfermann, A. W., Boy, H . M.,D o h , P. C., Hagedorn, W., Heins, M . , Wahl, J., and Reinhard, E. (1977). In “Plant Tissue Culture and Its Bio-technological Application” (W. Barz, E. Reinhard, and M. H. Zenk, eds.), pp. 125-141. Springer-Verlag, Berlin and New York. Alfermann, A. W., Schuller, I., and Reinhard, E. (1980). Pkuntu Med. 40, 218-223. Brodelius, P. (1978). I n “Advances in Biochemical Engineering” (T. K. Ghose, A. Fiechter, and N. Blakebrough, eds.), Vol. 10, pp. 75-129. Springer-Verlag, Berlin and New York. Brodelius, P., and Nilsson, K. (1980). FEBS Lett. 122, 312316. Brodelius, P . , Dew, B., Mosbach, K., and Zenk, M. H. (1979a). FEBS Lett. 103, 93-97. Brodelius, P., Deus, B., Mosbach, K., and Zenk, M. H. (1979b). Swedish Patent Application 7905615-6

26

P. BRODELIUS AND K. MOSBACH

Brodeliiis, P., Deus, B., Mosbach, K . . and Zenk, M. H . (1980). IT& “Enzyme Engineering” (11. H. Weetall and G. P. Royer, crls.), Vol. 5, pp. 373381. Plenum, New York. Chang, T. M .S . , ed. (1977). “Biomedical Application of Immobilized Enzymes and Proteins,” Vol. 1 and 2. Plenum, New York. Chibata, I. (1980). I n “Enzyme Engineering in Food Processing” (P. Linko and J. Larinkari, eds.), Vol. 2, pp. 1-26. Applied Science Publ., Barking. Felix, H., Brodelius, P., and Mosbach, K. (1981). A n d . Biochem. (in press). Cuilbault, G. G. (1976). I n “Methods in Enzymology” (K. Mosbach, ed.), Vol. 44, pp. 579-633. Acadcrnic Press, New York. Jones, A . , and Veliky, I. A. (1980). I n t . Fmnent. S y i n p . , Ma, July 20-25, London, Onturio Poster paper F-12.1.1.16. Kierstan, M., and Rucke, C. (1977). Biotechnol. Bioeng. 19, 387397. Kreuger, R. J., and Carew, D. P. (1978). Lloydiu 41, 327331. Kurz, W. G. W . , and Constabel, F. (1979). In “Advances in Applied Microbiology” (D. Perlmann, ed.), Vol. 25, pp. 209-240. Academic Press, New York. Larsson, P.-O., Ohlsson, S., and Mosbach, K. (1976). Nature (London) 263, 796-797. I h k o , P., and Larinkari, J., eds. (1980). “Enzyme Engineering i n Food Processing,” Vol. 2. Applied Science Puhl., Barking. Linse, L., Brodelius, P., Mosbach, M., and Mosbach, K. (1981). In prepration. .Martin, S. M. (1980). I n ”Plant Tissue Culture as a Source of Biochemicals” (E. J . Staba, ed.), pp. 149-166. CRC Press, Boca Katon, Florida. Mosbach, K., ed. (1976). “Methods in Enzymology,” Val. 44. Academic Press, New York. Mosbach, K., and Danielsson, B. (1981). Anal. Chem. 53, 83A-MA. Mosbach, K . , and Larsson, P.-0. (1970). Biotechnol. Bioeng. 12, 19-27. Mosbach, K., arid Mattiasson, B. (1978). In “Current Topics in Cellular Regulation” (B. L. Horecker and E. R. Stadtman, eds.), Vol. 14, pp. 197-241. Academic Press, New York. Mosbach, K . , and Mosbach, R. (1966). Actu Chem. Scand. 20, 2807-2810. Nilsson, K., and Mosbach, K. (1980). F E B S Lett. 118, 145-150. Reinhard, E. (1979). In ”Pflanzliche Zellkulturen und ihre Bedeutung fur Forschung und Anwendung” (W. Barz, ed.), pp. 47-58. Bundesministerium fur Forrchung und Technologic, Bonn. Rcinhard, E., and Alfermann, A. W. (1980). In “Advances in Biochemical Engineering” (A. Fiechter, ed.), Vol. 16, pp. 4 9 4 3 . Springer-Verlag, Berlin and New York. Scheurich, P., Schnabl, H., Zimmermann, U . , and Klein, J. (1980). Biochim. Biophys. Acta 598, f545-651. Srere, P. A , , Mattiasson, B., and Mosbach, K . (1973). Proc. Nut/. Acud. Sci. U.S.A. 70, 2534-2,%8. Staba, E. J , , ed. (1980). “Plant Tissue Culture as a Source of Biochemicals.” CRC Press, Boca Raton, Florida. Stockigt, J . (1979). Phytocheinishy 18, 965-971. Stockigt, J., and Zenk, M. H. (1977). J . C . S . C h e m Comin. 646-648. Stiickigt, J . , Treimer, J . , and Zenk, M . H. (1976). FEBS Lett. 70, 267-270. Veliky, I. A,, and Jones, A. (1980). I n t . Fennent. Sy7np. sth, July 20-25, London, Ontario Poster paper F-12.1.1.17. Zenk, M. H. (1978). In “Frontiers of Plant Tissue Culture” (T. A. Thorpe, ed.), pp. 1-13. Internatitrnal Association for Plant Tissue Culture. Zenk, M. H . , El-Shagai, H . , and Schulte, U. (1975). Pluntu Med. Suppl 79-101. Zenk, M. H., El-Shagai, H., Arens, H., Stockigt, J., Weiler, E. W., and Deus, B. (1977). In ”Plant Tissue Culture and Its Bio-technological Application” (W. Barz, E. Reinhard, and M. H. Zenk, eds.), pp. 2 7 4 3 . Springer-Verlag, Berlin and New York.

Genetics and Biochemistry of Secondary Metabolism

VEDPAL SINGHMALIK] The Upjohn Company. Kalamazoo. Michigan

28 I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31 I1. Illegitimate Genome Sequences . . . . . . . . . . . . . . . . . . . . . . . . . . 32 111. Enzymes of Secondary Metabolism . . . . . . . . ........ 34 A . Inducible Enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Compartmentalization . . . . . . . . . . . . . . . . . . . . . . . . . 35 36 C . Specificity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38 IV . Controlling Effect of the Environment . . . . . . . . . . . . . . . . . . . . 38 A . Nutrition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38 B . Trace Metals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39 C . Hydrogen Ion Concentration . . . . . . . . . . 39 D . Temperature and Aeration . . . . . . . . . . . . . . . . . . . . . . . . . . . 39 V. Genetics of Secondary Metabolism. . . . . . . . . . . . . . . . . . . . . . . . 41 A . Chromosomal Mapping . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42 B . Extrachromosomal Elements . . . . . . . . . . . . . . . . . . . . . . . . . 48 C . Genetic Engineering of Secondary Metabolites . . . . . . . . . 51 D . Reciprocal Genetics of Secondary Metabolism . . . . . . . . . . VI . Control of Secondary Metabolism . . ........ 53 55 A . Growth-Linked Suppression . . . . . . . . . . . . . . . . . . . . . . . . . 58 B. Multivalent Induction by Precursors . . . . . . . . . . . . . . . . . . 74 C . Feedback Inhibition and End Product Repression. . . . . . . 77 D . Catabolite Repression 81 E . Enzyme Modification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82 F . Factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82 G . Glutamine Synthetase . . . . . . . . . . . . . . . . . . . . . . . . 85 H . Energy Charge . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86 VII . Regulation of Autotoxicity . . . . . . . . . . . 87 A . Regulation of Permeability . . . . . . . . . . . . . . . . . . . . . . . . . . . 89 B . Modification of Target Site . . . . . . . . . . . . . . . . . . . . . . . . . . . 90 C . Biotransformation and Regulation of Catabolism . . . . . . . . VIII . Secondary Metabolism, S 94 Exoenzyme Formation . . 97 IX . Role of Secondary Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . 101 X . Epilogue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

'Present address: Philip Morris Research Center. P.O. Box 26583. Richmond. Virginia 23261.

27 ADVANCES IN APPLIED MICROBIOLOGY. VOLUME 28 Copyright @ 1982 by Academic Press. Ioc . All rights of reproduction 111 any form reserved. ISBN 0-12-W2628-7

28

VEDPAL SINGH MALIK

I. Introduction One must know all the literature, but not trust any of it.

RICHARDWILLSTATTER

In his classic text on the chemical activities of fungi, the great American microbiologist J. W. Foster (1949) conveyed the essence of secondary metabolism as perceived at that time: It would appear that the enzyme mechanisms usually involved in complete oxidation of the substrate Iwcome saturated, and the snbstrate molecules then are excreted and accumulate as such, or thcy are shunted to secondary or subsidiary enzyme systems which are able to affect only relatively ininor changes in the substance, which then accumulates in its transformed state.

Studies of the biosynthetic pathways of secondary metabolites have since shown that chemical changes of the starter substrate are much more complex than suggested by Foster (Queener et aZ., 1979; Martin and Demain, 1978). As a matter of fact, the synthesis of secondary metabolites is carried out by specific pathways involving enzymes coded by genes that are not involved in growth. Plant physiologists were the first to recognize that certain compounds, such as alkaloids, terpenes, camphor, and tannins, were obtained only from particular plant species. The distribution of these compounds was not universal among the plant kingdom, and they could not be assigned a specific fimction. These metabolites were dubbed as secondary products of metabolism (Ruhland, 1958). The epithet “secondary metabolism” already familiar to plant physiologists was further promoted by the English microbial chemist J . D. Bu’Lock (1961). In his elegant review, Bu’Lock exquisitely states, Given the gencrally acceptable view that there are basic patterns of‘general metabolism, on which t h r variety of organic systems imposes relatively minor modifications, we can defino secondary mctabolism as having, by contrast, a restricted distribution (which is almost species specific) and 110 obvious function in general metabolism.

Whereas primary metabolites (sugars, amino acids, vitamins, nucleic acids, and polymers derived from them) are both essential and ubiquitous, making their presence universal among all organisms,” secondary metabolites” is the term used to collectively define those naturally occurring organic compounds that are unique to a small number of organisms. Even though secondary metabolites are nonessential to the organism that produces them, many of them have interesting biological activity (Table I). Since time immemorial, man used such metabolite extracts of plants as medicine to relieve pain and to cure diseases, as poisons in warfare and hunting, and as narcotics, hallucinogens, or stimulants. These interesting biological activities motivated curious chemists, who isolated and characterized the active princi-

GENETICS AND BIOCHEMISTRY OF SECONDARY METABOLISM

29

TABLE I SOME EXAMPLES OF SECONDARY METABOLITES Secondary metabolite Ephedrine Ricinine Salicin Strychnine Coniine Rotenone Morphine Tetrahydrocannabinol Cocaine Caffeine Ceraniol Linalool Cinnamaldeh yde Eugenol Diallyl sulfide

Use Respiratory ailments Castor oil purgative Antipyretic Poison Poison Fish poison and natural insecticide Narcotic Hallucinogen Stimulant Stimulant Perfumes Perfume Spice Spice Spice

Source Ephedra plant Castor oil Willow bark Strychnos plant Hemlock

Opium Hashish; marijuana Cocoa Coffee Rose oil Lemon grass oil Cinnamon Cloves Garlic

ples from such plants. The structural complexity of secondary metabolites represents a great challenge to the synthetic chemists. Amino sugars, amino acids, quinones, coumarins, epoxides, alkaloids, glutarimides, glycosides, indoles, lactones, macrolides, naphthalenes, nucleosides, peptides, phenazines, polyacelylenes, polyenes, pyrroles, quinolines, and terpenoids are just a few examples of the diverse chemical structures represented by secondary metabolites. Unusual chemical linkages such as p-lactam rings, cyclic peptides, unsaturated bonds of polyacetylenes, polyenes, sterols, gibberellins, and the large rings of macrolides and ansamycins are peculiar to secondary metabolites. This diversity of a seemingly endless structural variety and complexity does not result from a multiplicity of basic building units but originates in a relatively small number of primary metabolites. Transformations of these precursors and their condensation with other moieties derived from central metabolic routes is responsible for the structural variety of secondary metabolites. Minor modifications of the number and the arrangement of carbon atoms of basic skeletons can yield a multiplicity of related chemical structures. Introduction of oxygen, nitrogen, chlorine, and s u l h r or changes in the oxidation level can further alter the hnctionality of the emerging metabolite. The study of secondary metabolism began in 1896 when Ernest Duchesne, a French medical student, discovered the low toxicity of extracts from Penicillium glaucum. These extracts probably contained penicillin, which was discovered in 1928 by Sir Alexander Fleming at St. Mary’s Hospital, London. However, it was not until World War I1 that research efforts to develop penicillin into a therapeutic agent were successful (Chain, 1980). H.

30

VEDPAL SINGH MALIK

W. Florey and Norman G. Heatley had worked with penicillin at Oxford. In 1941, England was undergoing air raids by the Nazi air force. Therefore, Florey and Heatley felt that penicillin work could be done better in the United States. They received financing from the Rockefeller Foundation to visit and enlist scientific collaboration of United States scientists. They contacted 0. E. May, the director of the Northern Regional Research Laboratory at Peoria, who received the following telegram from the United States Department of Agriculture headquarters: Thorn has introduced Heatley and Florey of Oxford, England, here to investigate pilot scale production of bacteriostatic material from Fleming’s penicillinm in connection with medical defense. Can you arrange irninrdiately for shallow pan srt-up to establish laboratory results. . . ?

0. E. May responded that the Northern Regional Research Laboratory was ready “to cooperate immediately.” Robert D. Coghill, Chief of the Fermentation Division, directed the penicillin project, which was terininated in 1945. Even though K. B. Raper, the mycologist on the project, isolated hundreds of strains of penicillin-producing fungi from soils collected by the Army Transport Command, the highest penicillin-yielding Penicilliurn chrysogenurri was isolated from a moldy cantaloupe obtained in a Peoria fruit market. During World War 11, industrial exploitation of the capability of hngi to produce penicillin received unprecedented attention. Commercial success of penicillin production not only laid the foundation for a prosperous billion-dollar fermentation industry but stimulated interest in the study of other microbial metabolites that might be of economic value (Rose, 1980). The Gascinating story of penicillin has been narrated in a superb manner by Chain and Raper and describes the efforts, good luck, and politics involved in commercializing the scientific observation that could otherwise have gone unnoticed for decades (Chain, 1980). During the early part of this century, Alsberg and Black 1912) reported the formation of an intriguing organic acid b y the cultures of Penicilliuin puberculun~They called it penicillic acid and noted its antiseptic action and moderate toxicity. More than a decade later Raistrick, working at the Nobel’s Explosives Company in Ardeer, Scotland, initiated investigations on the structures of fungal secondary metabolites. In 1929, Raistrick moved to the London School of Hygiene and Tropical Medicine, and he and his associates isolated more than 200 mold metabolites and determined the structure of penicillic acid. Since then the structures of numerous secondary metabolites have been determined and many such compounds are produced commercially. Thus, for example, almost 6000 antibiotics have been discovered and about 100 of them are the backbone of a multibillion-dollar, high-profit fermentation industry. Birkinshaw et al. (1936)showed that many secondary

GENETICS AND BIOCHEMISTRY OF SECONDARY METABOLISM

31

metabolites are formed during the stationary growth phase. Over the past 40 years, biosynthesis of secondary metabolites has been studied mainly by tracer methods. Mutants blocked in metabolic pathways have been used in only a few instances to establish the entire metabolic route of complex products originating from the intermediates of primary metabolism. Brute-force mutagenesis has been widely used to develop microbial strains that are being used for commercial production of many products. Technologies that exploit the metabolic plasticity of microbes will play an increasing role in the near h t u r e and solve the shortages of some chemicals, solvents, fuels, and energy. Many other products that are of intricate chemical structure and that are hard to synthesize chemically may be rnicrobially produced without generating toxic waste (Whitaker, 1980). Versatile metabolic machinery of diverse microbes also may be employed to economically produce valuable compounds and their derivatives (antibiotics, alkaloids, human interferon, insulin, ethanol, long chain alcohols, acetic glycerol, ethylene oxide, acrylic acid) from renewable resources and thereby reduce pollution that is generated by an energy-consuming, expensive chemical synthesis. The application of modern methods of molecular genetics and recombinant DNA technology to the study of secondary metabolism could provide some useful solid information quickly. There exist limitless opportunities in the exploration of secondary metabolism, the study of which can now be speeded up since new methodology can circumvent the biochemical and genetic studies that hardly exist for the organisms that produce such metabolites. The purpose of this article is to give a general perspective of secondary metabolism and to provide a stimulus for investigators to think deeply about the scientific issues and other interesting aspects of the biology of the organisms that produce industrially important secondary metabolites.

II. Illegitimate Genome Sequences Even though secondary metabolism is not universal, the same secondary metabolite is produced by many organisms in widely separated taxonomic groups. Therefore, secondary metabolite production may have no relevance to the taxonomic classification of organisms. The occurrence of gibberellins in plants and Fusariuin and of ergot alkaloids in morning glory as well as Claviceps purpurea indicates that genetic capabilities for the synthesis of these metabolites are distributed among organisms belonging to different taxonomic groups. The occurrence of the P-lactam nucleus in wild-fire toxin produced by Pseudomonas tnbuci (Stewart, 1971) and in many p-lactam antibiotics produced by various Streptomyces, Nocardia, Penicillium, and Cephalosporiuin species (Aoki and Okuhara, 1980; Gorman and Huber, 1979) could be taken as another suggestive piece of evidence of illegitimate

32

VEDPAL SINGH MALIK

genome mixing and reassortrnent among various organisms. Production of chemically similar antibiotics by taxonomically remote species (Wagman and Weinstein, 1980) is further evidence for the same mechanisms of synthesis of secondary metabolites and for the existence of common pathways of secondary metabolism in different species. Similar cellular metabolites are generally, but not always, derived via similar biosynthetic sequences. The same, but sometimes different, taxonomic groups utilize the same metabolic pathway and may possess the same or similar enzymes and regulatory mechanisms to assemble similar types of chemical structures. Many secondary metabolites, including antibiotics produced by taxonomically unrelated organisms, contain the same subunits or substituents (Malik, 1980~).The genes for the subunits that are present in various secondary metabolites may be carried on transposable elements. Mixing and reassortment of these transposons resulting from illegitimate dissemination of genome sequences between organisms of various taxonomic groups might have conferred the ability to produce hybrid molecules with novel biological activity. For example, rubradirins (Fig. 1)consist of subunits that are present in three M e r e n t families of antibiotics as diverse as ansamycins (rubransarol), novobiocins (coumarin), and the everninomicins (dipicolinic acid) (Hoeksema et a ] . , 1979). P-Lactamases are widespread in enterobacteria (Richmond and Sykes 1973; Sawai et al., 1980),nocardias (Wallace et ul., 1978), streptomycetes, micromonosporas, and other nonstreptoinycetes actinomycetales (Schwartz and Schwartz, 1979). Of the other antibiotic-inactivating enzymes coded by eubacterial plasmids, those that acetylate chloramphenicol (Wright and Hopwood, 1977) and those that adenylate, acetylate, or phosphorylate aminoglycosides are present in streptomycetes (Davies et al., 1979). Location of the genes determining the structure and synthesis of antibioticinactivating enzynies on transposable elements could have favored their dissemination by illegitimate recombination among various microorganisms. Many of these antibiotic-inactivating enzymes are coded by chromosomal genes (Wright and Hopwood, 1977; Davies and Smith, 1978) in Streptoniyces. The modern methods of recombinant DNA technology can be used to study the similarity among enzymes of diverse origin and to determine if these chromosomally coded enzymes in the streptomycetes are a result of the insertion of the antibiotic-resistance-determining transposons into the chromosomes.

111. Enzymes of Secondary Metabolism The enzymes associated with secondary metabolites can be divided into four classes:

-0

Yo,

CONH, Novobiocin

(Strepfomycesnioeus)

FIG. 1. Silnilar subunit structures found in unrelated antibiotics.

34

VEDPAL SINGH MALIK

1. Those of primary metabolism that yield the precursors of secondary metabolism. 2. Those that are specific for the synthesis of the secondary metabolites and that convert the branch-point intermediates into final secondary products. 3. Those that provide energy or cofactors, or that insert various functional groups into the biosynthetic intermediates. 4. Those that further metabolize the produced metabolite. Even though the general outline of the biosynthetic pathways leading to various secondary metabolites is known, the details of the biosynthetic sequences have been elucidated only in a few cases. Mutants blocked at each step of the entire pathway are hardly available for any given metabolite, and little is known about the enzymology of the reactions involved. Indeed, the enzymes of secondary metabolism have proved difficult to purfi. This may, in part, be attributed to the fact that often secondary metabolism is at its peak of activity when general metabolism is slowing down, when cells are dying and undergoing proteolysis.

A. INDUCIBLEENZYMES Even though the reaction mechanisms involved in secondary biosynthesis are not essentially different from those of general metabolism, each secondary metabolite is made by a unique pathway. The formation of many secondary metabolites occurs by multistep processes catalyzed by multienzyme complexes that usually are produced only during a certain growth phase. Thus some of the antibiotic-synthesizing enzymes are induced during a short period at the end of the logarithmic growth and the onset of the stationary phase (Martin and Demain, 1978). They are also unstable and are in many cases inhibited by the products of the reaction they catalyze. As a result, most of the published curves that show the relationship between enzyme activity, growth, and the secondary metabolite production could be misleading because the enzyme preparation used for plotting such curves was not extensively purified to remove inhibitory enzymatic reaction products. Anhydrotetracycline hydratase of tetracycline-producing Streptoinyces aureofaciens uses NADPH and oxygen to hydrate anhydrotetracycline. The specific activity of this hydratase enzyme in the high-tetracycline-producing strain was one order of magnitude higher than the low producer. The activity of the enzyme increases rapidly around 24 hr of growth. Toward the end of growth, the enzyme activity diminishes. The addition of phosphate to the tetracycline-producing medium caused a decrease in specific activity of the hydratase enzyme; this decrease corresponded with decreased tetracycline production (Z. Hostalik, Czechoslovak Academy of Sciences, Prague).

GENETICS AND BIOCHEMISTRY OF SECONDARY METABOLISM

35

Streptomyces griseus contains an enzyme for the formation of dihydrostreptosyl streptidine-6-phosphate from enzymatically prepared dTDP-Ldihydrostreptose and chemically prepared streptidine phosphate. The enzyme has a molecular weight of 70,000 and is composed of two apparently identical subunits. The enzyme requires 10-12 mM Mg2+and is stabilized by streptidine. Chemically phosphorylated streptidine is the best acceptor, but streptidine and streptamine can also function as acceptors of dihydrostreptose. This enzyme may be used to make analogs of streptomycin where streptidine could be substituted by other aminocyclitol subunits in the enzymatic reaction under altered incubation conditions. During the course of streptomycin production in the S . griseus fermentation, the activity of this transferase enzyme appears at 1day, peaks at 2 days, and then declines. This parallels the activities of dTDP-L-dihydrostreptose synthetase and aminotransferase involved in the formation of streptidine (Kniep and Grisebach, 1980). In Streptontyces erythreus, propionate kinase is increased in stationary phase of growth (Raczynska et al., 1973). The acetyl-CoA and propionyl-CoA carboxylase activities in a macrolide producer reached a maximum at the onset of antibiotic production and decreased about threefold during idiophase. Conversely, 6-methylsalicylic acid synthetase is induced in the logarithmic growth phase and yields 6-methylsalicylic acid, a precursor of patulin, which accumulates in the idiophase. In Cephalospwium acremonium, the enzymes involved in the ring expansion and cyclization reach maximum specific activity 13 hr after the fungus has stopped growing, whereupon the enzymes are rapidly inactivated (Demain, 1980). Toward the end of the logarithmic growth phase and shortly before the onset of tyrocidine synthesis, the antibiotic-synthesizing enzymes and carrier proteins required for transporting the constituent amino acids of tyrocidine are induced. In the later stages of the stationary phase, insoluble enzyme preparations for tyrocidine biosynthesis were obtained from Bacillus brevis and were unstable and sedimented with the particulate fraction. Although the antibiotic production was enhanced in the later phase (Lee et al., 1975), a change of solubility of the tyrocidine-producing enzymes with the appearance of forespore in B . brevis suggests a relationship between antibiotic production and spore formation. Spore membrane-bound enzymes produce tyrocidine enclosed in forespore and induce cell transitioning into sporulation from the vegetative phase (Lee et al., 1975). B. COMPARTMENTALIZATION The efficiency of microbial synthesis is attributable to genetically controlled enzymatic levels and special enzyme properties. Intracellular compartment-

36

VEDPAL SINGH MALIK

alization of enzymes, substrates, and cofactors makes the biosynthetic processes highly efficient. Multienzyme complexes with catalytic subunits arranged to catalyze successive steps of synthesis are known to be involved in the biosynthesis of secondary metabolites. The catalytic efficiency of these complexes is probably higher in the intact cell than in cell-free extracts because the intracellular intermediates are largely enzyme-bound during the biosynthetic process. Toxic enzyme-bound intermediates may not inhibit cellular functions because they are never free in the cytoplasm. In the cellfree extracts, on the other hand, this delicate order is disturbed with the ensuing dilution of the desired enzymatic activities.

C . SPECIFICITY Enzymes that carry out primary metabolic processes have evolved with strict substrate specificity because their product is essential to the growth of the organism. On the other hand, enzymes involved in the biosynthesis of secondary metabolites have not always been selected for rigid specificity because their products are not usually essential for the growth of the producing organism. Unnatural compounds that are formed as a result of the action of these loosely specific enzymes may serve as substrates for the synthesis of new structures. Because of the loose substrate specificity, secondary metabolites are produced as families of closely related molecules with minor modifications of a basic structure. For example, there are about a dozen natural penicillins, thienamycins, and olivanic acids, 3 neomycins, 20 rifamycins, 4 tyrocidines, 5 mitomycins, 10 bacitracins, 10 polymyxins, 20 actinomycins, 4 levorins, 4 polifungins, 13 bleomycins, and numerous sporedesmins. One Mi~romonospm-aproduces about half a dozen aminocyclitol antibiotics (Berdy, 1974). Streptomyces tenebrarius produces a complex of aminocyclitol compounds called nebramycin, with a dozen known chemical structures. The ratio of the components in the fermentation mixture varies greatly and depends on growth conditions. Several components have been characterized as apramycin, kanamycin B, and tobramycin (Stark et al., 1980). Caerulomycin is another major solvent-extractable antibiotic coproduced with nebramycin complex (Funk and Divekar, 1959). The number of antibiotic-like molecules, of which only a few are biologically active, is very large and is the result of a variety of biochemical and stereochemical transformations that occur during antibiotic synthesis. For instance, 72 tetracycline-type compounds can originate from a hypothetical nonaketide intermediate. Twenty-seven of them have been isolated (Hostalek et nl., 1979). In the synthesis of penicillins, the final acylation step is not specific. Many acids similar to phenylacetic acid can be utilized to synthesize penicillin G

GENETICS AND BIOCHEMISTRY OF SECONDARY METABOLISM

37

analogs. In the biosynthesis of some oligopeptides and polyketides, intermediates are formed by nonspecific reactions as any one of several alternative amino acids can be incorporated in the middle of the peptide antibiotics and none limits overall synthesis (Queener et al., 1978). The synthetases involved in the biosynthesis of bacitracin, polymyxin, and gramicidin can also incorporate amino acid analogs into the final molecules. Very frequently circular peptides are produced as a mixture of a variety of compounds thus making their purification discouragingly tedious. Synthetases are nonspecific to such a degree that they will accept and incorporate one of several amino acids and yield a mixture resulting from substitution of various amino acids in the final molecule. Enniatin, the cyclohexadepsipeptide antibiotic, has alternating residues of D-hydroxyisovderic acid and N-methyl amino acids, forming an 18membered ring. The enniatins A, B, and C contain the N-methyl derivatives of L-isoleucine, L-valine, and L-leucine, respectively. All three depsipeptide enniatins (A, B, and C) are synthesized by the same soluble multienzyme complex. The enzyme has been purified to a high degree from Fusarium and has a molecular weight of 250,000. Synthesis of specific types of enniatins is thus dictated by the intracellular concentrations of various amino acids that are subsequently assembled into different depsipeptides. Omission of S -adenosylmethionine results in the production of nonmethylated enniatins. The rate of formation of nonmethylated enniatins is only 15% of that of enniatins that require S-adenosylmethionine as a methyl donor. In biosynthetic schemes of certain secondary metabolites, even the exact order of reactions may not be important. Several parallel pathways may convert a given intermediate to the same end product. For example, several parallel routes probably convert lanosterol to ergosterol. An enzyme system of this kind can be used to carry out regiospecific one- or two-step transformations of unnatural sterols, which is of economic importance for biological transformation not only of sterols, but also of other molecules. If an enzyme that participates in the biosynthetic sequence as a group modifier is missing, then further enzymatic modifications still occur. For example, if either methionine or methylating enzyme is depleted from the cell, the demethylated tetracyclines are produced. Even though methylation is one of the earlier steps in tetracycline biosynthesis, further modifications of the demethylated precursor by succeeding enzymes proceed in the usual manner. The dehydrogenation step in the synthesis of many secondary metabolites is of economic significance. In the same fermentation, saturated and unsaturated compounds may occur as mixtures and their occurrence depends on fermentation conditions. Cholesterol and dihydrocholesterol, ergosterol and dihydroergosterol, gibberellins and dihydrogibberellins, fusaric acid and di-

38

VEDPAL SINGH MALIK

hydrofusaric acid, streptomycin and dihydrostreptomycin, spectinomycin and dihydrospectinomycin occur in varying ratios in the same fermentation. Dehydrogenation may be a reversible step, and understanding of the regulatory mechanism that controls this step may be used to direct the fermentation to minimize or to avoid undesirable species of metabolite. As a result of the loose specificity of enzymes involved in secondary metabolism, cellular synthetic machinery has been used to incorporate analogs of precursors for generating altered molecules (Malik, 197913).

IV. Controlling Effect of the Environment

A. NUTRITION Although good growth may occur in many media, secondary metabolites may only be produced in a specific medium. Thus Penicillium cyclopium forms penicillic acid on Raulin medium but not on Czapek-Dox medium (Bentley and Keil, 1962). Sometimes a given organism may produce one metabolite on one medium and a totally different one on another medium. For example, Penicillium griseofuluum produces griseofulvin on CzapekDox medium and fulvic acid on Raulin medium (Oxford et al., 1935). Variation in the chemical composition of the medium and its relationship to yields and type of secondary metabolites is well known. Development of a medium that produces high yields of a desired secondary metabolite is still empirical to a considerable degree (Demain, 1972, 1973). Distribution of precursors into various branch pathways may vary and may depend on the growth environment and the genotype of the organism. The flow of precursors can be increased in the desired direction by mutation or medium manipulation. Brewer and Frazier substituted gum (dextrin) for glucose to produce amphotericin B in preference to amphotericin A. The presence or absence of certain ions or of carbon and nitrogen sources can inhibit, activate, induce, and derepress certain enzymes, perturbing the normal channeling of key intermediates that support balanced growth (Weinberg, 1978). B. TRACEMETALS Depression of certain aromatic amino acid biosynthetic enzymes of Escherichia coli by growth in iron-deficient medium has been reported by McCray and Herrmann (1976). Iron-deficient medium is the key to the commercial production of citric acid. Trace metals play a remarkable role in secondary metabolism and may be involved in enzyme-coenzyme combinations. For example, a zinc-deficient culture of Rhizopus nigricans accumulates large amounts of fumaric acid. However, in the presence of small amounts of zinc, primary metabolism

GENETICS AND BIOCHEMISTRY OF SECONDARY METABOLISM

39

becomes predominant and no fumaric acid is produced (Foster and Waksman, 1939). In 1955, Ehrensvard noted that the major metabolite is 6-methylsalicylic acid when Penicilliuin urticae is grown on Czapek-Dox medium containing lo-* N Zn2+. However, with N Zn2+ in the same medium, the 6-methylsalicylic acid is not formed, but large amounts of gentisic alcohol, toluguinol, and patulin accumulate instead. The effect of the level of iron on the ratio of patulin to gentisyl alcohol production by a Penicillium was reported in 1947 by Brack. Multiple antibiotic moieties or totally different biologically active molecules can be produced in low yields during fermentation. In such cases, the use of various media results in diEerent antibiotic ratios. It becomes challenging to purify a highly active component that is produced in tiny amounts in a complex medium. Godfrey and Price (1972) identified many different components in a coumermycin fermentation. It was a stroke of good fortune to find that traces of cobalt ion directed the biosynthesis exclusively to couinermycin A , . Producing organisms may require vitamin B,2 in order to methylate antibiotic precursors and need traces of cobalt to ensure enough vitamin B,, to complete that sequence. The metabolite resulting from a fermentation in which biosynthesis has been directed mainly to the production of one component is easily isolated.

C. HYDROGEN IONCONCENTRATION p H has profound effects on both primary and secondary metabolism (Foster, 1949). For example, the best yields of itaconic acid by shake cultures of Aspergillus terreus are obtained when the organism is grown at a constant pH of 1.8. Any increase in the initial pH leads to increased cell mass but decreased itaconic acid. p H 2.3 is optimal in surface cultures, whereas below pH 2.2 and above p H 2.4 no itaconic acid is produced (Lockwood and Nelson, 1946). For maximum yield of kojic and citric acids, Aspergillus niger should be inoculated into a medium with pH 2 . 5 3 . 5 . A higher initial p H shifts the metabolism toward the formation of oxalic and gluconic acids.

D. TEMPERATURE AND AERATION Every fermentation process has an optimum temperature and aeration rate.

V. Genetics of Secondary Metabolism The rationality inherent in the techniques of modern genetics may disentangle many intellections about secondary metabolism. Genetics studies of secondary metabolites have been repeatedly

40

VEDPAL SINGH MALIK

scrutinized (Queener et al., 1978; Hopwood and Merrick, 1977; Malik, 1979a; AliKhanian and Danilenko, 1979), but experiments dealing with the mapping of genes determining defined enzymatic reactions are scarce. In fact, all biosynthetic steps, using blocked mutants and enzymes, have not been defined for any single secondary metabolite. Systematic and precise study of the biochemical genetics and regulatory systems of secondary metabolite-producing microbes are progressing rather slowly. Therefore, it is hard to formulate any sound hypothesis regarding regulation of secondary metabolism. Because of the economic importance of antibiotics, many efforts have been made to obtain antibiotic-overproducing strains by genetic recombination (AliKhanian and Danilenko, 1979; Malik, 1979a; Cape, 1979; Hopwood, 1978; Hopwood and Chater, 1980), but the results thus far have not been very encouraging. Synthesis of a secondary metabolite is a polygenic characteristic (Malik, 1979a). Vanek et al. (1971) estimated that more than 200 genes are involved in the synthesis of chlortetracycline. The genes directly governing synthesis of antibiotics from basic building subunits are involved, but those responsible for the synthesis of their precursors, coenzymes, cofactors, energy metabolism, transport mechanisms, cell permeability, architecture, and resistance to the produced antibiotic also affect final antibiotic yield. Various genetic methods developed with Streptomyces coelicolvr have been used by many investigators to study the genetics of various other streptomycetes (Hopwood and Merrick, 1977). AliKhanian and co-workers performed many experiments to unravel the genetic control of oxytetracycline biosynthesis in Streptomyces rim.osus, and they discovered that an auxotrophic mutation decreased or abolished antibiotic activity in S. rimosus. Deleterious and pleiotropic effects of auxotrophy on antibiotic production make it hard to map genes involved in antibiotic synthesis. Various investigators have used multiply marked auxotrophic strains in curing experiments to suggest involvement of plasmids in antibiotic synthesis. These results should be confirmed using a prototrophic, high-producing ancestor. Mutations that map at three different locations on the S. coelicolor chromosome are pleiotropic, because they nullify the production of methylenoinycin, actinorhodin, and aerial mycelium (Merrick, 1976). It would be useful to have a parallel attack on a few antibiotic-producing systems. The study of chlorainphenicol production by Streptomyces uenezuelae, of cephamycin production by S. griseus, of actinorhodin production by S . coelicolw, of tetracycline production by S. rimosus, and of j3-lactam production by several microbes have already been pursued to a level where meaningful experiments can now be designed to obtain answers to specific questions. Recombination occurs in many other secondary metaboliteproducing microbes (Hopwood and Merrick, 1977) but cannot as such be

GENETICS AND BIOCHEMISTRY OF SECONDARY METABOLISM

41

exploited to study metabolic regulation, because no practical means of performing a genetic complementation test exists in secondary metaboliteproducing organisms. Recombinant DNA technology will now facilitate the construction of merodiploids, allowing a complementation test and operon fusions. This will aid in the dissection and manipulation of regulatory mechanisms for commercial production of these metabolites (Malik, 1980b).

A. CHROMOSOMAL MAPPING Genetic material for primary pathways is organized into regulatory units, and genes of many pathways are clustered in one segment of the genome. A gene cluster that is involved in a single pathway is called an “operon.” Organization of genes in operon-like structures is efficient but not essential for coordinated regulation of a metabolic route. Genes of many pathways do not form single gene clusters (operons) but are scattered in various segments of the chromosome. When genes of a metabolic pathways form several gene clusters (minioperons) located at various sites on the chromosone, they still can have tight regulatory interdependence. All the minioperons that are coordinately regulated have been called “regulons” (Goldberger, 1979). By using complementation and cosynthesis, about nine structural genes involved in oxytetracycline synthesis have been located on the chromosome in two clusters (AliKhanian and Danilenko, 1979). The fact that about 200 genes may be involved in tetracycline biosynthesis makes it clear that a great deal remains to be done to completely understand the genetics of oxytetracycline production. In the meantime, by mutagenesis, the yield of tetracycline has been increased to more than 25 gmfliter. Another report of the chromosomal location of genes involved in secondary biosynthesis has been provided by Wright and Hopwood (1977). A series of 76 point mutations, causing blocks in actinorhodin production, map in a cluster on the S. coelicolor chromosome (Rudd and Hopwood, 1979), suggesting that most genes involved in actinorhodin biosynthesis are closely linked. Mutants blocked in actinorhodin biosynthesis fell into seven phenotypic classes on the basis of antibiotic activity, accumulation of pigmented precursors, and cosynthesis. Actinorhodin (Brockmann et al., 1966), kalafungin (Hoeksema and Krueger, 1976), nanaomycins (Omura et al., 1976), griseusins (Tsuji et al., 1976), granaticin, and the naphthocyclinones belong to the isochromane quinone class of antibiotics (Zeeck et al., 1974). Study of the genetics of actinorhodin biosynthesis by S. coelicolor could be relevant to the understanding of the biochemistry and genetics of the polyketides. Actinorhodin is a pH indicator. It is blue and very soluble in polar solvents above p H 7. Below pH 7, it is red and sparingly soluble. On certain media, this acid-base

42

VEDPAL SINGH MALIK

indicator pigment accumulates intracellularly in the red, acidic, waterinsoluble form (protoactinorhodin). The red protoactinorhodin is converted to the blue, water-soluble form by inverting the culture agar plates over ammonia solution in a petri dish lid. Mutants blocked in actinorhodin do not respond to ammonia fumes and do not change color from red to blue (Rudd and Hopwood, 1979). This simple assay, based on visual detection of colony color, makes genetics of actinorhodin rather easy. Another attractive feature of actinorhodin is the fact that this pigment is produced by S . coelicolur, whose genetics and molecular biology are by far the most advanced among all streptomycetes (Hopwood, 1980b; Chater, 1980). The genes that control biosynthesis of actinorhodin and another red pigment are closely linked in a single chromosomal cluster (Rudd and Hopwood, 1980).

B. EXTRACHROMOSOMAL ELEMENTS Although the knowledge of the genetic control of secondary metabolism is rather fragmentary, there are indications that plasmids may play a role in it. Because secondary metabolism is not essential for the survival of the producing organism and plasmids usually code for such secondary functions, it is possible that certain plasmid DNA sequences play a role in the synthesis of secondary metabolites (Kalakoutskii and Agre, 1976). This role may be direct. For example, structural genes for methylenomycin A biosynthesis in S . coelicolur appear to be plasmid borne (Hopwood, 1980). It is possible that the genes that code for the metabolic steps of secondary metabolite production are clustered in operons and are located on plasmids. There are four classes of evidence that suggest involvement of plasmids in the synthesis of antibiotics. They are considered in the following sections. 1, Natural Genome lnstability

Many researchers working with secondary metabolites have observed that metabolite production is frequently lost. Many such examples, including observation of unstable colony pleomorphism, have been reported (Malik, 1979a; Nakatsukasa and Mabe, 1978). The polygenic character of secondary metabolism may be partly responsible for the frequency of genetic degeneration of industrial strains. Another cause of variation in prokaryotes is associated with the extrachromosomal genetic elements known as plasmids. Location of genes on a plasmid ensures that (depending on the plasmid status of the cell) the cell gains or loses a block of genetic material. These additional novel genes could be of survival advantage to that cell in an existing environment or in adjustment to a new one. One example of a genetic element involved in genome instability is the

GENETICS AND BIOCHEMISTRY OF SECONDARY METABOLISM

43

element Tyl of yeast (Greer and Fink, 1979; Roeder et al., 1980; Scherer and Davis, 1980). A laboratory strain of Saccharoinyces cwevisiae contains 30 copies of Tyl per cell. It accounts for over 1%of the whole yeast genome. It is probably a virus of about 5 kilobases in length. It has duplications on the end that are called delta. The whole element, including duplications at the end, is transcribed, and the corresponding RNA accounts for 10% of the total RNA of yeast. Thus, Tyl constitutes a large fraction of the genome and produces a large fraction of RNA in the yeast. The function of Tyl is not known. It has no known phenotype. However, it can be tagged and then mapped by fusion to other genes of known phenotype (Roeder and Fink, 1980). Cameron et al. (1979) fused His3 gene in the middle of Tyl by in uitro manipulation and transformed the His3 -containing Tyl element into yeast, which integrated it into the chromosome by homologous recombination at many different locations. These integrated sequences are scattered over the whole yeast genome and recombine if they are located on different chromosomes; this results in scrambling of the yeast genome. The genetic element Tyl is probably responsible for most of the reciprocal translocation occurring in yeast (Chaleff and Fink, 1980). Gene conversion has been demonstrated by Scherer and Davis (1980) using the repetitive DNA of Tyl. These investigators fused a promoterless nonfunctional His3 gene into one Tyl element and another nonfunctional His3 gene with a deletion in the middle into another Tyl element. Both of these genes were inserted into the chromosomes of yeast to determine what events might occur to reestablish a functional His3 gene. A classical revertant should not restore function because both His3 genes are nonfunctional because of deletions. Some major rearrangement of the genome would be required for reestablishing function. In fact, these deletions were gene converted across chromosomal arms into a functional His3. These surprising results suggest that one gene sequence, without any change in its own sequence, can gene convert the other. Chromosomes with similar sequences can communicate with one another. One can even insert large pieces of DNA in the middle of His3. Davis inserted a galactose gene cluster into His3. That cluster was gene converted out to restore functional His3, suggesting that all that is required for gene conversion is flanking the DNA sequence homology at both ends of the sequence. Since repetitive DNA is very prevalent among microbes (Klein and Welch, 1980), this kind of phenomenon could rapidly rearrange genomes of organisms of industrial importance. Transposable element Tyl also affects gene expression. It does not affect gene expression if inserted in the middle or beside the gene. It only turns genes on if inserted at a distance. The frequency of these events are lo4. They act recessive and are cold sensitive.

44

VEDPAL SINCH MALIK

Other secondary metabolite-producing organisms like streptomycetes and fungi may have repetitive DNA sequences like Tyl. Therefore, they could also recombine and produce genome instability as a result of inversions, translocations, and deletions. These elements can also communicate with one another without being translocated or excised. This could result in gene cwnversions similar to those in yeast.

2 . Curing The naturally occurring instability of antibiotic production is further enhanced if a culture is treated with DNA chelating agents such as ethidium bromide or acridine orange. Antibiotic-negative variants occur at very high frequency (1to 10%or more) among the populations that have been exposed to UV, high temperature, novobiocin, or rubradirin. Clones that have been regenerated from protoplasts also yield a large proportion of antibiotic nonproducers. A number of plasmicl curing agents have been tried, and only treatment with novobiocin resulted in loss of ability of Streptomyces refuineus var. thermotolerans to produce anthramycin. The anthramycin nonproducers of S . refuineus were less resistant to anthramycin, had lost their typical leathery colonial appearance on plates, and produced a light-activated pink pigment not normally produced by the anthraniycin-producing strains. Similar plasmid DNA was present in both the anthramycin producer and the nonproducer. Novobiocin may cause deletions resulting in pleiotropic effects, because no reversion back to antibiotic production occurred even after repeated transfers (J. Stefan Rokem and Laurence H. Hurley, College of Pharmacy, University of Kentucky, Lexington, Kentucky 40506). The ability of Streptomyces kasugaensis to produce aureothricin, thiolutin, and kasugamycin is eliminated by treatment with curing agents. Two plasmids (6.8 and 15 megadaltons) have been isolated from S . kasugaensis, but further evidence is required to establish the role of these plasmids in antibiotic production. Curing studies with Streptomyces alboniger suggest that specific functions required for aerial mycelial formation, including pamamycin production, may be coded by extrachromosomal elements (Pogell, 1975; Redhaw et al., 1979). The curing action of acriflavine suggests the possible involvement of extrachromosomal elements in controlling aerial mycelia, pigment, and avermectin production in Streptomyces avermitilis. Other antibiotics that have been reported to be plasmid controlled are chloramphenicol (Akagawa et al., 1975), holomycin (Kirby, 1978), kasugamycin, aureothricin, methylenomycin (Bibb et al., 1980a), turimycin, chlortetracycline (Boronin and Sadovnikova, 1972) streptomycin (Shaw and Piwowarski, 1977), actinomycin (Ochi and Katz, 1978), neomycin, spiramy-

GENETICS AND BIOCHEMISTRY OF SECONDARY METABOLISM

45

cin (Omura et al., 1979), kanamycin (Chang et al., 1980; Hotta et al., 1977), and beromycins (Blumauerova et al., 1980b). Akagawa et al. (1975) have reported that upon acridine treatment of chloramphenicol-producing S. venezuelae, many nonproducer clones were obtained among the progeny. The fact of loss of chloramphenicol production at high frequency and further genetic analysis were used to suggest involvement of a plasmid in chloramphenicol biosynthesis (Akagawa et al., 1975). If chloramphenicol nonproduction in S. venezuelae is indeed attributable to permanent loss of a plasmid, then the cured strain should be incapable of reverting to chloramphenicol production. However, revertants of the strain claimed to have been cured of chloramphenicol production by Akagawa et al. (1975) have been selected (Malik, 1980~).These revertants simultaneously produce chloramphenicol (50 mg/ml) and are resistant to chloramphenicol(1OO mg/ml). This shows that the presumptive cured strain has not lost the genetic potential to synthesize chloramphenicol. Therefore, alternative interpretations have to be considered to explain this loss of the synthetic capacity in the pseudo-cured strain of Akagawa et al. (1975). S. venezuelae (VM3: his-leu-ade-cpp-) does not grow vigorously as some other chloramphenicol-producing Streptomyces (Malik, 1979b). The poor growth of S. venezuelae (VM,: his-leu-ade-cpp-) may be attributable to the presence of several auxotrophic markers and could influence the yield of chloramphenicol. A study of the effect of reversion to prototrophy on chloramphenicol production and growth might be of interest. Antibiotic production by Pseudomonas reptilivora as a result of phage conversion has also been reported (Martinez-Molina and Olivares, 1979). Nakano et al. (1980a) subjected Streptomyces lavendulae to curing agents and obtained unstable pleiotropic mutations at high frequency. Most of the auxotrophs obtained were arginine auxotrophs, required argininosuccinate for growth and either did not produce any P-Iactamase or produced a low level of p-lactamase. Arginine auxotrophs also failed to produce any aerial mycelia, formed small colonies, and showed increased sensitivity to benzylpenicillin. Nakano et al. (1980b) found only arginine auxotrophs at a frequency of 12%among plasmid-carrying S. kasugaensis cells that had been treated with acriflavine. Arginine auxotrophs did not contain any plasmid. Ethidium bromide treatment produced revertant prototrophs at a frequency of 1 0 -to ~ lo-'' and these revertants were shown to regain the plasmid. Arginine auxotrophs spontaneously reverted to prototrophs at a very low frequency. These authors suggest that an unstable genetic element regulating secondary metabolism inactivated argininosuccinate synthetase gene by transposition or insertion. Similar phenomena have also been reported in other

46

VEDPAL SINGH MALIK

Streptomyces (Pogell, 1975; Redshaw et al., 1979; Sermonti et al., 1978, 1980). Genes involved in chloramphenicol resistance, argininosuccinate synthetase, chromosome transfer, and aerial mycelium formation may be carried on a transposon SCTn,, which has two high transposition specifity regions on the S. coelicolor chromosome, one between cysA and metA and the other at the right of urgA (Sermonti et al., 1980). As in other bacteria, the chloramphenicol resistance on SCTn, is not attributable to chloramphenicol acetyltransferase, but the transposon may code for permeability alterations. This type of evidence for involvement of plasmids in antibiotic synthesis is only suggestive and not definitive. Other causes of genome instability in many organisms are well known (Malik, 1979a, 1980~).Tandem genome duplications, chromosomal rearrangement due to inversions, and insertions of DNA sequence all lead to instability. Other mechanisms analogous to control of phage variation in Salmonella or activation of mating type in Saccharomyces cerevisiae may be responsible for genomic instability in Streptomyces. Such mechanisms could be involved in regulation and synthesis of antibiotics and other secondary metabolites. The mechanism of DNA packaging in the Streptomyces spores could be analogous to DNA packaging in bacteriophage T4, resulting in circularly permuted genomes with tandem duplications. Certain agents (e.g., ethidium bromide) could induce chromosomal rearrangements, whereas others simply may select naturally occurring variants with spontaneous genome rearrangements. 3. Extrachromosomal D N A

A good system of genetic analysis can yield information suggesting involvement of plasmids in the biosynthesis and regulation of a secondary metabolite. Recombination frequency showing infectious transfer and no linkage to chromosomal markers was used to conclude that genes that determine chloramphenicol and methylenomycin biosynthesis are plasmid borne (Hopwood et d., 1980b). However, the demonstration of the presence of plasmid DNA in the antibiotic-producing strains and the absence of plasmid DNA in the antibiotic-negative strains would provide more evidence of involvement of plasmids in antibiotic production. Such correlations have not yet been demonstrated. Rearrangement of plasmid or insertion of plasmid into the chromosome with accompanying secondary metabolite production is another piece of evidence that could suggest plasmid involvement. Transformation of antibiotic production by the isolated plasmid DNA into an antibiotic-negative strain would be the best evidence, but such solid data have not yet been obtained for any antibiotic. The plasmid pUC3 was isolated from S. uenezuelae strain 3022a, which

GENETICS AND BIOCHEMISTRY OF SECONDARY METABOLISM

47

produces chloramphenicol. The plasmid was digested with the restriction endonuclease BamHI into four fragments with approximate sizes of 19, 5.7, 4.1, and 2.7 kilobase pairs. These fragments were cloned into Escherichia coli HBlOl using the plasmid pBR322 (having a single BamHI site) as a vector. None of the constructed E. coli clones produced chlorainphenicol in detectable amounts not did any of the clones express resistance to the 12 antibiotics tested. Marahiel (personal communication) has isolated a plasmid DNA from the cells of Bacillus brevis ATCC 9999 (gramicidin S producer) and of a gramicidin S-negative mutant. All of the plasmid DNA in the parent culture is associated with the chromosome and membrane, whereas the same plasmid is free in the cytoplasm of the mutant that does not produce gramicidin S. Interaction of plasmid with the membranes and chromosome may regulate genes involved in gramicidin S biosynthesis. The plasmid has a molecular weight of 41.2 x lo6 and does not seem to carry any resistance to antibiotics like lincomycin, novobiocin, kanamycin, or streptomycin. Microcins are low-molecular-weight (less than 1000) antibiotics produced by Gram-negative bacteria. They inhibit the growth of a wide range of bacterial genera by specific inhibition of methionyl-tRNA synthetase. Microcins also inhibit methionine synthesis because they act as competitive inhibitors of homoserine-0-transsuccinylase, an early enzyme involved in the biosynthesis of L-methionine. Plasmid codes for the synthesis of the microcin and resistance of the host cell to its action (Diaz and Clowes, 1980). I n no case has the antibiotic-synthesizing ability been transformed into a nonproducing Streptomyces by using purified plasmid DNA. Isolation of plasmids from macrolide-producing Streptomyces ( S . albogriseolus, S . griseoflavus, S. reticuli, and several others) was reported by H. Schrempf (Wirzburg, W. Germany). Cured variants of S . reticuli that have lost antibiotic resistance, antibiotic production, sporulation, and/or melanin production either have no plasmid DNA or contain plasmids with deletions and/or insertions. Expression of the tyrosinase gene was correlated with the presence of a 3-million-dalton plasmid fragment, which is lost in melanin-negative strains. However, only the transformation of melanin-negative strains to melanin producers by purified plasmid will provide the firm evidence that plasmid, indeed, directs melanin synthesis in S . reticuli. In some variants of S . reticuli, certain DNA sequences are amplified, and extensive reorganization of chromosomal sequences occurs. Similar observation of extensive genome instability and rearrangements was made in the neom ycin-producing Streptomyces faradiae. Strains of S . faradiae that have been cured of neomycin production contain somewhat different plasmids (Davis, 1980). One mutant (H3) that is resistant to high concentrations of neomycin yields five times more plasmid DNA and produces more phos-

48

VEDPAL SINGH MALIK

photransferase and neomycin . Restriction enzyme analysis of plasmid DNA korn H3 strains showed plasmid rearrangement. Parent plasmid was 52.1 megadaltons, but mutant H 3 plasmid was 56.4 megadaltons and had a 4-megadalton insertion near the two o'clock region. In other strains of S. furadiae that have different levels of resistance to neornycin, rearrangement occurs in the same region as in the parent plasmid. However, purified S. furucliae plasmid DNA did not hybridize with the purified genes of neornycin-3-phosphotransferase and neomycin-3-acetyltransferase that have been cloned in Hopwoods laboratory from the same S. furudiue strain. The coincidence of relationship between plasmid DNA, neomycin resistance, and neomycin production should be further examined by carrying out transformation of the neomycin-nonproducing streptomycetes with plasmid DNA.

c. GENETICENGINEERING OF SECONDARY METABOLITES Availability of host vector systems and several cloned genes provides probes for hybridization and selection, thus making molecular biology of these industrially important organisms amenable to further exploration. This knowledge is ripe for harvest-to achieve desired results in the fermentation industry. The new recombinant DNA methodology can be used to fuse genes and to construct merodiploids for performing a genetic complementation test yielding valuable information about cellular regulatory mechanisms. Several vectors can now be used to clone genes in streptomycetes. Fragments of S . coelicolor genome carrying methylenomycin resistance and various prototrophic alleles have already been cloned using SCPB* as a vector and S. coelicolor as a host (Malik, l980b). Such clones are highly unstable but can be stabilized if a fragment of SCP2* that carries a specific plasmid segregation fiinction is included in the recombinant plasmid. Hopwood arid associates cloned antibiotic resistance genes from S. farudiae and Streptomyces azureus into Streptomyces lividans. The S . lividans plasmid SLP1.2 was used as a vector to clone the neomycin-3phosphotransferase gene and the neomycin-3-acetyltransferase gene from S. farurliat.into S. livirlans. Using the same vector, thiostrepton resistance and erythromycin resistance from S . amreus and S . erythreus have also been cloned in S. liuidans. These antibiotic resistance markers can now be inserted into other vectors or combined together to build vectors with multiple resistances to generate a gene-transporting vehicle analogous to E . coli cloning vector pBR322. Many organisms, such as Drosophila, corn, and yeast (Stinchcomb et al., 1980), contain autonomously replicating sequences dispersed throughout their genome. Similarly, vectors can now be built totally out of chromosomal

GENETICS AND BIOCHEMISTRY OF SECONDARY METABOLISM

49

sequences of industrial organisms; these vectors will replicate autonomously or integrate into the host chromosome. Such vectors yield very high transformation rates (Struhl et al., 1979a). Some vectors are unstable, but DNA can be inserted to enhance stability. For example, centromere sequences make yeast vectors relatively stable. The temperate actinophage C31 has been developed as a suitable Streptomyces cloning vector (Chater, 1980). Like all known Streptomyces temperate phages, this actinophage resembles E . coli phage lambda in size and morphology and contains sticky ends. A 20-gene linkage map has been constructed by using temperature-sensitive plaque morphology and host range mutants. Heteroduplex analysis, denaturation, and restriction enzyme digestion have been used to align the linkage niap with the physical map of the linear 39-kilobase D N A of C31. Repressor gene ( C )is located in the middle of the genome and many genes for essential functions are located to the left of the C gene. Actinophages that are resistant to chelating agents have deletions that are in the center of the genome and reniove repressor gene or that are in the rightmost 25% of the molecule and remove phage attachment site to the chromosome. Partial Eco RI digests of E. coli plasmid pBR322 and actinophage deletion mutant C31Cts23 were ligated and transfected into S. lividans. Transfectants were selected by hybridizing clones to radioactive pBR322 DNA. The resulting C32-pBR322 recombinant molecule replicates as a phage in Streptomyces and as a stable plasmid in E . coli. Further modifications have produced a vector that has single restriction sites for BamHI and PStI and can be used to clone up to 7 Rb of foreign DNA. A C31-pBR 322 hybrid is restricted by S. albus P and does not form plaques on R-m+ S. albus G mutants if part of pBR322 is inserted in C31. This fact may be used to select deletion mutants. Ampicillin and tetracycline resistance genes of pBR322 may be expressed in Streptomyces if inserted in such a manner that they are transcribed from an actinophage promoter. Chloramphenicol acetyltransferase gene from E. coli has also been cloned in S. lividans using S . lividans plasmid SLP1.2 as a vector. This enteric bacterial gene is expressed in S. lividans and its transcription initiates from the promoter of either Streptomyces vector or E . coli gene controlling elements are being recognized by streptomycetes transcription machinery. These cloned antibiotic resistance genes provide a good selective marker for cloning D N A sequences in streptomyces. The convenient assay of chloraniphenicol acetyltransferase and P-lactamase can be further used to understand the molecular biology of Streptomyces promoters. In this way, systematic study of the molecular biology of Streptomyces may thus be exploited to generate hybrid antibiotics, to introduce useful genes for utilization of inexpensive carbon sources (e.g., cellulose) and for production of valuable human metabolites (e.g., insulin, interferon). Multicopy vectors with highly efficient constitu-

50

VEDPAL SINGH MALIK

tive promoters should soon be available to amplify and to express genes of interest in Streptomyces, making them very attractive for the production of valuable inetabolites. Certain thermophilic Streptomyces that grow in a very high cell mass on cheap carbon sources are another promising host for producing valuable metabolites (e.g., amino acids, organic acids, vitamins, enzymes, antibiotics, coloring pigments) at a reduced cost. Cell-free biosynthesis of antibiotics and other secondary inetabolites is yielding knowledge of their biosynthetic processes and regulation as well. Kleinkauf and co-workers (Technische Universitat, Berlin) have purified many multienzyme complexes from various Bacillus species; these complexes are involved in the synthesis of gramicidin S, tyrocidine, bacitracin, polymyxin, and many other peptide antibiotics. The purified enzymes can be used to make radioactive antibodies that may be used to develop radioimmunoassays useful in cloning genes that determine the synthesis of enzymes involved in antibiotic production. Enzymology of chloramphenicol biosynthesis in S. venexuelae unravels some very interesting phenomena (Malik, 1979b). Chloramphenicol is derived from chorismate, and the branching enzyme arylamine synthetase converts chorismate to p-aminophenylalanine. This enzyme is not presept in cultures unable to produce chloramphenicol and is repressed in producing cultures by addition of chloramphenicol. Arylamine synthetase has a subunit with aminotransferase activity. This aminotransferase subunit is multispecific and may be shared by anthranilate synthetase, p-aminobenzoic acid synthetase, and arylamine synthetase. If the same aminotransferase subunit is, indeed, common to the branching enzymes involved in biosynthesis of tryptophan, p-aminobenzoic acid, and chloramphenicol, then study of the regulation of the synthesis of this multispecific aminotransferase subunit may yield interesting information. Nucleotide sequences for this subunit may be isolated by utilizing cloned anthranilate synthetase gene of E . coli, Bacillus subtilis, or yeast as a hybridization probe. DNA sequencing could reveal the mystery and advance our knowledge of the regulatory biology of streptomycetes, exposing the structures of attenuators, leaders, operators, promoters, and other nucleotide sequences involving regulatory elements. Judicious manipulation of these sequences could result in a high level of gene expression that would increase yields of desired metabolites, ending up with a cheap cost of production. Several streptomycetes that produce novel /3-lactam antibiotics (thienamycin, cephamycin, nocardicin) have been isolated. Cloning of genomes of p-lactam-producing streptomycetes into P-lactam-producing hngi or vice versa could yield improved antibiotic fermentation processes or even new antibiotics. Because many genes that affect the growth are located in or near the ribosomal gene cluster in E . coli, isolation and sequencing of

GENETICS AND BIOCHEMISTRY OF SECONDARY METABOLISM

51

the ribosomal gene cluster of antibiotic-producing organisms may yield information pertinent to the regulation of antibiotic synthesis. Ribosomal genes are present in abundant copies in any organism and provide a vector that can be used to integrate any DNA sequence at many sites on the chromosome of the corresponding microbe. D. RECIPROCAL GENETICSOF SECONDARY METABOLISM

1 . Gene lsolation Methodology is available for the isolation of genes whose level of expression changes in response to the environment. Such genes are involved in secondary metabolism and are heavily transcriptionally regulated. Because these genes are not essential for growth, mutants in them cannot be isolated by conventional genetics. However, these genes can now be isolated and their transcriptional controls dissected. Genes, such as his-3 of yeast, that are constitutively expressed are not the best for isolation by this approach. The highly transcriptionally regulated sequences would be useful for manipulating other sequences to obtain a high level transcription of genes of commercial significance. There may not necessarily be any mutants available in secondary metabolism genes. These genes can be located, however, without tagging by mutation. DNA sequences that have abundant mRNA present in the cell in response to environment are selected. With this in mind, St. John and Davis (1979)looked for genes in Saccharornyces cerevisiae that responded to galactose. They isolated DNA sequences that were complementary to the RNA of galactose-grown S. cerevisiae but had no homology to the RNA of S. cerevisiae that had been grown on another carbon source. Rapid differential screening methods allowed easy isolation of such DNA sequences. It was not necessary to purify mRNA for this purpose, as crude extracts could be used. Total RNA was isolated from yeast grown on lactose, and another preparation of total cellular RNA was obtained from yeast grown on acetate. Both RNA preparations were labeled with 32P.Two replicas of the total genome DNA of many clones of yeast were made. One replica of each clone was hybridized with lactose RNA and the other one with glucose or acetate RNA. The clone that hybridized with lactose RNA and not with glucose RNA was the one of interest and had all the genes responsible for galactose metabolism, which are clustered. There are other genes that are not involved in direct catabolism of galactose but are inducible by galactose (a-glycosylase). These genes have also been isolated by this approach. Also, large sections of DNA belonging to the galactose metabolism region of chromosome have been cloned in a plasmid.

52

VEDPAL SINGH MALIK

2. In Vitro Mutugenesis and Chromosome Manipulation Rare mutational events down to can be screened for by inserting desired DNA fragments into bacteriophage h (Struhl and Davis, 1980; Struhl et al., 1980). The genes can also be mutagenized in vitro before they are put back into the chromosome. A deletion was made in the middle of h i s 3 gene by cutting with restriction enzyme, re-ligating, and then transforming yeast from His+ to His- to inanipulate the chromosome. This has been achieved by inserting the mutant gene in a vector that has a selectable marker, such ura-3. For example, transformants are selected for Ura-3+ and then screened for H i s X . If ura-3 is from a different microbe, the homologous recombination event at the ura-3 locus can be ignored. Most homologous recombinants occur at h i s 3 by forming duplicated unstable structures, giving His-3- and Ura-3+. After 10 generations, 1%of the cells lost the vector by homologous recombination, leaving the altered sequence and removing the wild type and vector. In this way, a genetic system based on homologous recombination and in uitro manipulation of DNA can be built where a genetic system did not exist. All kinds of mutants can be generated without examining the phenotype. Manipulation is termed “reciprocal genetics,” as the phenotype is identified after the genotype has been constructed. DNA of unknown function can be mutagenized and integrated into the chromosome, and the phenotype of the transformant characterized. This allows engineering of mutants without prior knowledge of phenotype and may have direct immediate application for manipulating genes involved in commercially important antibiotics. Three genes are needed for galactose metabolism: transferase, epimerase, and kinase. Accordingly, deletions were made in vitro in these genes, and the constructed genes were ligated to plasmids and put back into yeast as a duplicated structure. These strains were cultured until the vector did not spontaneously excise out at a detectable frequency. A method of scoring the rare excision event utilized a different vector (YRP15), which has tyrosinetRNA suppressor gene as a selectable marker in addition to the ura-3 gene (Struhl et al., 1979a). Only those transformants will grow on media of high osmotic strength that have lost the tyrosine-tRNA suppressor, whereas those containing suppressor tRNA do not grow because of a change in the cell wall (Thomas and James, 1980). This selection against the cells that contain tyrosine-tRNA suppressor allows detection of very rare events. By selecting for Ura-3, one obtains transformants that have received the mutant genes, hut by selecting against suppressor tRNA those transformants are selected where vector and suppressor tRNA have been excised. Such clones can be scored if they have replaced mutant gene for the wild type. This methodology of reciprocal genetics, developed by Davis, can be

GENETICS AND BIOCHEMISTRY OF SECONDARY METABOLISM

53

applied to a study of the genetics of secondary metabolism, because secondary metabolism is transcriptionally regulated and responds to the environment.

VI. Control of Secondary Metabolism Microbial productivity is exploited to a maximum in commercial fermentations of many metabolites (Woodruff, 1980). The channeling of metabolic intermediates in the desired direction is helped by the knowledge of the regulatory mechanisms and biosynthetic pathways. As mentioned earlier, antibiotic-producing microorganisms generally produce a variety of biologically active molecules with related chemical structure. The shifting of the component ratio, the repression of the synthesis of undesirable components, or the promotion of the yields of minor components could be enhanced by the knowledge of the regulation of the biosynthetic process. For example, originally cephalosporin C was a minor component of the original Cephalospurium strain of Brotzu. Even though the yield of cephalosporin has been raised to an industrially realizable level with brute force (mutagenesis and medium manipulation), the biochemical basis of superiority of most industrial strains is not known. The knowledge of the regulatory biology responsible for cephalosporin biosynthesis would also be of help in designing rational strain improvement programs. During microbial growth, regulatory controls operate at the level of transcription of DNA into RNA, the translation of RNA into polypeptides, and the activity of enzymes. However, in secondary metabolism and sporogenesis, additional specific control signals are superimposed on those regulating microbial growth and general metabolism. Several such signals could regulate transcription of genes for initiation of secondary metabolism:

1. Like vegetative genes, promoters of the secondary metabolism-specific operons could be available for transcription, but the specific RNA polymerase has to be altered in a manner so that it could transcribe these genes. The preferential inhibition of sporulation and antibiotic synthesis by the polypeptide antibiotic netropsin and by ethidium bromide suggests that the DNA composition of promoters and other controlling elements of genes involved in antibiotic synthesis and sporulation may be different from that for log phase genes (Keilman et al., 1976; Rogolsky and Nakamura, 1974; Sankaran and Pogell, 1975). If the promoters of genes governing secondary metabolism are indeed different from promoters of the genes ordinarily recognized by the vegetative

54

VEDPAL SINGH MALIK

RNA polymerase holoenzyme, the RNA polymerase may be modified to accommodate the promoter recognition specificity of the enzyme that transcribes genes responsible for secondary metabolism. However, if the promoters for secondary metabolism and vegetative genes are identical, then RNA polymerase could be altered to interact with putative regulatory factors that allow RNA polymerase either to bind to the promoters of the genes involved in secondary metabolism or that permit RNA polymerase to read through the blocks presented by negative effectors or special termination sites in genes that govern secondary metabolism. If the vegetative enzyme only abortively initiates RNA synthesis from genes determining enzymes involved in antibiotic synthesis, mechanisms similar to release from termination (Roberts, 1976) or attenuation could affect regulation of secondary metabolism. 2 . Promoters of these genes could be masked by some negative regulatory protein such as a hypothetical repressor. 3. The amount of positive regulatory effectors such as CAMP or ppGpp could be limiting, and in conjunction with a positive regulatory protein, some effectors (e.g., ppGpp) could be used to alter the capacity of these promoters when the repressor has been removed. These factors could be metabolic products that interact with RNA-binding proteins similar to the CAMP-CAPsystem involved in the regulation of the lac operon in E . coli or of glutamine synthetase. 4. Regulation may also occur at the level of translation, involving leaders, attenuators, terminator, and antiterminator sequences in mRNA. 5. Regulatory controls that affect the accumulation of starting precursors for secondary metabolites can indirectly affect initiation of secondary metabolism. The starting precursors have to be available at the time of elimination of the controlling mechanism. 6 . In parallel to synthesis, resistance development to autotoxic metabolites is regulated in the producing organism. 7. Metabolite transport and cellular permeability are also controlled so that high concentrations of secondary metabolite could accumulate outside

the cell. Practically no experiments have been done that could identify the type of transcriptional and translational apparatus that exists in cells actively engaged in secondary metabolism. Two types of transcriptional apparatus may exist simultaneously. One would transcribe log phase genes and the other would be capable of transcribing genes specific for secondary metabolism and sporulation. To gain an understanding of specific functions required for antibiotic formation, mutants that are resistant to antibiotics with defined site of specificity (streptolydigin, streptovaricins, rifampicin) and that grow normally but are aEected only in formation of secondary metabolism should be selected.

GENETICS AND BIOCHEMISTRY OF SECONDARY METABOLISM

55

Some mutants may have a genetic lesion at a specific stage in initiation of secondary metabolism. Study of the biochemical basis of such mutations may lead to an understanding of the role of RNA polymerase in secondary metabolite formation.

A. GROWTH-LINKED SUPPRESSION Although biphasic fermentations are common, they are not a sine qua non for secondary metabolism.

VININC(1979)

In 1961, Bu’Lock promoted the theme that many microbes excreted complex organic molecules particularly during stationary phase and not during active growth. In view of the different biosynthetic activity in the successive growth phases, the term “trophophase” has been coined to characterize the period of active cell multiplication and synthesis of primary metabolites. “Idiophase” is correlated with the synthesis of secondary metabolites when active growth is ceased (Bu’Lock, 1961, 1967). This division of growth into trophophase and idiophase brings some order, but it is an oversimplification of the phenomena taking place during the cell’s growth cycle. During the early part of this century, Natural product chemists studied secondary metabolism of fungi. As mentioned earlier, these fungi were usually grown as surface cultures. Stationary cultures develop as surface mycelial mats that cover the entire surface of the medium and are fully exposed to atmospheric oxygen, whereas the lower, partially anaerobic, surface is in direct contact with the growth medium. The kinetics of such growth are different and more complex than the kinetics in shaken cultures. There is often a heterogeneity of metabolic environment in fungal cultures. On the other hand, shake cultures of many streptomycetes and fungi develop as a uniform mass or as pellets of mycelium but are still not completely homogeneous. However, shake cultures were not routinely studied, and the careful experiments that reveal relationships between growth and secondary metabolite formation were not performed. As knowledge of the biochemistry of inicrobiol growth becomes more complete, the data support the view that the relationship between growth and secondary metabolite formation is not quite as simple as originally thought by Bu’Lock (1961) and other naturalproduct chemists. Although the synthesis of some secondary metabolites is an example of the distinct sequence of trophophase and idiophase (Bu’Lock, 1961; Gaworowska et al., 1975), synthesis of many other secondary metabolites does not follow a distinct biphase growth pattern. Most of the chloramphenicol, etamycin, and 6-methylsalicylic acid are produced during the active growth period. Synthesis of rifamycins begins in the early logarithmic

56

VEDPAL SINGH MALIK

growth phasc of Nocardiu mediterranei, and addition of barbital to the culture medium prolongs the logarithmic growth phase and, at the same time, stimulates the synthesis of the antibiotic about twofold (Ruczaj et al., 1972). Synthesis of secondary metabolites also overlaps growth phases in many cultures that are slowly growing in nutritionally poor, chemically defined medium. The induction of upecific enzymes for secondary inetaholism is attributcd to removal of an override mechanism that is operational during the preceding balanced and coordinated growth. The ainidinotransferase in streptomycin fermentation, the acyl transferase and the phenylacetate activating enzyme in penicillin production, arid both enzymes in gramicidin S production are known to be produced in the later part of the growth phase. Phenoxazinone synthetase, involved in actinomycin biosynthesis, is not produced until after growth (Martin and Demain, 1978). Even though the formation of secondary enzymes is generally repressed during the logarithmic growth and is derepressed during suboptimal or stationary growth, and even though their synthesis is controlled by regulatory mechanisms, initiation of secondary metabolism frequently coincides with the exhaustion of some major ingredient of the growth medium and cessation of growth. Penicillin production by Penicillium chrysogenurn 8176 is most pronounced after the exhaustion of both glucose and acetate (Jarvis and Johnson, 1947), and synthesis of anthraquinone pigments of Penicilliuin islariclicuiri begins after the exhaustion of the nitrogen source (Gatenbeck and Sjoland, 1964). Synthesis of ergot alkaloids by Claviceps purpuren and of candicidin by S. griseus begins after all the phosphate has been utilized pining and Taber, 1979; Martin, 1978). A limited supply of oxygen necessary for oxidative metabolism may also induce antibiotic formation (Seddon and Fynn, 1973; Flickinger and Perlman, 1979). Medium that causes slow growth or depletion of an essential nutrient from a rich medium that supports rapid cell proliferation also cause disruption of integrated cellular regulatory mechanisms. This disruption is then followed by relaxation of the growth-associated overriding mechanism that suppresses secondary metabolism. Borrow et al. (1961)have tried to define the factors that initiate secondary metabolism in shake cultures of Gibberella fujikuroi. They have designed media that allow gibberellin production to commence upon exhaustion of any one of several ingredients. They found that gibberellin production occurs during a certain phase of growth of G.fujikuroi, and they defined the following four growth phases (Borrow et n l . , 1964a,b). 1. Balanced phase. This is the period of rapid growth and nutrient uptake that begins at the time of inoculation and ends at the exhaustion of the first

GENETICS AND BIOCHEMISTRY OF SECONDARY METABOLISM

57

nutrient. Cell multiplication and most mycelial mass increase occurs until nitrogen, carbon, phosphorus, magnesium, or some other nutrient has been exhausted from the medium. 2. Storage phase. Cell proliferation stops at the beginning of this phase. However, unutilized residual carbon source is converted into mycelial fats and carbohydrates, resulting in increased mycelial dry weight. Other nutrients are consumed and the production of gibberellins begins. 3. Maintenance phase. Constant and maximum mycelial dry weight characterize this phase. Carbon uptake and gibberellin production continue. 4 . Terminal phase. Carbon source has been utilized and mycelium depleted of internal fat and stored carbohydrate. Reserve materials break down, resulting in protein turnover and mycelial lysis. Taber and Vining (1963) have shown that accumulation of ergot alkaloids

by Claviceps purpurea occurs during a prolonged transition phase that is characterized by the accumulation of nitrogenous but not carbyhydrate storage material. This phase falls between the balanced and storage phase of Borrow et al. (1964b), where magnesium and phosphorous were limiting nutrients. Mycelial proliferation continues, but cellular composition changes from that of the balanced growth phase. During growth in some media that do not support alkaloid production, cells pass from balanced into storage phase and store carbohydrates, polyols, and lipids (Spalla et al., 1978; Udvardy, 1980). Vining and Taber (1979) postulated that during logarithmic growth, an unknown growth-linked override mechanism represses the synthesis of the enzymes responsible for the formation of ergot alkaloid by Claviceps purpurea. Ergoline is formed from tryptophan, mevalonic acid, and a methyl group derived from methionine (Maier et al., 1980). Tryptophan plays both a regulatory and a precursor role in this biosynthesis (Otsuka et al., 1980). The former, but not the latter, role can be mimicked by thiotryptophan, which induces dimethylallyltryptophan synthetase, the first pathway-specific enzyme of ergoline biosynthesis. During rapid growth, tryptophan normally accumulates in the mycelium, but the induction of enzymes by accumulated amino acid does not occur until phosphate depletion disrupts growth, thus culminating in ergoline alkaloid production. At this time, some growth-linked override regulatory mechanism ensures that the transcription of genes involved in ergoline biosynthesis is no longer prevented. A high growth rate may alter the amount of some regulatory molecules (e.g., a highly phosphorylated nucleotide) that participates in transcription. Transcription of genes involved in alkaloid biosynthesis may not begin even in the presence of inducing levels of tryptophan, which has probably removed the repressor. It is possible that when exhaustion of nutrients (such as phosphate) reduces the growth rate and adjusts the intracel-

58

VEDPAL SINCH MALIK

lular concentration of a regulatory molecule, the overriding regulatory mechanism, which is growth rate-linked and represses induction of ergoline biosynthesis, is lifted, and genes specific for ergoline synthesis are expressed. An alteration of RNA polymerase may also be one of the pleiotropic controls that govern the expression of genes specific for secondary metabolism. The case of a single secondary metabolite would not be a sound base for attempts at making a model of regulation. Accumulating experience with some of the better-studied systems, such as chloramphenicol, ergot alkaloid, f3-lactarn antibiotics, and actinomycin D biosynthesis, have to be accommodated. There is heterogeneity in these systems. What appears to be the same secondary metabolism of Bu’Lock (1961) may turn out to have different genetic and biochemical bases in different classes of compounds. A thorough examination of the control of many other secondary metabolites may exhibit the same kind of heterogeneity.

B. MULTIVALENTINDUCTION BY PRECURSORS Besides the diversity of their chemical structure and biological activity, secondary metabolites are formed from intermediates or end products of primary metabolic processes. Therefore, their biosynthetic origin could be a basis for examining their regulation. When the grow-linked override mechanism is lifted, the synthetases of secondary metabolism can be produced. However, levels of intracellular substrates for the synthetases determine their activity and rate of secondary metabolite formation. For example, 6-methylsalicylic acid was present in the growing cells of a patulin producer, but 6-methylsalicylic acid was not formed until growth ceased (Bullock et al., 1975). This inactivity of synthetases could be attributable to a limiting supply of substrates and cofactors required for the reaction. A schematic outline of the relationship between primary and secondary metabolism is depicted in Fig. 2. It can be seen that the branch-point intermediates (acetyl CoA, shikimate, malonate, mevalonate, a-aminoadipate) or end products (cysteine, valine, tryptophan, pentose) are usually starting precursors of secondary metabolism (Drew and Demain, 1977). For example, chorismate is a precursor not only of aromatic amino acids and the vitamins but also of chloramphenicol and pyocyanin (Malik, 1979b). Malonyl-coenzyme A is a branch-point intermediate between fatty acids and griseofulvin, tetracycline, patulin, and cycloheximide. Mevalonate is an intermediate that can be channeled either to sterols or to the gibberellins, helvolic acid, fusidic acid, p-carotenes, xanthophylls, terpenes, and ergot alkaloids. a-Aminoadipate is the precursor of lysine, penicillins, and cephalosporins. Acetolactate can also be a precursor of primary and secon-

GENETICS AND BIOCHEMISTRY OF SECONDARY METABOLISM

Neomycin Gentamycin Kanamycin Tobramycin Ribostamycin Paromomycin Streptomycin Spectinomycin

DNA, RNA

*

Hexose phosphate

-t

Pentose phosphate

-

Tetrose phosphate

Shikimate

Perimycin

Pyocyanin Tetracycline

Serine c -Phophoglycerate

Penicillin

Cephamycin

I I

I

rcysteine

Phosphoenolpyruvate

I

f

Valine c -Pyruvate

/

I

59

Novobiocin Anthramycin Sibiromycin Lincomycin Rubradirin

4 \f Proprate

Rifamycin Streptovaricin

Psilocybin Lysergic acid Ergoline

Pyrrolnitrin lndolmycin

Cephalosporin

Phenylalanine

1

7

Gliotoxin Maldnate (Malonyl.CoA)

1

Polyketides

4

'

wKetdutarate

1

Showdomycin Oxazinomycin Formycin Pyrazofurin

J

I \

ltaconate

lsoprenoids Ergosterol Cholesterol Gibberellin

Oosperin Orsellinic acid 6-Methylsalicylate Patulin Griseofulvin Aflatoxin Erythromycin Tetracycline Rifamycin Streptovaricin Streptolydigin Cycloheximide

C.c.r,,,rl I.-."._

Carot enoid Trisporic acids

FIG.2. Relationships between primary and secondary metabolism.

dary metabolites. Many secondary metabolites are produced by branched pathways: one branch going to the secondary metabolite, the other branch going to a primary metabolite such as an amino acid. Intermediates of branch pathways could be toxic to the organism if they accumulated in high concentrations intracellularly. Elaborate regulatory mechanisms have evolved that prevent such branch pathways from hnctioning when the end product of the branch has increased up to a certain level. When several metabolites are derived from branches of a common pathway, mutation in one of the branches often leads to the overproduction of the end product of an interconnecting branch. Regulatory mechanisms that control the synthesis of these key branchpoint intermediates are important for increasing the production of secondary metabolites. Common regulatory mechanisms probably control steps early in the branching sequence from primary metabolism. Under growth conditions leading to the accumulation of certain key branch-point intermediates

60

VEDPAL SINGH MALIK

above normal levels (when enzymes of primary metabolism have been completely saturated), the accumulated substrate induces secondary enzyme systems only if the overall override mechanism that is associated with growth has been lifted. Various degrees of nutritional limitation coincide with the cessation of growth and relaxation of the override mechanism. Simultaneously, primary metabolites and intermediates accumulate because many pathways utilizing them slow down and their synthesis is only poorly controlled by either feedback inhibition or repression. In addition, ATP could accumulate because it is not needed for the energy-requiring growth processes. Glucose catabolites, such as acetyl-CoA, accumulate and are not h r t h e r utilized for primary metabolism. ATP inhibits citrate synthase and other enzymes that metabolize the products of glucose metabolism. Under these circumstances, the growth-associated override mechanism is lifted and acetyl-CoA is channeled toward fatty acid or polyketide synthesis by inducing subsidiary pathways. 1. Acetate Polyinalonate Pathway

In 1893, Collie suggested “the manner in which the group -CH2-CW (keten) can be made to yield, by means of simplest reactions, a very large number of interesting compounds; the chief point of interest being that these coinpounds belong to groups largely represented in plants.” Even though Collie’s ideas with respect to the synthesis of terpenes, fatty acids, and aromatic compounds were correct, his work was ignored for more than two decades. In 1919, Raistrick and Clark wrote, “So long ago as 1893 and, more recently, in 1907, Collie, who proposed the term polyketides for the series of compounds containing -CH2COgroups, pointed out the importance for biological chemistry of this class of compounds. These observations do not seem to us to have received from the biochemists the consideration that they deserve.” In 1947, Lipmann discovered coenzyme A, and in 1951, Lynen identified the active form of acetate as acetyl coenzyme A. By this time, availability of radiotracer has helped the biochemists to establish the role of acetate in the biosynthesis of steroids, cholesterol, fatty acids, and other reactions of primary metabolism. Based on the known importance of acetate in the biosynthesis of fats and terpenoids, Birch and Donovan (1953) examined the oxygenation pattern of many natural aromatic products. They found that the position of the oxygen atom indicated that the aromatics were assembled by cyclization of a polyketoinethylene acid, CH,(COCH,),COOH; polyketomethylene acid could be formed by head-to-tail condensation of acetate units. In 1955, Birch et al. provided the first experimental proof of the polyacetate hypothesis. Radioactive 6-methylsalicylic acid was prepared by feeding CH,COOH to cultures of Penicillium griseofulvuni. The labeling

GENETICS AND BIOCHEMISTRY OF SECONDARY METABOLISM

61

pattern of isolated 6-methylsalicylic acid as determined by chemical degradation supported the polyacetate hypothesis of polyketide synthesis. Polyketides are all derived from a P-polyketide chain. The polyketide chain consists of acetate or propionate units. It is now known that in fungi the polyacetate pathway is, in fact, an acetate polymalonate pathway. AcetylCoA in the starter position is condensed with malonyl-CoA, accompanied by loss of CO,. Malonate is derived by carboxylation of acetate. Polyketides in which methylmalonyl (propionate) units are the starter units and are extensively reduced [as in macrolides (erythromycin)] are characteristic of Streptumyces. In higher plants, aromatic amino acids serve as starter units of flavanoids and other polyketides. Flavanoids are synthesized in plants by condensation of one acetate-malonate-derived monobenzenoid nucleus with one shikimic acid-derived monobenzenoid nucleus. Polyketide antibiotics can be divided into two groups: (1) Antibiotics in which the carbon skeleton is derived from acetate, propionate, and butyrate (e.g., nanaomycins); (2) Antibiotics in which sugars and amino acids contribute to the carbon skeleton (e.g., spiramycin, tylosin). Polyketides and the common fatty acids have a common biogenic constitution because both are chains of acetate-derived subunits. Because of the participation of special carrier proteins, intermediates and end products are not in equilibrium with other cell components. For instance, acetoacetylCoA that is involved in fatty acid synthesis does not equilibrate with acetoacetyl-CoA that is intermediate in orsellinic acid synthesis because both are bound to their respective synthetases and are not free in cytoplasm. However, in streptomycetes antibiotics, the primer propionyl-CoA condenses with methylmalonyl-CoA. As a result, 3-carbon units are added, resulting in methyl groups attached to the growing polyketide chain. Aglycone (erythronolide) of erythromycin is synthesized by this route, which involves condensation of propionate and methylmalonic acids. Malonyl-CoA serves as a primer for tetracycline biogenesis and supplies the C-2 carboxyl group. The remaining carbon atoms of the ring may also come from either the polyketide route or chorismate. The C-6 methyl group is introduced prior to ring closure and is donated by methionine. The nitrogen atom at C-4 arises via transamination. The C-6 hydroxyl and C-5 hydroxyl are derived from molecular oxygen. Malonyl-CoA for the biogenesis of chlortetracycline in S. aureofaciens may not be derived from acetate but from oxaloacetate (Hostalek, 1978). Acetyl-CoA carboxylase is not present in high-chlortetracycline-producing strains during antibiotic production. The low activity of acetyl-CoA carboxylase in S. aureujkciens during chlortetracycline production is correlated with low activity of pyruvate kinase and pyruvate dehydrogenase complex. This indicates that the conversion of phosphoenolpyruvate to acetyl-CoA is

62

VEDPAL SINGH MALIK

suppressed when chlortetracycline is being produced. Phosphoenolpyruvate carboxylase is very active, yielding oxaloacetate, which is further channeled toward malonyl-CoA synthesis, a precursor required for chlortetracycline biogenesis. A wide variety of both aromatic and nonaromatic molecules are produced by various microbes via acetate-malonate condensation. Different enzyme complexes exist to form different oligoketide-derived products with different affinities for the acetyl and malonyl moieties. Relative lack of NADPH during certain phases of growth might favor the formation of oligoketides (orsellinic acid, 6-methylsalicylic acid, alterionol) at the expense of fatty acids, which require substantial reducing power in the form of NADPH for their synthesis. In the polyketide route, the keto groups generated during condensation of malonate and starter molecule are not reduced as in fatty acid biosynthesis. Thus, the concentration of reducing cofactor (NADPH) could be regulating diversion of acetate to fatty acid or polyketide. Gatenbeck and Hermodsson (1965)examined the possibility of NADPH as a regulatory factor involved in determining the fate of acetyl and malonyl donors in cell-free extracts of Alternuria tenuis. In the present of NADPH, utilization of acetyl-CoA was large for lipid synthesis relative to alternariol formation. Enzymatic reactions involved in the genesis of secondary metabolites compete for the same coenzymes and ions that catalyze basal metabolic reactions. Subtraction of these compounds from the common pool d e c t s other processes in which these coenzymes and ions take part. For instance, synthesis of rifamycins is accompanied by a distinct shift in the oxidizedl reduced nicotinamide adenine dinucleotide (NADINADH) and pyruvate/ lactate ratios toward the oxidized forms. This suggests a requirement for hydrogen equivalents in rifamycin synthesis and indicates an effect on the oxidation processes in N . meditmunei (Ruczaj et al., 1972).The occurrence of rifamycins in forms showing different degrees of oxidation of the molecule suggests that rifamycin may play the role of an oxidoreduction agent in the metabolism of the microorganism that produces this antibiotic. Hostakek et al. (1969) have called attention to the deficiency of acetate units in the synthesis of oligoketide antibiotics. These investigators have shown that intense energy production is unfavorable for the synthesis of tetracyclines by S. aureofuciens. High activity of the enzymes of the citric acid cycle oxidize acetate and diminish the pool of acetate, a building block of oligoketide antibiotic tetracycline. Roszkowski (1972) correlated carboxylation of acetate and propionate with polyene biosynthesis. In a high-producing strain of Streptomyces noursei var. polijungini, the total pool of acyl-CoA was fivefold greater than in a

GENETICS AND BIOCHEMISTRY OF SECONDARY METABOLISM

63

low producer, when cells were grown on carbohydrates or lipids. Mutants that produce increased amounts of polifungin have twice the activities of acetyl-CoA and propionyl-Cod carboxylases as compared to the parent. Carboxylase activity toward both acyl-CoA derivatives and polyene production also correlates in low-producing strains. Rafalski and Raczynska (1972, 1973) also observed that synthesis of the nystatin-type antifungal antibiotic polifungin and of lipids in S. noursei var. polijungini is correlated with increased activity of acetyl-CoA carboxylase and propionyl-CoA carboxylase. These enzymes produce malonate and methylmalonate, which are units for elongation of oligoketide. Mutants blocked in the fatty acid synthesis pathway leading from malonyl-CoA should further increase the flow of malonyl-CoA to oligoketides, increasing their yields and altering cell membrane permeability properties simultaneously (Malik, 1979a). Catabolism of exogenous fatty acids via P-oxidation increases the cellular pool of acetyl-CoA. Fatty acid esters also reduce further oxidation of acetylCoA by inhibiting citrate synthetase. A high supply of carbohydrates also leads to elevated levels of acetyl-CoA, which is channeled toward formation of polyketides. Glucose inhibits fatty acid catabolism by catabolite repression. Both tylosin synthesis and oleic acid oxidation were inhibited simultaneously by glucose, but enzymes involved in the synthesis of mycarose, the sugar moiety of tylosin, were not affected. Cerulenin is an inhibitor of the condensing enzyme that condenses acetyl-ACP and malonyl-ACP to forin P-ketoacyl-ACP, a precursor of fatty acids. The initial enzyme involved in the biosynthesis of polyketides is similar to the condensing enzyme of fatty acid biosynthesis as biosynthesis of polyketides is also inhibited by cerulenin. Mutant strains that are resistant to cerulenin may overproduce polyketides. As a matter of fact, daunorubicin, an anthracycline antitumor antibiotic, is overproduced by a ceruleninresistant streptomycete mutant (McGuire et al., 1980).

2 . Shikimate and Chorismate Although the acetate polymalonate pathway is prominent in eukaryotes, the shikimic acid pathway is generally used for synthesis of secondary metabolites in prokaryotes. Pyocyanin, candicidin, corynecins, chloramphenicol, and aromatic amino acids are synthesized by the shikimic acid pathway. In 1954, Tatum and Gross noted that a mutant of Neurospora that was blocked in the conversion of deh ydroshikimate to shikimate accumulated protocatechuate. Accumulation of dehydroshikimate induced a secondary pathway channeling into the formation of protocatechuate. Another example of a fungal secondary metabolite derived from shikimate is the

64

VEDPAL SINGH MALIK

terphenylyuinone volucrisporin. Chandra et al. (1966) showed that ni-hyclroxyphenylpyruvateis the intermediate between shikimate and volucrisporin. Ansamycins are characterized by an aliphatic, oligoketide-type bridge, which connects two different positions to an aromatic moiety (e.g., streptovaricins, rihamycins). The 7-carbon amino unit of the naphthoquinone part of rifamycins is derived from a shikimic acid-like precursor. Mutants of AT. mediterruiiei, one blocked in the transketolase and another in shikimate kinase, accumulate o-ribulose and shikimate, respectively, in amounts equivalent to the rifamycin B produced by the parent strain. This suggests that primary metabolites (like ribulose) are channeled in equimolar amounts to synthesis of secondary metabolites. By manipulating regulation of primary pathways, one could, therefore, increase yields of antibiotic and other fermentation products. Synthesis of chloramphenicol by S . venezuelue is subject to control by integrative regulatory mechanisms at several levels. End-product inhibition modulates the amount of chorismic acid entering into the chloramphenicol branch of the aromatic pathway. The first enzyme (3-deoxy-Darabinoheptulosonate phosphate synthetase) is neither repressed nor feedback-inhibited by the intermediates and end products of the pathway. Synthesis of none of the other enzymes involved in aromatic amino acid synthesis is repressed by these amino acids or the intermediates of the pathway. L-Phenylalanine and L-tryptophan inhibit prephenate dehydratase and anthranilate synthase activities, respectively. Thus, induction of chloramphenicol synthesis could occur when the pool of chorismate at the branch point is diverted from the multiple branch pathways leading to the various aromatic metabolites. This type of regulatory pattern is called “multivalent induction” because the end products of multiple branch pathways control the level of chorismate, the intermediate at the inducing branch point (Malik, 1980a). Numerous chloramphenicol analogs that could provide tools for genetic and biochemical analysis of the system have been synthesized. The mechanism of chromosome transfer in S . venezuelae has been revealed, and circular genetic maps have been constructed (Francis et a l . , 1975). This organism also harbors a plasmid that can be used as a vector for transporting recombinant DNA molecules (Malik and Reusser, 1979). Transducing phages for this streptomycete have also been reported. Chloramphenicol is produced in well-defined medium, and the total RNA isolated from cells that are engaged in chloramphenicol synthesis can be used as probe to select genes that are expressed during chloramphenicol production. Addition of aromatic amino acids stimulates chloramphenicol synthesis, but the amount of chloramphenicol produced is also limited by the sensitiv-

GENETICS AND BIOCHEMISTRY OF SECONDARY METABOLISM

65

ity of the producing organism to the produced antibiotic. Therefore, resistance of the organism to the antibiotic should first be increased before other approaches to yield improvement are initiated. Chloramphenicol is commercially produced by chemical synthesis. The cost of chloramphenicol may be decreased if a successful fermentation for its production could be developed. The fact that production of chloramphenicol parallels growth of S . venezuelae may be exploited to develop a continuous culture commercial fermentation for chloramphenicol. Tryptophan is an essential amino acid with a 50-million-dollar market. Yield of tryptophan by microbial fermentation has been poor. However, many microbes that produce chorismate-derived metabolites may be manipulated to excrete tryptophan. Such microbes have evolved with mechanisms to accumulate intracellular pools of chorismate, which could be diverted toward the synthesis of tryptophan, eliminating secondary metabolite production. Several chorismate-derived metabolites (e.g., candicidin, rifamycin, and ergot alkaloids) are being produced in quantities of up to 10 gmfliter in commercial fermentations. Organisms that excrete ergot alkaloids could be manipulated to develop a commercial tryptophan fermentation.

3. Mevalonate and Isoprene Unit The number of secondary metabolites derived from mevalonate is rapidly increasing. In addition to its role as a precursor of various secondary metabolites, mevalonate is often a precursor of side chains of one or more isoprene residues. Two commercially important mevalonate-derived fungal products are the gibberellins and sterols (Rose, 1980).

4 . Amino Acids as Precursors Amino acids that are derived from the tricarboxylic acid cycle intermediates form the starting points for a number of alkaloids, peptides, and p-lactam antibiotics.

a. Peptides. Peptide antibiotics (gramicidin, tyrocidine, valinomycin, bacitracin) are synthesized by multifunctional enzymes utilizing the protein thiotemplate mechanism. Such multienzyme complexes have high reaction efficiencies as a result of cellular compartmentation of intermediates. A growing peptide chain is. synthesized by a phosphopantotheine carrier in a series of transpeptidation and transthiolation steps (H. Kleinkauf, Technische Universitat, Berlin). Peptide bond formation occurs without tRNA, mRNA, and ribosomes. The amino acid sequence in the peptide antibiotic is dictated by the specificity inherent in multienzyme complexes. The longest peptide synthesized by such a mechanism in vitro is alamethicin, which consists of 20 amino acid residues. However, the biosynthesis of

66

VEDPAL SINGH MALIK

cyclic decapeptide antibiotics, gramicidin S and the tyrocidines, has been well worked on. Two multifunctional polypeptide chains known as the heavy and the light enzyme are involved in the biosynthesis of gramicidin S: cyclo(D-Phe-Pro-Val-Orn-Leu).Neither of these two gramicidin S synthetases can be dissociated into subunits by protein-denaturing agents, which suggests that several functional domains on a polypeptide chain are covalently bound. Gramicidin S synthetase probably carries 24 different catalytic functions. The activation center for proline and the transfer region for phenylalanine may have regulatory functions affecting the biosynthesis of gramicidin S. Binding of proline stimulates the other reaction centers of the multienzyme, thus regulating the efficiency of the biosynthesis. This effect is strongly increased if phenylalanine is transferred to the initiation site of the synthetase by the light enzyme. Studies on the activation of amino acids show that binding of substrates is random in formation of the aminoacyl adenylates, except in the case of ornithine, where evidence for an ordered mechanism has been obtained. Rate of formation and stability of enzyme-bound adenylates is lower than in corresponding aminoacyl-tRNA ligases.

b. P-Lactarn Antibiotics. All P-lactams have common amino acid precursors (Fig. 3). Both penicillin and cephalosporin ring systems are synthesized from L-cysteine and L-valine. Penicillin N is produced in all cephalosporin C fermentations. However, the reverse is not true. Relative yields of penicillin N and cephalosporin C are changed inversely by varying aeration of C. acremonium or by addition of methionine or carboxymethyl-L-cysteine to washed mycelial suspensions. Hydrophobic benzylpenicillin needs La-aminoadipic acid (L-MA) as an obligatory “precursor,” which is finally exchanged with the coenzyme A ester of the side chain acid. 6-Aminopenicillanic acid (6-APA), the penicillin nucleus, is not an intermediate but is formed as a shunt product when no side-chain precursor is present. Isopenicillin N is an intracellular hydrophilic intermediate. The pathway is as follows: L-AAA

+ L-CYS+ L.-AAA-L-CYS

LLD-trlpCptidC’

L-Val

i 1 1 isopiwicillin N

ptwicillin G

+

ph2nylact:tyl

L-AAA

COA

In Cephalospurium, the cyclization of LLD-tripeptide to isopenicillin N is stimulated by Fez+.Isopenicillin N is converted to penicillin N by inversion of its L-AAA side chain to D-AAA. Penicillin N is then subjected to ring

GENETICS AND BIOCHEMISTRY OF SECONDARY METABOLISM 0-KETOGLUTARATE

1

1

1

7

EXTRACELLULAR METHIONINE

I

HOMOCITRATE SY NTHETASE

f

HOMOCITRATE

1

cis-HOMOACONITATE

1 1

HOMOISOCITRATE

U-KETOADIPATE

aF d

72

I

ACID SYNTHETASE

U-~-OIHYOROXYISOVALERATE

1

S-ADENOSYL METHIONINE

S-ADENOSYL HOMOCYSTEINE

1

I-

%

PYRUVATE

METHIONINE

-1

67

KETOISOVALERATE

HOMOCYSTEINE

W 0

L.o. AMINOADIPATE

6 ADENYL-0-AMINO AOIPATE

J

\ I

~-IL-u-AMINOADIPYL)-CYSTEINE

n-AMINO-8-ADIPY L-SEMIALOEHYOE

t

l-

[L-VPILII~T I

AMINOADIPY LJ-L-~YSTEINYL-&VA~INE

s-~L-

SACCHAROPINE ISOPENlClLLlN N

PENICILLIN G

DEACETOXYCEPHALOSPORIN C

DEACETYLCEPHALOSPORIN C

1 i

CEPHALOSPORIN C

7-METHOXYCEPHALOSPORI"

FIG. 3. The biosynthetic pathway to p-Iactam antibiotics.

expansion to deacetoxycephalosporin C . Early-blocked Cephalospwium mutants, which do not produce penicillin N or cephalosporin C in fermentation, catalyze cell-free ring expansion. Late-blocked mutants produce only penicillin N and do not catalyze cell-free ring expansion. The ring expansion reaction is stimulated by Fez+,ascorbate, and ATP. The terminal steps of the biosynthetic pathway include the oxidation of deacetoxycephalosporinC by a dioxygenase to deacetylcephalosporin C. The dioxygenase is stimulated by Fez+,ascorbate, and a-ketoglutarate. Deacetylcephalosporin C is acetylated to cephalosporin C (Baldwin et al., 1980; Sawada et al., 1980a,b,c). Recently prokaryotic streptomycetes have been shown to produce not only

68

VEDPAL SINGH MALIK

hydrophilic penicillin N and 7-methoxycephalosporins but several new p-lactams. Total synthesis of 1-oxacephalothin and 1-carbacephalothin establish that sulfur can be replaced without loss of biological activity by oxygen and -CH2-, respectively. Naturally occurring compounds of this type with no sulfur-containing, hsed-ring system may well be produced by streptomycetes. Pathways for the biosynthesis of a-aminoadipic acid differ in fungi and streptomycetes. In streptomycetes, degradation of lysine provides a-aminoadipic acid (Stirling and Elson, 1979) whereas in fungi, a-aminoadipic acid is the precursor of lysine. Two other compounds with the penicillin ring system (8aminopenicillanic acid and isopenicillin N) are produced by P . chrysogenum. However, in addition to producing penicillin N, Cephalospurium species produce cephalosporin C. Cephalospurium species have never been found to produce either the nucleus of cephalosporin C, 7-aminocephalosporanic acid, or any other p-lactam antibiotics that have substituded D-a-aminoadipic acid. Stages in the biosynthesis of clavulanic acid by Streptomyces clavuligerus (Elson and Oliver, 1978) do not fit in the scheme proposed for the biosynthesis of p-lactam antibiotics in eukaryotic fungi (see Fig. 2). The penicillin and cephalosporin precursor, &(a-aminoadipy1)cysteine-valine is not involved in the biosynthesis of clavulanic acid, which lacks a 6-amino group and aminoadipyl side chain and possesses an oxazolidine rather than a thiazolidine or dihydrothiazine ring. Glycerol is incorporated intact into the three p-lactam carbons of clavulanic acid; the remaining five carbons being derived from a-ketoglutarate. These studies with the biosynthesis of clavulanic acid in S. clavuligerus assert that different pathways for biosynthesis of p-lactam ring do exist in procaryotic streptomycetes and eukaryotic hngi. c. Lysine Effect. Regulatory processes that control the supply of basic building blocks exert a direct influence on antibiotic formation. a-Aminoadipic acid is the branch point for lysine and benzylpenicillin biosynthesis. Lysine could inhibit its own synthesis from a-aminoadipic acid, causing a rise in the level of the latter in the cell and diverting it toward p-lactam synthesis. However, a different situation exists in penicillin-producing fungi. When lysine is added to the fermentation medium, homocitrate synthetase is inhibited and repressed. This depletes the intracellular level of a-aminoadipic acid and thereby decreased production of penicillin (Luengo et al., 1980). Because of impermeability of prototrophic Cephalosporium, antibiotic synthesis is neither inhibited by lysine nor promoted by a-aminoadipic acid. However, a lysine auxotroph blocked after a-aminoadipic acid grows well, but because of feedback inhibition of homocitrate synthase, forms no cephalosporin

GENETICS AND BIOCHEMISTRY OF SECONDARY METABOLISM

69

(Lemke and Nash, 1972). Addition of lysine to streptomycetes fermentations provides a-aminoadipate and thereby stimulates production of 7-methoxycephalosporins. In a cephamycin producer, S. clauuligerus, the activity of the aspartokinase is subjected to concerted feedback inhibition by lysine and threonine. Mutants resistant to lysine analog S -(2-aminoethyl)-~-cysteineproduced from 1.5 to 4 times as much antibiotic as the analog-sensitive parent. The aspartokinase activity of the enzyme obtained from analog-resistant mutants was not inhibited by the concerted effect of lysine and threonine.

d. Valine. The configuration of the valine moiety in p-lactams is D; and valine provides the carbon skeleton of the penicillamine residue of the p-lactam antibiotics. Addition of valine to complex fermentation media does not affect penicillin yield. However, ~-valineincreases the rate of penicillin formation by washed mycelium. The conversion from pyruvate to acetolactate is the initial step in valine formation and is catalyzed by acetohydroxy acid synthetase. This enzyme is subject to feedback inhibition by ~ - v a l i n ein wild-type strains. Acetohydroxy acid synthetase from a high-penicillin-yielding mutant was much less sensitive to feedback inhibition by L-valine. Furthermore, the mutant enzyme had only one binding site for valine, compared to the two binding sites for the ancestral enzyme. Enzyme content in the superior strain was also twice that in the parent (Goulden and Chattaway, 1968, 1969). The specific activity of glutamate dehydrogenase was derepressed in a high-yielding mutant, whereas low-yielding mutants were repressed for the synthesis of this enzyme (Queener et al., 1978). The altered regulation pattern for glutamate dehydrogenase may enhance nitrogen assimilation for cephalosporin C synthesis. An inverse relationship between vegetative mass and cephalosporin C was observed by Queener et al., suggesting that conditions that are best for vegetative development are usually worst for antibiotic production. Superior antibiotic producers of Streptomyces lipmunii lack control in the Ile-Leu region, suggesting that valine synthesis can be a rate-limiting step in antibiotic production (Godfrey, 1973). e. Methionine and Sulfur Metabolism. The effect of the metabolism of sulfur-containing compounds on p-lactam antibiotics has been extensively investigated (Drew and Demain, 1977). Methionine can be used as the sole source of nitrogen or sulfur by P-lactam-producing organisms. Furthermore, methionine exerts a stimulatory effect on the yield of cephalosporin if added during vegetative growth of C. acremonium. Because other sulfurcontaining compounds (such as cysteine), which are efkient precursors of

70

VEDPAL SINGH MALIK

the sulfur atom of cephalosporin, are without stimulatory effect on the antibiotic yield, methionine must also have a regulatory role. Several groups (Drew and Demain, 1977) have used mutants blocked in sulfur metabolism to elucidate the role of methionine in cephalosporin C formation. In an early-blocked mutant that grows on cysteine, methionine, cystathionine, and homocysteine, but not on sulfate, the magnitude of cephalosporin production stimulation was methionine > cystathionine > cysteine. If methionine was stimulating cephalosporin production only as a result of being the precursor of sulfur for the antibiotic, the sulfur amino acids should stimulate cephalosporin C production in the reverse order of that observed by Drew and Deinain (1975c, 1977). This opposite order of stimulation experimentally obtained suggests that cysteine and cystathionine are converted via transsulfuration to methionine, which then exerts the regulatory effect and stimulates cephalosporin C synthesis. To support this point, Drew and Demain (1975~) blocked transsulfuration from cysteine to methionine by mutating an early-blocked mutant in sulfur utilization. This double mutant did grow on methionine but not on cysteine or sulfate and produced little antibiotic in the presence of excess cysteine and low enough levels of methionine just to allow normal growth. This double mutant produced cephalosporin in the presence of excess methionine. Furthermore, a nonsulfur-containing analog, norleucine, could replace excess methionine, supporting the regulatory role of methionine. Methionine may also be the inducer of cephalosporin formation in C. acremonium, but the exact nature of the molecular mechanism involved here is hard to visualize. A methionine auxotroph of C. acreinonium produced cephalosporin yields greater than its parent when supplemented with methionine. A CIBA mutant blocked in the sulfate reduction pathway prior to sulfide formation assimilated more exogenous methionine and synthesized four times more cephalosporin than its parent. High levels of cephalosporin C production with a non-sulfate-utilizing mutant could be a result of its inability to synthesize cysteine, the repressor of methionine permease. Mutants of C. acreinonium that efficiently synthesize cephalosporin C from sulfate have been isolated. One mutant is similar to the cys-3-mutant of Neuraspura crussa in which synthesis of sulfate permease as well as aryl sulfatase is coordinately controlled. This mutant utilized sulfate as effectively as methionine for cephalosporin C synthesis. A natural isolate of C. acremoniuin was derepressed for aryl sulfatase and synthesized cephalosporin preferentially from methionine, deriving sulfur via transsulfuration. Aryl sulfatase repression in the mutant may be attributable to accumulation of sulfide, a corepressor of sulfatase in fungi. Another mutant efficiently utilized sulfate and produced double the amount of cephalosporin produced

GENETICS AND BIOCHEMISTRY OF SECONDARY METABOLISM

71

by its parent. Cephalosporin production by this mutant was sensitive to methionine. A methionine synthase is only operative in sulfate-mediated cystathionine formation that is repressed by high concentrations of exogeneous methionine but stimulated by an excess of sulfate. This is why mutants with an operative alternative route produce increasing cephalosporin C from sulfate and have limited tolerance for methionine. The increase in cephalosporin C production from sulfate and methionine sensitivity may both be the regulatory consequences of a single mutational event. A mutant lacking cystathionine y-lyase did not grow on methionine, homocysteine, and cystathionine, but did grow well on cysteine and inorganic sulfur. Methionine did not stimulate cephalosporin production in synthetic medium but decreased the yield to about 20%ofthe antibiotic production of the parent. Furthermore, in the sulfate-supplemented, methionine-free medium, the antibiotic potency of the mutant was lower than the parent. These results demonstrate the importance of cystathionine y-lyase for methionine-stimulated antibiotic synthesis and indicate that cystathionine may be a prerequisite for antibiotic synthesis. The cleavage of cystathionine may induce the transfer of a cysteine moiety into a peptide intermediate involved in p-lactam synthesis (Treichler, 1979). Cystathionine synthesis from inorganic sulfide occurs by two alternative routes. Both of these routes were blocked in a mutant defective in hoinoserine 0-acetyltransferase. This 0-acetylhomoserine auxotroph was resistant to methane selenol. Because of block in the synthesis of 0acetyl-L-homoserine (the common acceptor substrate for both metabolic routes leading to cystathionine), this mutant was unable to synthesize cystathionine from cysteine or inorganic sulfur. However, the mutant grew on methionine, homocysteine, and cystathionine, but not on cysteine or inorganic sulfur. In synthetic medium supplemented with low levels of rnethionine and excess cysteine or sulfate, this mutant did not produce increased cephalosporin. The parent or revertants of the mutant produced cephalosporin C in good yields on addition of excess cysteine or sulfate. This mutant required high levels of methionine to produce cephalosporin C. The parent, but not the mutant, produced considerable amounts of cephalosporin C in media supplemented with cysteine or sulfate. Blocking both routes to cystathionine virtually eliminated cephalosporin C production from cysteine or inorganic sulfur. This decreased antibiotic productivity suggests that cephalosporin is derived through cystathionine by means of sulfide fixation utilizing 0-acetylhomoserine sulfhydrylase (methionine synthase) and cystathionine p-synthase (Treichler, 1979). Simultaneous operation of both routes to cystathionine biosynthesis may have an additive effect on cephalosporin production. To understand this, the other route of anabolic cysteine synthesis was also blocked by mutagenesis of

72

VEDPAL S I N G H MALIK

a strain with impaired cystathionine y-lyase; the parental marker was removed by reversion. These double mutants grew on cysteine but not on inorganic sulfur, methionine, homocysteine, or cystathionine. All these mutants were methionine- and homocysteine-sensitive. This inhibition was only reversed by cysteine. In synthetic media, no difference in productivity was found between mutant and parent when methionine was used as the sulfiir source. The impairment of anabolic enzyme O-acetylserine sulfhydrylase had a positive effect on cephalosporin C production with excess sulfate in the medium. A block in the anabolic synthesis of cysteine must encourage conversion of sulfide to cystathionine by the alternate route. Addition of methionine to the fermentation medium has no effect on penicillin yield in P . chrysogenurn because in penicillin sulfate is reduced into cysteine via cysteine synthetase. However, in cephalosporin, cysteine is derived exclusively from methionine via reverse transsulfuration. Both O-acetylhomoserine sulfhydrylase and methionine synthase, but no O-acetylserine sulfnydrylase (cysteine synthase), were present in cell-free extracts of P . chrysogenurn during penicillin production in sulfate-containing synthetic and complex medium. This suggests that sulfide to cystathionine via hoinocysteine is the main route for optimal p-lactam synthesis in P . chrysogenum. Wild-type Penicillium strains use sulfate for antibiotic synthesis because they utilize this alternative route. Cephulosporiuni acremoniuin possesses potent O-acetylserine sulfhydrylase, the activity of which exerts an inhibitory effect on the operation of the alternate route. Therefore, mutants in this enzyme will utilize an inorganic sulfur source by an alternate pathway. The stimulation or inhibition of the alternate route could be attributable to many factors. A C. acrenzonium mutant with an enhanced potential to utilize sulfate for cephalosporin C production (Komatsu and Kodaira, 1977) exhibited elevated cystathionine P-synthase activity. O-acetylserine sulfhydrylase activities in the parent and the mutant were similar. Mutations enhancing levels of enzymes involved in the alternate route of cystathionine biosynthesis can increase p-lactam yield. The common nonprotein amino acids, such as sarcosine and ornithine, are frequent components of antibiotics. Synthesis of these amino acids follows known pathways. For instance, ornithine, a constituent of bacitracin, is formed during antibiotic production both from glutamate, as intermediate in the synthesis of arginine, and as the degradation product of arginine. Enzymes catalyzing anabolic as well as catabolic production of ornithine from arginine are induced during sporogenesis and synthesis of bacitracin in B. subtilis (Pass et a l . , 1974). The nonprotein amino acid constituents are also limiting in the synthesis of peptide antibiotics, a , y-Diaminobutyric acid and ornithine affect synthesis of colistin and bacitracin, respectively (It0 et a l . , 1970; Pass et al., 1974).

GENETICS AND BIOCHEMISTRY OF SECONDARY METABOLISM

73

Mutation that prevents ornithine degradation by ornithine 6-transaminase enhances antibiotic production and simultaneously decreases the glutamate and proline pools. Both of these amino acids are formed from ornithine (Pass et al., 1974).

5 . Sugar Derivatives These compounds originate from pentoses and hexoses, which during growth are incorporated into polysaccharides and nucleic acids. During trophophase of P. clirysogenum, glucose is metabolized primarily by the hexose monophosphate shunt, yielding large amounts of NADPH. However, in the idiophase, glycolysis is predominant. The carbon skeleton of glucose is incorporated either intact into aminoglycosides (e.g., streptomycin) (Kirby, 1980)or partially as a glycoside moiety attached to a carbon skeleton derived by another pathway (as in macrolides, e.g., erythromycin). Deregulation of the hexose monophosphate shunt generates from glucose excess intracellular ribose, which is the precursor for nucleosides and aminoglycoside antibiotics. Glucose provides acetate, propionate, and NADPH for the successive reduction steps of the highly oxidized polyketide chain. Glucose is also involved through the pentose phosphate cycle in the biosynthesis of the p-aminoacetophenone moiety of candicidin. In the chlortetracycline-producing strains ATP glucokinase attained peak activity after about 12 hr of incubation and then declined in parallel to the decrease in cellular ATP level. The activity of this enzyme was the lowest during the stationary phase of growth. Glucose-6-phosphate formation showed the presence of an alternate mechanisms of phosphorylation of hexoses that supply building blocks for antibiotic synthesis, i. e., polyphosphate glucohnase, which was present in the culture only during antibiotic production and after ATP glucokinase activity had diminished. Cellular polyphosphate was maximum after the drop in ATP level. This is connected with the shift from the adenylate phosphorylation mechanism to the polyphosphate system. Production culture possessed 10% adenylates of the polyphosphates. In S. coelicolm, uptake and metabolism of arabinose, glycerol, fructose, and galactose were repressed by glucose, cellobiose, mannose, and nonmetabolized 2-deoxyglucose (Hodgson, 1980). Mutants that can grow on glycerol, arabinose, etc., in the presence of 2-deoxyglucose have lost repression by glucose of many sugar-metabolizing pathways. Most mutants that are resistant to 2-deoxyglucose probably are missing glucose kinase, and the corresponding mutations map at two chromosomal locations. A small number of 2-deoxyglucose-resistantmutants utilize glucose and may be altered in a protein involved in carbon catabolite repression. None of the 2-deoxyglucose-resistant mutants overproduced actinorhodin or methyl-

74

VEDPAL SINGH MALIK

enomycin. However, they sporulate potently, suggesting that glucose represses sporulation. Romano and Margiotta (unpublished results) have used the nonmetabolizable analogs 2-deoxy-D-glucose (2-DOG) and 6-deoxy-~-glucose(6-DOG) to study the properties of the D-glucose transport system of Streptomyces griseus MA45. Glucose-grown vegetative cells accumulate both these analogs against a concentration gradient. The transport system has an apparent K , of 24 p M for 2-DOG and 27 p M for 6-DOG and is competitively inhibited by D-glucose with a K , of 6 p M . 6-DOG is accumulated exclusively as the free sugar. 2-DOG is transported as the free sugar but is accumulated intracellularly both as freesugar and as sugar phosphate, the latter as a result of hexokinase activity. Toluenized cells showed ATP-dependent phosphorylation of glucose and 2-DOG, but no phosphoenolpyruvate(PEP)-dependent phosphorylation of these sugars. Thus, these sugars are accumulated by an active transport system and not by a PEP:hexose phosphotransferase system. The transport system is inhibited by respiratory inhibitors such as NaCN and by protonconducting ionophores such as carbonyl cyanide-m-chlorophenyl hydrazone, which prevent both the establishment of a proton motive force and the synthesis of ATP. It is not inhibited by reagents that prevent ATP synthesis only, such as arsenate of N,N’-dicyclohexylcarbodiimide.These data indicate that the glucose transport system is energized by a proton motive force and not by ATP directly. The system is specifically inducible by glucose and shows high specificity in its activity. Thus, fructose- or galactosegrown cells do not transport 2-DOG or 6-DOG. Fructose or galactose do not show activity for the glucose transport system in glucose-grown cells, either as inhibitors of 2-DOG uptake, or as uptake substrates.

c. FEEDBACK INHIBITION AND ENDPRODUCT REPRESSION Secondary metabolites are often excreted into the environment and rarely accumulate intracellularly. In such situations, excessive extracellular product may not act directly by interacting with the allosteric branching enzymes or regulatory proteins (Vining, 1979). The biosynthetic intermediates or degradation products of the excreted metabolite could be the real regulators of these secondary pathways. Several secondary metabolites are reported to exert controlling effects on their own synthesis. 6-Methylsalicylic acid synthetase, a multienzyme complex, is responsible for the synthesis of 6-methylsalicylic acid (6-MSA) by Penicillium uriticae from acetyl-CoA, malonyl-CoA, and NADPH. Like yeast fatty acid synthetase, it is composed of four identical multifunctional

GENETICS AND BIOCHEMISTRY OF SECONDARY METABOLISM

75

polypeptides. Each subunit has a molecular weight of 790,000. Two types of thiol groups (cysteine and cysteamine) and two partial activities (palmityl transferase and dehydratase) are present. The immunological cross-reaction between both 6-MSA synthetase and fatty acid synthetase suggests functional similarity between these two enzymes. Despite the resemblances, they are different proteins coded b y separate genes. Patulin inhibits the total activity and condensation step catalyzed by 6-MSA synthetase. Because polyketide synthesis depends on the formation of malonyl-CoA from acetyl-CoA, it will be affected by the same regulations of the carboxylase reaction that control fatty acid synthesis, including allosteric activation by citrate and feedback control by long-chain fatty acids. Mechanisms of this kind may explain the adverse effects of lipid-antifoams on the yield of griseofulvin and other polyketides. In the chloramphenicol-producing cultures, arylamine synthetase, the branch-point enzyme, is not feedback inhibited but is repressed by increasing concentrations of chloramphenicol (Jones and Westlake, 1974; Malik, 1979b). Because chloramphenicol is excreted into the medium and never accumulates intracellularly, some intermediates of the biosynthetic pathway probably repress transcription of the arylamine synthetase gene. Nakano and co-workers (1974) have shown that synthesis of corynecins, which are structurally closely related to chloramphenicol, was repressed by the p-aminophenylpropanoid intermediate. Addition of tryptophan to the candicidin-producing cultures of S. griseus represses and feedback-inhibits p-aminobenzoic acid synthetase. This curtails the amount of cellular p-aminobenzoic acid, which is the aromatic moiety of the polyene, macrolide, antifungal antibiotic candicidin (Martin and Demain, 1978). Both p-aminobenzoic acid and tryptophan are derived &om chorismic acid. Anthranilate synthetase and p-aminobenzoic acid synthetase may share a protein subunit, and regulation of the synthesis of the common subunit could be affected by tryptophan. Production of aurodox in Streptomyces goldiniensis is controlled by feedback inhibition (Unowsky and Hoppe, 1978; Liu et al., 1979). High yields of aurodox were obtained by reversion of a nonproducer followed by selection of mutants resistant to aurodox. Reversion of nonproducer to aurodox production could have altered the first biosynthetic enzyme of the branch pathway, resulting in a protein desensitized to feedback inhibition. First enzymes of the branch pathways are known to be involved in regulation of metabolic routes (Crawford and Stauffer, 1980). Resistant strains of S . goldiniensis able to grow on 2 gm/liter of aurodox produced higher than 2.5 gmAiter of aurodox. These resistant strains were further improved for antibiotic production by mutagenesis. Strains so

76

VEDPAL SINGH MALIK

selected maintained aurodox resistance even when passed many times in the absence of antibiotics. In these mutant strains, resistance to aurodox could have become constitutive as compared to the inducible aurodox resistance of the parent culture, and constitutive aurodox iesistance may partially account for the increased antibiotic production. The speed with which aurodox inhibits its own synthesis suggests allosteric inhibition of a biosynthetic enzyme rather than repression of enzyme synthesis, the likely mechanism of aurodox regulation. Other reports of feedback inhibition and end product inhibition have appeared in the literature. Agroclavine and elymoclavine inhibit dimethylallyltryptophan synthetase. Anthranilate synthase is feedback-inhibited by elymoclavine (Floss et al., 1974). In protoplasts of Cluuiceps, elymoclavine (0.2 m M ) inhibits the incorporation of tryptophan into the elymoclavine. The end product (gramicidin S) adversely affects the activity and stability of the gramicidin S synthetases from Bacillus ln-evis. End product inhibition is very frequent, but end product destabilization has rarely been reported. Two serine racemases have been partially purified from the Dcycloserine-producer Streptomyces garyphalus. One of them is not inhibited by D-cycloserine. Cycloheximide inhibits its own synthesis when added to producing cultures of S. noursei (Spizek et al., 1965)or S . griseus (Kominek, 1975). Accumulation of penicillin by P. chrysogenuin is inhibited by supplements of penicillin to the growing culture (Gordee and Day, 1972) but the mechanism of inhibition by penicillin of its own biosynthesis is not known. Sulfur metabolism also influences the yields of p-lactam antibiotics. Sulfur for penicillin biosynthesis can be derived from sulfate via the sulfate reduction pathway and from methionine via reverse transsulfuration (Drew and Demain, 1977). Hihg-penicillin-yielding mutants like 4176 take up inore sulfur from the medium than the wild type (Tadrew and Johnson, 1958). Blocked mutants of C. acreinoniuin demonstrate the difference in sulfur metabolism between P. chrysogenum and C . ucremoniuin (Treichler, et al. 1979). Anabolic synthesis of cysteine occurs as a result of the reaction of O-acetylserine with sulfide in the presence of O-acetylserine sulfhydrylase. Cysteine is also synthesized by an alternative route of sulfide fixation in the presence of O-acetylhomoserine sulfhydrylase (methionine synthase). This yields homocysteine, which is transposed to cysteine by the catabolic transsulfuration enzymes, cystathionine P-synthase and cystathionine y-lyase. Both pathways influence antibiotic yield. A mutant of C. acwmonium was boosted in L-serine sulfhydrylase level and utilized an increasing amount of sulfate for cephalosporin biosynthesis. The mutant was sensitive to methionine because it maintained a large pool of cysteine (Komatsu and Kodaira, 1977).

GENETICS AND BIOCHEMISTRY OF SECONDARY METABOLISM

77

D. CATABOLITE REPRESSION The term “catabolite repression” has been used to explain effects of rapidly utilizable energy sources on the expression of dispensable catabolic pathways. The mechanism of catabolite repression has been exhaustively investigated with the lac operon of E. coli. Lac operon in E . coli is not derepressed by inducers such as lactose in the presence of readily utilizable sugars such as glucose. Besides control of the lac operon by repressor, catabolite repression exerts an additional overriding control. Exhaustion of glucose or rapidly metabolizable carbon source results in slow growth rate and increased intracellular level of cyclic 3’,5’-adenosine monophosphate. This nucleotide is an effector molecule, which together with a catabolite activator protein (CAP) controls the lac operon positively by binding to the operator and making it available for transcription, provided repressor is removed by inducer. What controls CAMP levels under a variety of growth rates is not known. Catabolite repression is a phenomenon where certain nonessential enzymes, which may be necessary only in a given environment at a particular time, are repressed by catabolic products of a readily utilizable carbon source. Whatever the nature of mechanisms, they are probably operative in delaying and reducing antibiotic production by a rapidly used carbon source such as glucose. Production of many antibiotics is inhibited when glucose is used as a carbon source. However, this does not show that the inhibitory effect of glucose on antibiotic synthesis is attributable to catabolic repression, as in the case inhibition ofp-galactosidase in E . coli (Magasanik et al., 1974). The negative effect of glucose has been observed on the yield of penicillin (Johnson, 1952), actinomycin (Gallo and Katz, 1972), streptomycin (Demain and Inamine, 1970), indolmycin (Hurley and Bialek, 1974), kanamycin (Satoh et al., 1976), and puromycin (Redshaw et al., 1979). 0-Demethyl puromycin 0-methyltransferase, which catalyzes the final step in puromycin biosynthesis, is repressed by glucose (Sankaran and Polgeli, 1975). However, the effect of glucose on antibiotic synthesis is not universal. Most aminoglycosides are derived from glucose and their synthesis is not repressed by glucose. Chloramphenicol synthesis is not repressed on glucose and depends on the nutrient combination of the growth medium. Neither glucose nor phosphate represses chloramphenicol production (Malik, 1972). Aharonowitz and Demain (1978) examined the cephalosporin production by S . clavuligwus. Growth on preferred carbon sources such as glycerol and maltose produced high biomass but the specific production of cephalosporins

78

VEDPAL SINGH MALIK

decreased as carbohydrate concentration was increased. Maximum cephalosporin yields were not obtained under conditions supporting highest biomass production. Poorer carbon sources such as starch, a-ketoglutarate, and succinate yielded more cephalosporin, and antibiotic production was closely associated with growth. The negative effect of glucose on bacitracin production was also first thought to be attributable to catabolite repression, but later proved to be attributable to acid production and lowered pH (Haavik, 1974). Coumermycin A can be produced in fermentation broths that comprise a wide variety of assimilable sources of carbon and nitrogen. Under certain conditions, glucose or ammonium salts do not suppress the biosynthesis of antibiotic. Improved yields result from the addition of phosphates and chlorides (Godfrey and Price, 1972). Phenoxazinone s ynthetase catalyzes the synthesis of actinocin, the chromophore of the antibiotic actinomycin. The de nouo synthesis of this enzyme occurs late in the growth cycle when glucose has been exhausted from the medium. In general, those carbon sources that support vigorous cellular growth are most effective in suppressing actinomycin synthesis. The repressive effect of glucose is insignificant once actinomycin synthesis has been well established. Several sugars, acetate, citrate, and pyruvate repress phenoxazinone synthetase formation. This transient inhibition of actinomycin synthesis once antibiotic production has begun may not only be attributable to catabolite repression as it operates in control of lac operon of E . coli but may also involve glutamine synthetase and nitrogen metabolism (Katz, 1967, 1968). Industrial microbiologists have solved the problem of catabolite repression of antibiotic production by medium manipulation. One way is to use two carbon sources in the fermentation. Low concentration of readily utilizable carbon source allows good growth of the organism. A second, nonrepressive, slowly utilizable carbon source is metabolized during antibiotic production. Another way is the continuous feeding of the repressive carbon source at such a slow rate that the inhibitory effect is not exerted. In fact, today most penicillin is no longer made with lactose (Salter0 and Johnson, 1953) but is made with continuous slow feeding of low levels of glucose (Demain, 1968). The p-lactam antibiotic produced in S. clauuligerus (Ahronowitz and Demain, 1978) and C . acremoniunz (Mehta et al., 1980) is limited by glycerol. The inhibitory efyect of glycerol may be attributable to altered membrane structure and permeability and not attributable to catabolite repression. The macrolide chroinophore of polyenes is synthesized from acetate and propionate via the polyketide pathway. The aminosugar moieties found in polyene macrolides are derived from glucose. Glucose is the preferred carbon source for the production of the polyene antibiotic candicidin (Martin

GENETICS AND BIOCHEMISTRY OF SECONDARY METABOLISM

79

and McDaniel, 1975). However, in pilot fermentations, high initial concentrations of glucose retarded growth and produced abnormal fermentation patterns. Slow feeding of glucose as in the penicillin fermentation (Pirt, 1976) increased the yield of the candicidin and candihexin antibiotics. Increase in yield was similar when glucose level was maintained at 5 or 15 gm/liter. Respiration rate was higher when glucose was slowly fed, with most polyene remaining attached to the producing cell. Maximal growth rates and final cell mass accumulations were lower, but glucose utilization rates were higher in fermentations where glucose feeding was slow. Tereshin (1976) increased the rate of nystatin production several fold by carbohydrate feeding of various ages of the producing organism. Tereshin (1976)found that glucose and mannose support identical growth and candicidin production in a synthetic medium. Galactose, fructose, arabinose, maltose, sucrose, and lactose decreased candicidin yield. Disaccharides were also poor carbon sources for growth and mycoheptin production by Streptoverticillium mycoheptinicum. Starch and glucose proved to be the best carbon sources for mycoheptin production. Specific antibiotic production was lower using starch as a single carbohydrate source, but biomass yield was higher with starch than with glucose. Lower alcohols (methanol, ethanol, propanol) stimulate polyene production. Intermittent addition of glucose to fermentation increased the yield of amphotericin B and heptaene. Isolation of constitutive mutants for secondary metabolite production is difficult because of the fact that precursors also have to accumulate above a certain level even if growth-linked repression is lifted. However, it should be possible to obtain mutants resistant to catabolite repression. As a matter of fact, the report by Light that a mutant of Penicillium patulum can be obtained that produces 6-methylsalicylic acid synthetase during growth rather than after growth is encouraging. Certain interconverting enzymes of antibiotic synthesis are repressed by glucose, such as mannosidostreptomycinase or mannosidase, which converts undesirable mannosidostreptomycin to streptomycin (Demain and Inamine,

1970). Ragan and Vining (1978) have tested the general hypothesis that overall control of secondary metabolism is mediated by catabolite repression. They measured CAMPlevels in a culture of S . griseus that produced streptomycin during stationary phase of growth (Cella and Vining, 1975). Concentrations of CAMP were highest during active growth when no streptomycin was being produced. The level of CAMP had declined 90% by 5 hr before streptomycin production was initiated. The low concentration of CAMP was found during streptomycin accumulation. These results of Ragan and Vining (1978) suggest that a catabolite repression-type mechanism correlated with an in-

80

VEDPAL SINGH MALIK

crease in the intracellular cyclic 3',5'-adenosine monophosphate concentration does not directly mediate onset of streptomycin biosynthesis in S. griseus. In E . coli, catabolite repression controls inducible catabolic pathways. Secondary metabolism is not a catabolic, but a biosynthetic process. Time required for catabolic enzyme production after peak CAMP levels are attained is about 3 min. However, streptomycin appears in the medium 1 hr after the increase in the specific activity of a biosynthetic transaminase (Horner, 1964). The delay in the appearance of streptomycin in the medium is probably attributable to the lack of accumulation of precursors from which streptomycin is produced. Endogenous accumulation of starting precursors and derepression (induction) of enzymes responsible for the synthesis of secondary metabolites may have to coincide for onset of secondary metabolism. When endogenous levels of starting precursors begin to decrease, synthetases involved in secondary metabolism may start to decay and the rate of accumulation of secondary metabolites in the medium starts to decrease. The experiments of Ragan and Vining (1978) show that streptomycin accumulation in the medium begins 5.5 hr after the peak intracellular CAMP concentration in S. griseus. These studies suggest that the CAMPlevels and streptomycin production are not directly linked but do not rule out an indirect cascade mechanism mediated by effectors. Involvement of CAMP in turimycin production by Streptomyces hygroscopicus has been suggested by Gersch et al. (1978).These authors recorded B decrease in the concentrations of both CAMP and cyclic 3',5'-guanosine monophosphate at the start of turimycin biosynthesis. The intracellular CAMP concentration was claimed to be directly correlated with the amount of growth and inversely correlated with turimycin production. However, intracellular CAMP and cGMP concentration increased when turimycin production was discontinued (Gersch, 1980). Guanosine 5'-diphosphate 3'-diphosphate (ppGpp) and guanosine 5'triphosphate 3'diphosphate (pppGpp) are important pleiotropic signal molecules in a control system that senses an amino acid deficiency and redirects various cellular activities in response (Lagosky and Chang, 1980). In E . coli, they are synthesized by the ATP:GTP pyrophosphate transferase; stringent factor protein (relA gene product) on ribosomes as a result of the binding of a codon specific for uncharged tRNA during amino acid starvation. In B . suhtilis and E . coli, their intracellular level increases during amino acid starvation and carbon stepdown. Because of the importance of the these nucleoside oligophosphates in metabolic regulation in bacteria, it is possible that changes in their intracellular concentration may be correlated with the onset of secondary

GENETICS AND BIOCHEMISTRY OF SECONDARY METABOLISM

81

metabolism. With this in mind, An and Vining (1978)measured ppGpp and pppGpp in S. grieseus during logarithmic and stationary growth phase. They concluded that initiation of streptomycin production in S . griseus is not directly controlled by ppGpp. During exponential growth of S . griseus, the levels of ppGpp and pppGpp were several fold higher than in the stationary phase when antibiotic was produced. Levels of these nucleotides decreased sharply when the culture entered stationary phase. Production of streptomycin started several hours after concentrations of ppGpp and pppGpp had fallen. High ppGpp level does not inhibit streptomycin production, because streptomycin production is not delayed in a glucose starvation medium despite a rising ppGpp concentration. Ragan and Vining (1978) suggest that depletion of phosphate from the medium may be the cause of the sharp decrease in intracellular cAMP concentration as S . griseus enters stationary phase. Exhaustion of phosphate is a prerequisite for initiation of streptomycin biosynthesis, and the decrease in cAMP levels may just be a response to the metabolic switching, rather than a cause of initiation of streptomycin production.

E . ENZYMEMODIFICATION Irreversible inactivation of specific enzymes can regulate flow of precursors via competing metabolic pathways. In vivo degradation and covalent modification of enzymes can diminish wasteful cycling of metabolites through unproductive pathways (Switzer, 1977). Some examples are phosphorylation of pyruvate dehydrogenase in Neurospora crassa (Wieland et al., 1972); the irreversible dissociation of glutamine synthetase induced by NH4+ions in Candida utilis (Sims et al., 1974);the adenylation of glutamine synthetase in Streptomyces and in E . coli (Tyler, 1978; Streicher and Tyler, 1980); the deacylation of citrate lyase in Rhodopseudomonas gelatinosa (Giffhorn and Goltschalk, 1975); the oxidation of an iron-sulfur center of glutamine phosphoribosylpyrophosphate amidotransferase in B . subtilis (Turnbough and Switzer, 1975); and the proteolysis of uridine nucleosidase in yeast (Magni et a l . , 1978; Holzer and Heinrich, 1980; Laskowski and Kato, 1980). Besides an enzyme being inhibited or inactivated, it can be modified (glutamine synthetase), or altered (RNA polymerase), or degraded. Regulation of the quantitative aspects of enzyme production may present a problem. Concentrations of cellular enzymes are regulated so that neither too much nor too little is produced by normal cells. It has not been proved that the “correct” amount of enzymes are made by cells engaged in secondary metabolism.

82

VEDPAL SINGH MALIK

F. FACTORS Mutants of S. griseus that produce little streptomycin and do not sporulate regain the sporulation and antibiotic production if a small quantity of broth of the parent culture is added. The active substance in broth, called factor A, is 2s-isocapryl-oyl-3R-oxymethyl 6-butyrolactone (Kleiner et d., 1976). All industrial strains of S. griseus used for streptomycin production make factor A, traces of which stimulate sporulation, transamiriating enzymes, and streptomycin production up to a level of grams per liter. The molecular formula for factor A is CI:3H'1.t04.All S. griseus strains require factor A for expression of their normal life cycle. The factor A is also produced by Streptoinyces bikiniensis, which produces streptomycin. However, as streptomycin-producer Streptoinyces alhus does not produce factor A, it is not always required for streptomycin production. Only traces of it are needed for a thousand-fold stimulation of antibiotic synthesis; therefore, its role is not of a precursor (Ensing, 1978). The mutants deficient in factor A production bind factor A. Factor A activates an enzyme that hydrolyzes NADP. As a result of the splitting of NADP, adenosine diphosphoribose phosphate (a specific inhibitor of glucose-6-phosphate dehydrogenase) accumulates, resulting in the altered pattern of glucose metabolism in a streptom ycin-producing organism. Another protein, called factor C, is produced late in the growth phase and stimulates sporulation in S. griseus. This cytodifferentiation factor stimulates RNA synthesis and reverses the inhibition by actinomycin D of RNA and protein synthesis in E . coli, B . subtilis, and S. griseus. Factor C raises the T , of the DNA and affects its structure in such a way that mRNA production from latent cell differentiation genes is increased. The characterization of factor C and its mode of action could provide valuable information on the regulation of sporulation in streptomycetes. Some terpene-like extractable molecule effects development of S. alboniger (Pogell, 1975). G. GLUTAMINESYNTHETASE Glutamine is a donor of amino groups in the biosynthesis of all amino acids, the purines, pyrimidine nucleotides, and complex carbohydrates. This central role of glutamine in cellular metabolism is in keeping with the diversity and flexibility in the allosteric control of glutamine synthetase activity (Magasanik, et al., 1974; Tyler, 1978). Glutamine synthetase [L-glutamate: ammonia ligase (ADP-forming), E C 6.3.1.2) catalyzes the synthesis of glutamine from glutamic acid and ammonia.

GENETICS AND BIOCHEMISTRY OF SECONDARY METABOLISM

glutamate

83

+ NH3 + ATP + glutainine + ADP + Pi

In E . coli and other enteric bacteria, the synthesis of glutamine is regulated at two levels:

1. The level of transcription of the glnA gene, which is inversely proportional to the availability of nitrogen in the medium, regulates the amount of glutamine synthetase protein in the cells (Tyler, 1978). 2. Covalent modification of glutamine synthetase protein controls the activity of glutamine synthetase to synthesize glutamine (Prusiner and Stadtman, 1973) The addition or removal of an AMP moiety inactivates or activates the enzyme. Increase in the level of glutamine synthetase resulting from ammonia limitation is responsible for the activation of synthesis of enzymes that supply the cell with ammonia and glutamate. Ammonia limitation in cells growing with glucose as a source of carbon results in the increase in the intracellular ratio of 2-ketoglutarate to glutamine, stimulating deadenylylation and consequent derepression of glutamine synthetase. This shift is ultimately responsible for glutamine synthetase-mediated induction of enzymes that produce ammonia and glutamate from other sources. When nitrogen is in excess, covalent modification by adenylylation of E . coli glutamine synthetase converts this enzyme to a less active form. Adenylylation is catalyzed by the enzyme glutamine synthetase adenyltransferase and protein PII. The protein PII is also covalently modified by uridylation, catalyzed by uridyl transferase. In 1974, Boris Magasanik and his collaborators, working with Klebsiella aerogenes, noted that mutant strains GlnB (PI,)and GlnD (uridyl transferase) possessed little glutamine synthetase that was highly adenylated. On the other hand, GlnE (adenyl transferase) mutants contain elevated levels of glutamine synthetase that was not adenylated. Based on these results, it was suggested that glutamine synthetase controls transcription of its own structural gene (glnA),and that adenylation of glutamine synthetase modifies it as a regulator of transcription. The results of Garcia et al. (1977) threw a monkey wrench in the hypothesis of Magasanik when they reported that the product of a newly identified gene (glnF)is involved in the synthesis of glutamine synthetase in Salmonella and probably also in E . coli. GlnF is located far away from glnA and glnD genes of Salmonella and does not map in the region of the Salmonella chromosome that corresponds with GlnB (PII)and glnE (adenyl transferase) genes of Klebsiella. GlnF extracts contain normal amounts of all

84

VEDPAL SINGH MALIK

proteins involved in covalent modification of glutamine synthetase and glnF may be involved in the regulation of nitrogen metabolism. As a matter of fact, most glutamine synthetase preparations used by co-workers of Magasanik were not absolutely pure and might have contained glnF product as a minor contaminant. If glnF product is indeed a regulatory protein, then only a few molecules per cell would be enough to exert the regulatory effect. In Gram-positive bacilli, the mechanism or regulation of nitrogen metabolism has not been well explored. Covalent modification of glutamine synthetase in B. subtilis (Deuel et al., 1970)and Bacillus stearotherrnophilis (Hachimori et al., 1974; Wedler and Hoffman, 1974) does not occur. However, Streicher and Tyler (1980) have demonstrated that the activity of glutamine synthetase in a Gram-positive, filamentous, spore-forming bacterium Streptomyces cattleya (Kahan et al., 1979)is regulated through covalent modification, as in enteric bacteria. S. cattleya produces a p-lactam antibiotic thienamycin. Radiolabeling experiments of Streicher and Tyler (1980) demonstrate that addition of ammonium chloride to S. cattleya cells growing under nitrogen limitation conditions leads to rapid adenylylation and inactivation of glutamine synthetase. As in E . coli, the adenylylation reaction in S. cattleya crude extracts required ATP and was stimulated by glutamine and inhibited by a-ketoglutarate; the ratio of these two metabolites regulated the adenylylation state of glutamine synthetase. Tronick et al. (1973) have reported that glutamine synthetase of Streptoinyces rutgwsenis and Streptomyces diastatochromogenes is not adenylated. However, when S. cattleya was grown under conditions of Tronick et al. (1973), the glutamine synthetase was present in a very low adenylylation state and escaped detection by the methods used. The glutamine synthetase of these two streptomycetes is probably adenylylated and should be further examined by the methodology and growth conditions used by Streicher and Tyler (1980). Aharonowitz (1979, 1980) found that the level of glutamine synthetase in the S. clavuligerus cells responds to the source of nitrogen in the growth medium. Streicher and Tyler (1980) found more than a 20-fold increase in glutamine synthetase protein levels in derepressed cultures of S. cattleyu. The increased glutamine synthetase protein levels in depressed S. cattleya cells could be attributable to an increased rate of transcription of the S . cattlqa glnA gene or to decreased rates of degradation of glutamine synthetase protein or gZnA mRNA. The role of glutamine synthetase in the control of the onset of thienamycin production in S. cattleyu is not known, but definitely deserves consideration. In the presence of ammonia, the active octameric glutamine synthetase of

GENETICS AND BIOCHEMISTRY OF SECONDARY METABOLISM

85

the yeast Candida utilis is converted to the less active tetramers and then into inactive monomers (Sims et al., 1974). Sanchez et a2. (1980) suggest that glutamine rather than glutamate is the amino donor for the synthesis of amino acids that are involved in penicillin synthesis. High concentrations of ammonium ion prevents glutamine synthetase formation and thereby results in a decreased intracellular glutamine pool. During penicillin production, irreversible transamination catalyzed by glutamine transaminase could produce penicillin precursors L-cysteine, L-valine, and L-a-aminoadipic acid from the a-keto acids glyoxylate, a-ketoisovalerate, and a-ketoadipate, respectively. Leucomycin production by Streptomyces kitasatoesis is inhibited by high concentration of ammonium ion but not so much by inorganic phosphate. Addition of 0.5 to 2%water-insoluble magnesium phosphate to the leucomycin production medium stimulated leucomycin production. Omura et al. (1979) claim that magnesium phosphate stimulates conversion of media glycine into L-serine. These authors further speculate that magnesium phosphate stimulates leucomycin production by trapping free ammonia from the media. H. ENERGYCHARGE The biosynthesis of excessive amounts of secondary metabolite may be energetically favorable for the utilization of metabolites accumulated as a result of a faulty regulation of primary metabolism. The so-called energy charge has been used to evaluate the intensity of energy metabolism and its regulatory role. This parameter, defined as (ATP) (MADP)(ATP) (AMP) reflects the energy state of the cell. Phosphate (0.3-300mM) supports extensive microbial growth but a concentration of inorganic phosphate of 10 m M and above exerts a depressive effect on the synthesis of many secondary metabolites belonging to different biosynthetic groups. Many antibiotics are industrially produced at growthlimiting concentrations of phosphate (Martin, 197%). Phosphate addition inhibits antibiotic synthesis by stimulating growth of the nongrowing candicidin-producing S . griseus. Accompanying the inhibition was a rapid increase in intracehdar ATP concentration (Martin et al., 1979~).A rapid decrease in intracellular ATP could be associated with the onset of antibiotic synthesis. The intracellular level of all adenylates in the wild-type isolate was 10-fold higher compared to that in the chlortetracycline production strain. The production culture was suppressed overall in adenylate synthesis. However, the values of the energy charge were similar in the production and the

+

+

86

VEDPAL SINGH MALIK

wild-type cultures. The decrease during antibiotic production may not be attributable to a low level of ATP but may be the result of an increase in ADP and AMP. The signifcant rise in the A M P level in the wild-type strain may reflect increased AMP synthesis. The rise in the AMP level could also be attributable to the splitting of diphosphate bonds in ATP and ADP. The phosphatase increased sharply during chlortetracycline production although ATP was minimal. This nonspecific phosphatase, which splits all diphosphates is repressed by inorganic phosphate and probably generates phosphate during phosphate limitation while metabolism shifts from the adenylate phosphorylation system to the polyphosphate phosphorylation system. With increased concentration of inorganic phosphate in growth medium, the ATP concentration is maintained at a high level throughout the cultivation, and the energy charge values are considerably lower than under phosphatelimiting conditions. Cell growth is favored, and cellular polyphosphates increase. Phosphatase enzymes involved in antibiotic synthesis are repressed. The level of anhydrotetracycline hydratase, an inducible enzyme of the tetracycline biosynthetic pathway, catalyzing the hydration of anhydrotetracycline to 5a, 1la-dihydrotetracycline, is sharply diminished on increasing orthophosphate concentration above the optimum value. Starting precursors of antibiotic synthesis in S . aureofaciens could be synthesized by specific pathways different from those yielding the same intermediates for growth. Intensity of antibiotic production occurs under conditions not the best for growth. Besides synthesis of enzymes of antibiotic biosynthesis, unfavorable growth environment induces other metabolic changes, e.g., the activation of regulation mechanisms governing Pi regeneration. The process represents an overall economizing rearrangement of the culture metabolism immediately after growth termination.

VII. Regulation of Autotoxicity Many secondary metabolites have no biological toxicity against the organism that synthesizes them because they lack a target site and can, therefore, be exempted from the penality of toxicity. On the other hand, many prokaryotes produce autoinhibitors and must have mechanisms of selfdefense against their own autotoxic metabolites (Lisivinova et a l . , 1979). Various mechanisms of antibiotic tolerance in producer organisms have been extensively reviewed (Demain, 1974b; Vining, 1979; Malik, 1979b) and will not be discussed here. The ability of an organism to produce an autotoxic metabolite can be curtailed by the level of sensitivity of the producer to the autoinhibitor that it produces. In many cases, resistance is inducible and does not even require

GENETICS AND BIOCHEMISTRY OF SECONDARY METABOLISM

87

the synthesis of the autotoxic metabolite. For example, chloramphenicol resistance in S . venezuelae is induced by externally added chloramphenicol under conditions when no chloramphenicol is produced (Malik, 1979b).This suggests that resistance and synthesis of chloramphenicol in S . venezuelae are independently controlled and may even constitute separate operons. The first few molecules of endogenously produced chloramphenicol also induce resistance. Like many other antibiotic resistance markers, chloramphenicol resistance in the producer S . veizezuelae is very unstable and may be coded on a translocatable element. The close coupling between the regulation of biosynthesis and development of resistance to autoinhibitors could be of evolutionary significance. However, no general rules can be formulated to predict the location of genes involved in resistance and synthesis of these autoinhibitors. Genetic inapping in s. coelicolor suggests that genes involved in the synthesis of methylenoinycin A and those for resistance are located on plasmids. A. REGULATION OF

PERMEABILITY

During production of antibiotics, a marked development of mesosome-like membranous structure usually occurs. Although mesosoines are known for their important primary metabolic energy-producing processes, membrane-bound areas and vacuoles could be associated with production, accumulation, and release of antibiotics froin the producing cell. This cellular compartmentalization resulting in localization of antibiotics in such areas may help to separate the toxic antibiotic from potentially sensitive sites in the cell. If they accumulate in veiscles formed by invaginations of inembrane, they could then be secreted through cell walls and never reenter the cell. Enzymes catalyzing terminal steps of antibiotic synthesis could be associated with membranes as in many other microbiol enzymes. Aurodox inhibits both protein synthesis and the release of the elongation factor Tu from the ribosomes of the producing organism S . goldiniensis (Unowsky and Hoppe, 1978). The inducible resistance to aurodox in the producer organism is probably attributable to alteration of membrane permeability (Liu et al. , 1979) and similar to development of resistance to tetracycline inhibition of protein biosynthesis. Chloramphenicol resistance in S. venezuehe is not attributable to chlorarnphenicol acetyltransferase but to a permeability barrier. Study of the genetic control of resistance to chloramphenicol in producing streptomycetes offers the opportunity to understand control of membrane permeability in these industrially important organisms. The role of the peptide antibiotic bacitracin in a producer organism has

88

VEDPAL SINGH MALIK

been investigated and the environmental regulation of sporulation, expecially with respect to manganese has been elaborated. The exact mechanism of resistance of the producer to bacitracin remains to be discerned, but the resistance phenotype is dependent on the expression of bacitracin synthetase genes. Bacillus brevis becomes impermeable and resistant to edeine during antibiotic production. Nascent edeine exists in an inactive form in postlogarithmic-phase producer cells; it is bound via a thioester linkage to a fast-sedimenting fraction containing polyenzyines of edeine biosynthesis and D N A membrane complex (Borowska and Szer, 1976). In industrial fermentation, fatty acids and their esters are added to growth media to stimulate tylosin production. Exogenous fatty acids alter the fatty acid composition of cellular phospholipids. Cells grown in the presence of oleic acid show different transport properties for valine. Alteration in permeability could influence the excretion of an antibiotic. The stimulation of neomycin and streptothricin production by exogenous oleic acid is attributable to alteration of membrane permeability (Arima et al., 1973; Okazaki et al., 1974). Even though neomycin is not a polyketide-derived antibiotic, its final yield in fermentations of a S. faradiae mutant that requires oleic acid for neomycin formation depends on the cellular fatty acid spectrum (Atima et al., 1973; Okazaki et d.,1974) The high antibiotic production capability of certain phage-resistant strains could be attributable to their altered membranes (Malik, 197913). Modification of cell membrane may release cellular metabolites so that they are diluted into the production media and do not exert regulatory controls (Demain and Birnbaum, 1968). Polyene-resistant mutants could be altered in cell membrane; they secrete large amounts of desired products (Fisher, 1980). Mutants of Penicillium stolonifmum resistant to amphotericin B, filipin, and nystatin were altered in sterol composition (Wilkerson et al., 1978). Some mutants produced altered amount of ergosterol and unique sterols. One mutant produced increased amounts of ergosterol and mycophenolic acid, a metabolite partially derived from farnesylpyrophosphate. Mutants of C. acremonium or Cephalosporium polyaleuruin resistant to nystatin, kabicidine, or trichomycin secrete more than 10 gm/liter of cephalosporin C. A kabacidine-resistant mutant of Fusurium secretes high yields of ergosterol and of alkaline protease (Suzuki et al., 1974). Another kabicidineresistant mutant of Trichoderma reesei is derepressed in production of cellulase (Gallo, 1979). Study of the structure of the membranes of these hyper-secreting strains could establish the type of membrane alterations that allow metabolite secretion.

GENETICS AND BIOCHEMISTRY OF SECONDARY METABOLISM

89

B. MODIFICATION OF TARGETSITE Prokaryotic streptomycetes produce many antibiotics that are inhibitors of protein synthesis and act at the level of the prokaryotic 70 S ribosome. Ribosomes from some of the autoinhibitor-producing streptomycetes are modified in such a manner that the autoinhibitor no longer binds to their ribosomes (Vining, 1979). 1. Methylation of Ribose

Resistance of S. azureus to its own polypeptide antibiotic thiostrepton is attributable to methylation of the ribose of its 23 S ribosomal RNA. Whether the methylase responsible for resistance is inducible or constitutive is still unresolved (Cundliff and Thompson, 1979). Methylation of 23 S rRNA occurs before assembly of the 50 S ribosomal subunit is completed. Thiostrepton-resistant methylase of S. nzureus is an RNA-ribose methylase that introduces a single methyl group into adenosine in 23 S rRNA. Ribose methylation is a novel mechanism of antibiotic resitance and high frequency of eukaryotic rRNA methylation may determine resistance to thiostrepton.

2. Methylation of Adenosine Base Two other examples involve base methylation as a mechanism of posttranscriptional modification of rRNA. 1. Kasugamycin-resistant mutants of E . coli possess undermethylated 16 S rRNA and lacks N6,N6-dimethyladenosine normally located near the 3’ terminus (Helser et d., 1971). 2. Overmethylated 23 S rRNA is responsible for the so-called MLS phenotype that is found in Staphylococcus aureus, Streptococcus, and Streptomyces erythreus (Weisblum and Graham, 1979; Horinouchi and Weisblum, 1980).

The presence of N6,N6-dimethyladenosine in the 23 S rRNA of S . aureus is responsible for coresistance to macrolide, lincosamide, and streptogramin B-type antibiotics (MLS resistance) (Weisblum and co-workers). MLS resistance in S. aureus and Streptococcus pyogenes is encoded on plasmids. In fact, a fragment of plasmid DNA from S. pyogenes that codes for the 23 S rRNA methylase cross-hybridizes with the plasmid coding for MLS resistance in S. aureus (Weisblum et al., 1979). Antibiotic resistance genes could have originated in the producing organism (streptomycetes, in most cases) and somehow were transferred to the

90

VEDPAL SINGH MALIK

true bacteria (Benveniste and Davies, 1973). Graham and Weisblum (1979) showed that an erythromycin-producing strain, S. erythreus NRRL 2338, has the same MLS resistance phenotype as that observed in S. aureus and S. pyogenes. This culture contained N N ‘-dimethyladenosine in its 23 S rRNA, which was not the case in several other species of streptomycetes, including some other macrolide producers (Cocito, 1979). Their studies, however, did not suggest the existence of any extrachromosomal DNA in S. mythreus. By methylating its 23 S rRNA, S. erythreus becomes resistant to macrolide-lincosamide-streptogramin B(MLS)-type antibiotics. Horinouchi and Weisblum (1980) have cloned the inducible erythromycin resistance gene from S. uureus into B. suhtilis. The cloned 1442-base pair TAQ1A fragment codes for a leader peptide (19 amino acids) and a “29-K protein” that consists of 243 amino acids. The promoter region for 29-K protein has 4 complementary inverted repeat sequences named “1, 2, 3, and 4.” The C-terminal half of the leader peptide is coded by sequence 1, which is complementary to 2. Sequence 2 is complementary to 3, and sequence 3 is complementary to 4. Ribosome binding site for the synthesis of 29-K protein lies in a loop formed by the complementarity of sequence 3 4. Like attenuators (Keller and Calvo, 1979; Rosenberg and Court, 1980; Johnston et al., 1980), the promoter of 29-K protein does not have transcription termination signals. Ribosomes engaged in leader peptide synthesis could be partially inhibited b y optimal inducing levels of erythromycin, resulting in accumulation of partially blocked ribosomes in sequence 1. Horinouchi and Weisblum (1980) postulate that accumulation of a high level of stalled ribosomes in sequence 1can perturb the translationally inactive double-stranded inverted repeats between sequences 1 and 2 and sequences 3 and 4, resulting in hydrogen binding of sequence 2 with 3. This results in induction of erythromycin resistance, because the ribosome binding site for the synthesis of 29-K protein, which was otherwise not available because of the association of sequence 3 with 4, is free. Cerulenin resistance in a cerulenin-producing fungus is attributable to a cerulenin-insensitive fatty acid synthetase (Kawaguchi et al., 1979).

‘,

+

c. BIOTRANSFORMATIONAND REGULATION OF

CATABOLISM

Most organisms can also inactivate the produced secondary metabolite. Mechanisms that control antibiotic resistance and inactivation in the producing organism have commercial significance because their manipulation is used to increase the yield of antibiotics in industrial fermentations. Enzymes that convert antibiotics into biologically inactive compounds in-

GENETICS AND BIOCHEMISTRY OF SECONDARY METABOLISM

91

fluence the final yield of antibiotic accumulated in the fermentation broth. For instance, lincomycin is oxidized to lincomycin sulfoxide by an oxidoreductase of Streptomyces lincolnensis (Argoudelis and Mason, 1969). Polymixin E is decomposed by a protease produced by Bacillus polymyxa, which synthesizes this antibiotic (Woyczikowska et al., 1973). Chloramphenicol is degraded by the producing streptomycetes, which contain the enzyme chloramphenicol hydrolase (Malik and Vining, 1970). The mikamycin-producer Streptomyces mitakaensis contains the enzyme mikamycin P-lactonase, which causes low titers of the produced antibiotic in the medium. A kinase from streptom ycin-producing S. griseus will phosphorylate streptomycin at the 3 position of the N-methyl-L-glucosamine ring. Inactivation of the streptomycin by phosphorylation is controlled by the level of phosphate in the medium. The kinase may be responsible for the resistance of the producing organism to streptomycin. A second phosphorylating enzyme (ATP:dihydrostreptomycin-6-P-3a-phosphotransferase) phosphorylates the 3a’ position of the dihydrostreptose moiety of dihydrostreptomycin. A third enzyme, streptomycin-6-kinase (ATP:streptomycin-6-phosphotransferase), is found in Pseudomonas aeruginosa (Kida et al., 1975) and S. bikiniensis (Miller and Walker, 1969). Recently, Piwowarski and Shaw (1979) have suggested that resistance to streptomycin in S. bikiniensis may be the result of phosphorylation of streptomycin at the 6 position of the streptidine subunit by a plasmid-borne kinase. This enzyme is not present in logarithmic phase cells, which are not yet committed to streptomycin production and are susceptible to 25 pglml of streptomycin. The kinase activity appears in stationary phase cells 12 hr before streptomycin is detected in the medium. Stationary phase cells are producing streptomycin and are resistant to more than 200 pglml of streptomycin. Certain isolates of S. bikiniensis that have lost the ability to produce streptomycin were selected after treating the culture with acriflavine or ethidium bromide. These cultures were inhibited by 10 pg/ml of streptomycin throughout their growth, did not inactivate streptomycin, and lacked streptomycin-8kinase. These results suggest that phosphorylation by streptomycin-6-kinase plays an important role in resistance to streptomycin in S. bikiniensis. The streptomycin-6-kinase could be so located on plasma membrane that it inactivates the Streptomycin that is trying to enter the cell via the polyamine transport system (Holtje, 1978, 1979). Cellular phosphatase may convert the inactive phosphorylated antibiotic into streptomycin before its secretion from the cell. As a result, streptomycin accumulates in the medium and not the phosphorylated derivative. Phosphate inhibits formation of alkaline phosphatases required for the synthesis of streptomycin

92

VEDPAL SINGH MALIK

(Miller and Walker, 1970b), viomycin (Pass and Bojanowska, 1969b), and vancoinycin (Mertz and Doolin, 1973). Dephosphorylation is the final step in the biosynthesis of these antibiotics. In the presence of phosphate, inactive phosphorylated derivatives of streptomycin and neomycin accumulate in the media (Miller and Walker, 1970b; Majumdar and Majumdar, 1970). Streptomyces glaucescens produces hydroxystreptomycin, tetracenomycins, an a-amylase inhibitor, and a bacteriocin. It is also resistant to streptomycin (1 pg/ml) and secretes melanin. In S. glaucescens, genetic determinants for streptomycin resistance, secretion of melanin and chitinase, synthesis of laminarinase, and production of spores are highly unstable (Suter et al., 1978). A frequency of spontaneous mutant phenotypes of up to 1% can be obtained for any of these markers. The mutation frequency can be increased to more than 10% by prolonged cold storage or by cultivation on medium supplemented with ethidium bromide. Mutants in several of the aforementioned traits occur simultaneously. Genetic analysis shows that such phenotypes are attributable to different independent mutations and are not the pleiotropic effects of single mutations. Mutants resistant to high levels of streptomycin (100pglml) occur infrequently. However, mutations that cause increased sensitivity to streptomycin (<1 pg/ml) are common but unstable (Freeman and Hopwood, 1978). Stable streptomycin-sensitive strains can be obtained by several cloning steps of streak purification. Sensitivity of the mutant strains is due to increased accumulation of streptomycin (Hutter et al., 1979). Genes responsible for streptomycin sensitivity and melanin formation have been mapped on the chromosome. The DNA of the mutant and parent strains were labeled with [3H]thymidine and [14C]thymidine,respectively. After digestion with restriction enzymes BainHI and SalGI; the DNAs were mixed and analyzed by agarose gel electrophoresis. The determination of the 14C:3Hratio in various DNA bands indicated the absence of a 13.5-kilobase DNA fragment from the DNA of a streptomycin-sensitive, melanin-negative mutant. This 13.5-kilobase DNA fragment can now be cloned and used as a probe to hybridize to the various DNA fragments of the parents that have been resolved by electrophoresis. This type of analysis may reveal if streptomycin sensitivity is attributable to the participation of a translocatable and insertion element. Other examples of chromosomal instability are oxytetracycline sensitivity in S. rirnosus (Hopwood, 1978; Borisoglebskaya et al., 1979), chloramphenicol sensitivity in S. coelicnlor (Freeman et at., 1977; Sermonti et at., 1977, 1978, 1980; Micheli, 1980), and chloramphenicol sensitivity in S. uenezuelae (Malik, 1979b). S . coelicolor and chloramphenicol-producing S . venezuelae are resistant to chloramphenicol but do not possess chloramphenicol acetyltransferase (Freeman et al., 1977).

GENETICS AND BIOCHEMISTRY OF SECONDARY METABOLISM

93

An inhibitor of chloramphenicol acetyltransferase is produced by certain streptomycetes (Miyamura et al., 1979). Many variants of chloramphenicol acetyltransferase have been found in many non-chloramphenicol-producingstreptomycetes (Shaw and Hopwood, 1976) and bacteria (Shaw et al., 1979; Liddel et al., 1978; Shaw, 1979). Chloramphenicol resistance due to chloramphenicol acetyltransferase is determined by a transposable genetic element (Tn9), which consists of two direct repeats of the insertion sequence IS1 flanking a region of 1102 base pairs (Chandler et al., 1979). Transposition ofTn9 results in the duplication of a %base pair sequence of the insertion site. One copy of the %base pair sequence is located at each end of inserted element. Alton and Vapnek (1979) have sequenced the 1102-base pair region between the directly repeated IS 1 sequences encoding chloramphenicol acetyltransferase of E . coli. The gene product ofTn9 is expressed in Saccharomyces cerevisiae when the chloramphenicol-resistance determinant is inserted into a 2-pm yeast plasmid. Expression of the bacterial chloramphenicol acetyltransferase gene also occurs in streptomycetes when ligated to a streptomycetes promoter (Bibb et al., 1980b). With the available methodology, it should now be possible to clone and sequence the genetic determinants of chloramphenicol acetyltransferase of streptomycetes. If streptomycetes chloramphenicol acetyltransferase is encoded by a transposable genetic element similar to Tn9 of E . coli, then it could be useful for studying mutagenesis and genetics in streptomycetes. A gene coding for neomycin and butirosin phosphotransferase from a butirosin-producing Bacillus circulans has been cloned and expressed in E . coli (Davies et al., 1979). Many aminoglycoside-modifying enzymes have been described in both producing organisms and in plasmid-carrying bacteria (Dowding, 1979b). Many P-lactamases of Gram-positive bacteria (Imsande, 1978) and penicillin-biiiding proteins of p-lactam-producing Streptomyces cacaoi, Streptomyces olivaceus, and S . clavuligerus have been examined (Ogawara and Horikawa, 1980a,b). Another example of antibiotic inactivation by the producing organism is the metabolism of nebramycin by S. tenebrarius (Stark et al., 1980). Nebramycin activity declines in the medium after reaching a peak. However, nebramycin inactivation does not occur if ample energy source is present in the media. The nature of the inactivation product, when known, could be important if nebramycin is degraded to subunits that could be used to prepare analogs of nebramycin. The control of the enzymatic hydrolysis of cephalosporin C to deacetylcephalosporin C occurs in the producing organism. When either glucose, maltose, or sucrose is fed to a C.acremonium mutant, the formation of an

94

VEDPAL SINGH MALIK

extracellular carboxyesterase acting on cephalosporin C is suppressed (Hinnen and Nuesch, 1975). Derepression of cycloheximide degradative enzymes upon exhaustion of glucose from the medium of cycloheximide-producing S . griseus has been suggested by Kominek (1975).

VIII. Secondary Metabolism, Sporulation, and Exoenzyme Formation Synthesis of antibiotics in the stationary growth phase, which in Bacillus and Streptomyces is the phase of cell differentiation and formation of spores, suggested that antibiotic synthesis and sporogenesis might be linked. However, these two processes are not inseparably interrelated; for example, Pseudornonas strains produce antibiotics but do not form spores. Sporulation and secondary metabolite formation are initiated at the end of logarithmic growth when certain nutrients in the growth medium are depleted. The fact that secondary metabolism and sporulation are initiated at the end of growth makes analysis of the specific genes very difficult. Many genes are released from growth-linked repression at the end of the growth phase, and it is difficult to differentiate between these derepressed gene functions and those that are specific for growth, sporulation, or secondary metabolite formation. The genetics of secondary metabolism and sporulation is much more complex than the genetics of growth because the expression of genes involved in these processes is superimposed on the expression of logarithmic phase genes. Identification of any primary products for any of the genes involved in secondary metabolism or sporulation is yet to be achieved. Another inherent difficulty caused by the pleiotropic nature of most mutations affecting secondary metabolism and sporulation makes it difficult to approach this problem by modern methods of molecular genetics. Bacterial sporulation is a time-ordered sequence of biochemical and morphological events resulting in the conversion of a vegetative cell into a spore. At the end of exponential growth when sporulation begins, cells elaborate several proteolytic enzymes and antibiotics (Bose et al., 1979). Certain temporal and genetic relationships exist between these two early molecular events. However, the exact nature of the relationship remains elusive. In B . subtilis, sporulation was initiated by the presence of excess glucose, ammonium ions, and phosphate; by guanine deprivation of a guanine auxotroph; or by the addition of decoyinine, an inhibitor of GMP synthetase. Under these circumstances, ATP generation and substrate synthesis pro-

GENETICS AND BIOCHEMISTRY OF SECONDARY METABOLISM

95

ceeds by glycolysis, and citric acid cycle enzymes are not required. Glucose repressed the synthesis of inducible enzymes even when guanine deprivation triggered sporulation. However, enzymes typically found during sporulation were the same whether sporulation was induced by nutrient limitation or by deprivation of guanine nucleotide in the presence of excess glucose, phosphate, and ammonium ions (Vasantha and Freese, 1980). Sporulation and secondary metabolism are favored under similar growth conditions. This may be attributable to some regulatory event that triggers both processes. Many best producers of secondary metabolites are asporogenous mutants. Such mutants could be impaired in the events occurring after the regulatory signal that is common to both sporulation and secondary metabolite formation. In this way, precursors and energy would be diverted to secondary metabolism instead of sporulation. The production of extracellular proteases and antibiotics has been intimately linked with the initial stages of sporulation. Sporulation, serine protease, and cephamycin production in Streptomyces lactamdurans is well coordinated (Ginther, 1979). Most protease-less and antibiotic-less mutants are also asporogenous and many result from pleiotropic effects of mutations (Schaeffer, 1969). Extracellular protease is probably not required for sporulation but intracellular protease may convert coat precursor to coat protein and modify and degrade other enzymes. Antibiotic production may be an earlier event than sporulation in cellular differentiation. Most industrial strains that are potent producers of antibiotics sporulate poorly. High productivity of these strains could be attributable to the fact that precursors of sporulation are channeled into antibiotic formation instead. The subunit pattern in the RNA polymerase from sporulating cells is quite different from that in vegetative cells. The subunit of RNA polymerase from cells engaged in secondary metabolism and vegetative growth have not been studied. Modification of RNA polymerase can control the transcription of a number of scattered genes simultaneously, but what controls the modification of RNA polymerase is not known. Knowledge of regulatory mechanisms that control genes coding for RNA polymerase subunits of genes controlling enzymes that modify subunits is needed. To understand the expression of sporulation genes during developmental sequence, Losick (1981) cloned a B . subtilis gene whose transcription is activated at an early stage of spore development. This gene encodes an RNA of about 400 bases and is called the “0.4-kilobase gene.” The transcription of this gene, which maps near the origin of replication of the B . subtilis chromosome is restricted in mutants that are blocked at the early stages of sporulation. A modified form of RNA polymerase that transcribes the 0.4kilobase gene has been isolated from early sporulating cells of B . subtilis.

96

VEDPAL SINGH MALIK

This modified RNA polymerase lacks (T factor but contains a novel polypeptide subunit. This new subunit, which replaces the (T factor of RNA polymerase, has a molecular weight of 37,000 and is known as P3‘. About six genetic loci known as the “Stage 0” genes control initiation of sporulation in B . subtilis. Mutations in Stage 0 genes SpoOA, SpoOB, SpoOE, SpoOF, and SpoOH block sporulation at the first morphological step, Stage 0, and diminish transcription of the 0.4-kilo11ase gene in vivo. Pa?may be the product of a Stage 0 gene because it turiis on early sporulation genes by modifying RNA polymerase. The “0.3 kilobase gene” of Bacillus subtilis is transcribed at an intermediate stage of sporulation. The promotor of this gene is not recognized and transcribed by the Sigma factors isolated from vegetatively growing cells, but is recognized and transcribed by the RNA polymerase activity isolated froin sporulating cells. In order to determine its function, a recombinant DNA plasmid (~1419) has been constructed to introduce an insertion mutation in the “0.3 kilobase gene.” P1419 replicates autonomously in E . coli and confers resistance to tetracycline and chloramphenicol. However, this recombinant plasmid does not replicate autonomously in B . suhtilis. The plasmid does confer resistance to chloramphenicol, however, if by homologous recombination it can integrate into B . suhtilis chromosome. Cloning of a specific B . subtilis gene into p1419 provides the homology required for integrating into that particular gene. A 68-base-pair fragment of the “0.3 kilobase gene’’ was cloned into ~ 1 4 1 9and , the integer was transformed into €3. subtilis. Plasinid integrated cloqe to the chromosomal site of the “0.3kilobase gene.” Phenotypes of these insertion mutants appears to affect cortex formation. These experiments are of great significance since they provide novel strategies for isolating genes, mutating them in vitro, introducing them into the genome by homologous recombination, and then studying the phenotypic effect of the gene on the constructed strain. This approach of reciprocal genetics has immediate application to the study of secondary metabolism and should therefore be pursued. Because PS7is present in both vegetative and sporulating cells, it may direct transcription of many classes of genes. However, other Stage 0 gene products may affect specific participation of P37in sporulation RNA synthesis. In this way, one or more regulatory protein subunits of RNA polymerase that interchange may be involved in control of sporulation. Different forms of RNA polymerase containing distinct regulatory subunits may control sporulation and secondary metabolism. Little is known about the molecular mechanisms in initiating sporulation or antibiotic formation. Pleiotropic mutations at any one of nine distinct genetic loci block sporulation in B . subtilis at early stages without affecting

GENETICS AND BIOCHEMISTRY OF SECONDARY METABOLISM

97

vegetative growth (Piggot and Loote, 1976). These pleiotropic mutations scattered throughout the B. subtilis chromosome are also impaired in Stage O functions, such as antibiotic and protease production, competence for transformation, and sensitivity to surface active antibiotics and phage. Trowsdale et al. (1978) examined the ribosomal composition of these pleiotropic Stage 0 mutants and of the same mutants bearing an additional mutation in another locus abrB, which suppresses the pleiotropic mutant phenotype without suppressing the sporulation defect. Suppressed strains were resistant to polymyxin and wild-type antibiotics and produced extracellular proteases. Some of them produced antibiotic and were nonpermissive for two bacteriophages that only replicate in Stage 0 mutants. All mutants have alterations in different ribosomal proteins, suggesting that a group of structural genes specifying several ribosomal proteins are located in the abrB region near the origin of replication on the genetic map of the B . subtilis chromosome. Without allowing sporulation, these ribosomal alterations permit reading of messages that specify proteins concerned wtih proteases, antibiotics, and other early functions in sporulating cells that are masked in Stage 0 mutants. In wild-type sporulating strains, this mRNA may be read when a change takes place on the ribosome early in sporulation. Stage 0 gene products alter the ribosomes so that they translate certain species of message. Early in sporulation, changes take place in ribosomeassociated proteins (Trowsdale et al., 1978).

IX. Role of Secondary Metabolism Genetics and molecular complexity of various secondary metabolites suggests that their synthesis requires specific enzymes. Because they are accumulated in significant amounts, a significant pool of materials of primary metabolites and energy is used in their formation. Secondary metabolism must, therefore, have a function, to prevent its elimination during evolutionary selection. Like other organisms, microbes have evolved over millions of years. Certain microbes, such as E . coli, are metabolically very efficient. Their metabolism is well regulated and various metabolic steps are always so coordinated that they are in step with each other. Whenever concentrations of any cellular products increase above certain levels, efficient organisms switch off corresponding operons and utilize other well-evolved regulatory mechanisms to prevent accumulation of end products and intermediates. Such tightly regulated microbes with balanced metabolic circuits are of little use to industrial microbiologists because they do not overproduce cellular metabolites. However, in many microbes, certain primary metabolic reactions lack

98

VEDPAL SINGH MALIK

partial or complete regulation. As a result, intermediates and end products of primary metabolism are overproduced. Such organisms have evolved with an alternative strategy to well-coordinated cell regulatory mechanisms (Malik, 1980a). They have developed additional pathways that channel these oversynthesized cellular products into secondary metabolites. This has probably occurred in a series of sequential steps.

1. As a result of some faulty cellular regulatory mechanism, cellular products of primary metabolism probably accumulated. 2. Some cellular enzyme with loose cofactor and substrate specificity acted upon the accumulated primary product and converted it to the poor substrate of a second cellular enzyme. In this manner, multistep pathways for converting overproduced metabolites into secondary metabolites have evolved. 3. These accumulated intermediates were probably poor substrates for the cellular enzymes. As a result, genes for these enzymes were tandemly duplicated. At this stage in evolution, one gene was selected for rigid enzymatic specificity and became fixed in the operons participating in primary metabolism. However, a sister gene coded for enzyme with loose specificity and, as a result of chromosomal rearrangement and reorganization, got fixed in the clusters that participate in transformation of accumulated products into secondary metabolites. In this way, such organisms possess, in addition to genes for growth, genes for production of secondary metabolites. Regulation is an essential condition of life. Metabolic regulation is not a late development somehow grafted on to preexisting metabolic sequences; the sequence and their regulation necessarily evolved together, with tnutation and selection acting in most cases on the same molecules in the development of catalytic efficiency and of regulatory response. Hardly any reaction or pathway can be imagined to be adaptive in the absence of regulation. Evolution of every inetabolic sequence has been accompanied by evolution of control characteristics that cause the sequence to respond appropriately to the needs of the organism under a wide range of internal and external conditions. Just as the individual reaction has meaning only in terms of the sequence in which it participates, the sequence is useful only by virtue of its integration into the overall metabolism of the cell or organism. The hnction of regulatory enzymes (secondary metabolite production) is to affect this integration. Complex regulatory responses result from the simultaneous occurrence of numerous regulatory interactions, each relatively simple in principle. Secondary metabolites might have evolved as a viable option to switching

GENETICS AND BIOCHEMISTRY OF SECONDARY METABOLISM

99

off metabolic pathways by various control mechanisms and may be beneficial for the organism. In this way, intermediates and end products of the primary metabolism, which would normally accumulate in the cell, are excreted, whereby their regulatory effect is diminished or completely abolished. Some of these secondary metabolites accidentally happen to be biologically active; some are antibacterial and are even toxic to the organism that produces them. Producing organisms have then evolved with mechanisms that protect against the toxic effects of their own metabolites (Demain, 1974a; Davis, 1980; Vining, 1979; Malik, 1972, 1979b). Ensing (1980) suggested that an antibiotic is involved in the maintenance and control of dormancy of spores in Streptomyces viridochromogenes. The antibiotic is present in spores and prevents spore germination by inhibiting Na+,K+-activated ATPase. As a result, dormant spores oxidize glucose but produce no ATP. Upon initiation of germination, the antibiotic is released from spores, leaving ATPase free from inhibition. It has been suggested that streptomycin is a component of the cell walls of producing S . griseus. This idea was tested using a mutant strain that produces streptomycin when streptidine is added to the medium. The rate and amount of growth was essentially the same when streptomycin was or was not produced. Cell walls from organisms grown in the presence or absence of streptidine were equally susceptible to lysozyme. Spores formed in both conditions were equally dormant and germinable. Cell walls from organisms growth with [14C]streptidinecontained label. This label was removed by a 1.0 M NaCl and was attributable to [14C]streptidine.Therefore, the antibiotic was ionically bound and not a structural component of the walls. No function for streptomycin at the level of cell walls was found (Ensing, 1980). The ticklish question of the role of secondary metabolites cannot be easily answered. The diversity of their chemical structure and biological activity suggests that the search for a general answer may be naive. Philosophical solutions to this problem were attempted many years ago. Although Waksman (1943, 1956) suggested that antibiotics are a metabolic waste product, these substances confer a competitive advantage upon the producing organism (Krassilnikov, 1958). B . brevis produces two types of peptide antibiotics: the linear gramicidins and cyclic tyrocidines. Most antibiotics that are produced during the early stages of sporulation are potent inhibitors of the vegetative growth of the producing organism. Gramicidin-negative mutants of B . breuis produce defective spores unless supplied with gramicidin within a critical period during transition from growth to sporulation. This period coincides with alteration in the specificity of transcription and suggests that peptide antibiotics may inhibit promoter recognition by RNA polymerase of genes required for veg-

100

VEDPAL SINGH MALIK

etative growth but not for sporulation (Sarkar et aZ., 1977). However, mutants that are defective in gramicidin S synthesis have been found to sporulate normally (Deniain and Piret, 1979). Sporulation is inversely related to increase in gramicidin titer. In a chemostat under carbon limitation, spores are formed at a dilution rate lower than that required for formation of gramicidin S synthetase. Antibiotic negative mutants sporulate well and most antibiotic-producing industrial strains are poor sporulators. Antibiotic synthesis and sporulation are two independent processes that compete for a common pool and respond to a common regulatory mechanism. The various aspects of secondary metabolism are very close to those of sporulation and production of oligopeptides antibiotics. Production of typical secondary metabolites is one of the earliest events in sporulation. There is a discrete point in time during outgrowth of spores at which spores overcome the inhibitory effects of the peptide antibiotics. In a medium lacking nitrogen, R . brevis cells at this stage undergo sporulation in the presence of both tyrocidine and gramicidin. In vitro tyrocidine binds to DNA and inhibits initiation of transcription. Gramicidin binds to RNA polymerase and inhibits transcription in the absence of tyrocidine. However, when both gramicidin and tyrocidine are present, gramicidin reactivates a tyrocidine-inhibited transcription system. In this way, both peptide antibiotics may play an antagonistic gene regulatory role during sporogenesis in B . breuis. Serine protease of Bacillus lichenijmmis releases antibiotic bacitracin in oitro. The extracellular proteases have been separated into three distinct serine enzymes on carboxymethyl cellulose (CMC) columns. Bacitracin forms a complex with the specific protease, CMC fraction I11 in uivo and inhibits the activity of the enzyme responsible for its activation in vitro. Coincidence chromatography and gel filtration experiments suggest a direct peptide-protein interaction. Bacitracin appears to exert product inhibitiontype control on the protease (Vitkovic and Sadoff, 1977a). An antibiotic was produced in vitro by proteolysis of the vegetative cell protein with the CMC 111protease. This antibiotic was shown to be identical to bacitracin in its UV spectra, biological activity, and chromatographic properties in a multiplicity of systems. Similarities exist between ethanol-extractable peptides excreted by sporulating cells and those produced in uitro, in their number, in their R f values on chromatography, and in their labeling pattern with D,L-[ 14C]ornithine. Crude bacitracin synthetase does not appear to contain nor docs it require CMC I11 protease for activity. Unequivocal involvement of peptide antibiotics in inducing sporulation in Bacillus would require genetic proof, because the catalytic concentration of the antibiotic sufficient to induce sporulation may be unmeasurable. Evidence accumulating in recent years casts doubt on the essential role of

GENETICS AND BIOCHEMISTRY OF SECONDARY METABOLISM

101

antibiotics for sporogenesis. Mutants of many organisms exist that sporulate well but produce no antibiotics (Demain, 1980). Antibiotics, like extracellular protease, are regarded as the markers of the first stage of sporulation, and in accordance with the theory of sequential induction are taken to be inducers of the next stage of sporulation. In no case, however, are sporulation and antibiotic synthesis quantitatively correlated. Many industrial strains of Streptomyces and antibiotic-producing molds sporulate poorly. Furthermore, selection of suitable culture conditions can intensify one of these processes at the expense of the other (Bernlohr and Novelli, 1960).

X. Epilogue Synthesis of a secondary metabolite is a highly ordered and coordinated event. Negative and positive regulatory control mechanisms operative during balanced growth have to be lifted, with the simultaneous accumulation and availability of initiating precursors. Concentration of acetyl, amino, amidino, and other group donors further influences the emerging metabolites. Mechanisms of secretion, and resistances to certain metabolites also have to be developed. Exploration of the molecular biology of secondary metabolism is now possible as a result of the availability of methods of recombinant DNA technology and reciprocal genetics. The changes in the transcription of genome responsible for initiation of secondary metabolism should be characterized. In the majority of cases, the enzymes catalyzing different stages of secondary metabolism are not known. Multiple enzymatic complexes are often involved in secondary metabolism and are not very stable. Work along these lines is in progress with representatives of the genera Bacillus, Streptomyces (Hopwood, 1980a; Malik, 1979b), yeast (Davis, 1980), and PenicilZium (Demain, 1980). Mutants that are deficient in proteases (Ohman et al., 1980) and nucleases may be used for isolating enzymes and plasmids from these organisms. Reciprocal genetics and recombinant DNA technology can circumvent the efforts required to understand genetics and molecular biology of industrial microorganisms and can yield most meaningful results that will contribute to the understanding of the regulation of the economically important metabolic pathways involved in secondary metabolism. HEFERESCES Aberhart, D. J. (1977). Tetrahedron 33, 1545. Abraham, E. P . , and Fawcett, P. (1974). In “Industrial Aspects of Biochemistry.” (B. Spencer, ed.), pp. 319334. North-Holland Publ., Amsterdam.

102

VEDPAL SINGH MALIK

Aharonowitz, Y. (1979). In “Genetics of Industrial Microorganisms.” (0.K. Sebek and A. I. Laskin, eds.). Amer. Soc. for Microbiology, Washington, D.C. Aharonowitz, Y. (1980). Annu. Rev. Microbial. 34, 209. Aharonowitz, Y . , and Demain, A. L. (1978). Antimicroh. Agetits Chemothw 14, 159. Aharonowitz, Y., and Demain, A. L. (1979). Can. J . Microhiol. 25, 61. Aharonowitz, Y., and Demain, A. L. (1980). Biotechnol. Bioeng. 22 (Suppl. l), 5. Akagawa, H., Okaniski, M., and Umezawa, €1. (1975). J. Gen. Microbiol. 90,336. AliKhanian, S. I., and Danilenko, V. N. (1979). Hind. Antihiof. Bull. 21, 125. AI’Nuri, M. A , , and Egorov, N. S. (1968). Mikrobiologiya 37, 413. Alsberg, C. L., and Black, 0. F. (1912). Proc. Soc. E x p . Biol. M e d . 9, 6. Alton, N. K., arid Vapnek, D. (1979). Nature (London) 282, 864. An, G., and Vining, L. D. (1978). Can. J. Microbiol. 24, 502. Aoki, H., and Oktthara, M. (1980). Annu. Reu. Microbiol. 34, 159. Argoudelius, A. D., and Mason, D. J . (1969). J . Antibiot. 22, 289. Arima, K., Okazaki, H., Ono, H., Yarnada, K., and Beppu, T. (1973). Agric. B i d . Chem. 37, 2313. Arnstein, H. R. V., and Morris, L). (1960). Biochem. J. 76, 357. Arnstein, H. R. V.. Artman, N., Morris, D., and Toms, E. J. (1960). Biochem. J. 76, 353. Atkinson, D. E., and Walton, G. M. (1967). J . Biol. Chern. 242, 3239. Audhya, T. K., and Russell, D. W. (1975). J . Gen. Miwohiol. 86, 327-332. Baldwin, J. E., Singh. P. D., Yoshida, M., Sawada, Y., and Demain, A. L. (1980). Biochem. J. 186, 889. Ball, W. J . , and Atkinson, D. E. (1975). J. Bacteriol. 121, 085. Bandyopadhyay, S . K., and Majumdar, S. K. (1974). Antirniwob. Agents Chernuther. 5,431. Banerjee, A. B., and Bose, S. K. (1974). J . Ractcriol. 87, 1397-1406. Barahas, G. (1980). Folia Microbiol. 25, 270. Basak, K., and Majumdar, S. K. (1973). Anfirnicrob. Agents Chernother. 4, 6. Battersby, A. H. (1978). Ezperientia 34, 1-13. Behal, V., and Vanek, Z. (1970). Folia Micrubiol. 15, 354. Behal, V., Cudlin, and Vanek, Z. (1969))). Folia Microbiol. 14, 117. Behal, V., Jechova, V., and Vanek, Z. (1977). Phytochernistry 16,347. Benedict, R. C., Lindenfelser. L. A , , Stodola, F. H., arid Traufler, D. H. (1951). J. Bacteriol. 62, 487. Bentley, R., and Campbell, I. M. (1968). I n “Comprehensive Biochemistry; Metabolism of Cyclic Compounds” (M. Florkin and E. H. Stolz, eds.), Vol. 20, p. 415. Elsevier, Amsterdam. Bentley, R., and Keil, J. G. (1962). J . B i d . Chern. 237, 867. Bcnveniste, R., and Davies, J. (1973). Proc. Natl. Acad. Sci. U . S . A . 70, 2276. Berdy, J . (1974). Adu. Appl. Microbiol. 18, 309406. Berdy, J. (1980). Process. Biochem. 15(7), 28. Bernlohr, R. W., and Novelli, G. D. (1960). Arch. Biochern. BiophyY. 87, 232. Bernlohr, R. W., and Novelli, 6. D. (1963). Arch. Biochem. Biophys. 103, 94-104. Bibb, M. J., Ward, 1. M., and Hopwood, D. A. (1978). Nature (London) 274, 398. Bibh, M. I , , Schottel, J . , and Cohen, S. N . (l98Oa). Nature (London) 284, 526. Bibb, M. J.. Ward, J. M.,and Hopwood, D. A. (1980b). Dcu. Inn. Microbiol., in press. F.I IW. , (1953). Aust. J. Cheiri. 6, 360. Birch, A. J., and D O ~ O V ~ Birch, A. J., Massey-Westropp, R. A,, and Moye, C. J. (1955). Ailst. J . Chem. 8, 539. Birkinshaw, J. H., Oxford, A. E., a d Raistrick, H . (1936). Biochem. J. 30, 394. Block, K., and Vance, D. (1977). Antzu. Reo. Biochem. 46, 2m-293. Blumauerova, M., Mateju, J., Stajner, K., and Vanek, Z. (1977). Folia Mimobid. 22, 275.

GENETICS AND BIOCHEMISTRY OF SECONDARY METABOLISM

103

Blumauerova, M., Podojil, M., Gauze, G. F., Maksimova, T. S., Panos, J., and Vanek, Z. (1980a). Folia Microbid. 25, 207. Blumauerova, M . , Podojil, M., Gauze, G. F., Maksimova, T. S., Panos, J., and Vanek, Z. (1980h). Folia Mimohiol. 25, 213. Bollon, A. P . , and Vogel, H. J. (1973). J. Bacteriol. 114, &32. Borisoglebskaya, A. N . , Perebityak, A. N., and Boronin, A. M . (1979). Antibiotiki 24, 883. Boronin, A. M . , and Sadovnikova, L. G . (1972). Genetics (USSR) 8, 174. Borowska, Z. K., and Szer, W. (1976). Biochiin. Biophys. Acta 418, 63. Borrow, A., Jeffreys, E. G . , Kessell, R. H. J., Lloyd, E . C., Lloyd, P. B., and Nixon,, I. S . (1961). Can. J. Micohiol. 77, 222. Borrow, A , , Brown, S . , Jeffreys, E. G . , Kessell, R. H. J . , Lloyd, E . C., Lloyd, P. B., Rothwell, A., Rothwell, B., arid Swait, J. C. (1964). Can. J. Mimohiol. 10, 407. Borrow, A., Brown, S., Jefferys, E. G . , Kessell, R. H. J., Lloyd, E. C., Lloyd, P. B., Rothwell, A., Rothwell, B., and Swait, J . C. (l964b). Can. J. Mimohiol. 10, 445. Bose, R . , Ray, B., and Bose, S . K. (1979). Foliu Mirruhiol. 24, 373. Brack, A. (1947). IZela. Chiin. Actu 30, 1. Brandsch, R. Hefco, E., Brandsch, C . , Rotinberg, P., and Kelemen, S. (1980). J. Antibiot. 33, 1204. Brockinann, H., Zeeck, A,, Menve, A., and Muller, W. (1966). Justus Liehigs Ann. Deo. Chem. 698, 209-229. Bruehl, G . W . , Millar, R. L., and Currfer, B. (1969). Can. J. Plant Sci. 49, 235. Bu’Lock, J . C. (1961). Ado. A p p l . Microhiol. 3, 293. Bu’Lock, J. D. (1967).“Essays in Biosynthesis and Microbiol Development. Wiley, New York. Bu’Lock, J . D. (1974). “Industrial Aspects of Biochemistry,” (A. J. Spencer, ed.), Vol. 1. NorthHolland Puhl., Amsterdam. Bu’Lock, J . D. (1975a). Dec. l n d . Microhiof. 16, 11. Bu’Lock, J. D. (1975h). 111 “The Filameritous Fungi” (J. E. Smith and D . R. Berry, eds.), Vol. 1, pp. 33-58, Wiley, New York. Bu’Lock, J. D., Hainilton, D., M U ~ I WM. , A,, Powell, A. J . , Slnalley, H . M.,Shepherd, D., and Smith, G . N. (1975). Can. J . Microhiol. 11, 765-778. Byiig, 6. S . , and Turner, J. M. (1977). Biochetn. J . 164, 139. Cameron, J. R., Loh, E. Y . , and Davis, R. W. (1979). Cell 16, 739. Cape, R. E. (1979). CHEMTECZZ 638. Carson, M . , and Jensen, R. A . (1974). J . Bacteriol. 117, 312. Catlin, E. R., Hassall, C. H . , and Pratt, B. C. (1968). Biochim. Bioph!/s. Acta 156, 109. Cella, R., and Vining, L. C. (1975). Cun. J. .Vfic.rohiol. 21, 463. Chain, E. B. (1980). CHEMTECII. Chalcff, D. T., and Fink, G . R. (1980). Cell 21, 227. Chandler, M.,Boydelatorir, E . , Willeins, D . , and C h o , L,. (1979). A40l. Gen. Genet. 176, 221. Chandra, P . , Read, G . , and Vining, L. C. (1966). Can. J. Biochein. 44, 403. Chang, L. T., Behr, D. A., and Elander, R. P. (1980). Deo. Inn. hficrobiol. 21, 233. Chapman, A. G., Fall, L., and Atkinson, D. E. (1971). J . Bacteriol. 108, 1072-1086. Chassy, B. M., and Suhadolnik, R. J . (1969). Biochiiti. Bioph!ys. Actu 182, 307. Chatrr, K. F. (1980). Dco. lnrl. Microbial. 21, 65-74. Chater, K. F., and Merrick, M . J. (1979). I n “llevelopmental Biology of Prokaryotes” (J. H . Parish, ed.). Blackwell, Oxford. Chopra, I., and Howe, T. G. 9. (1978). Microhiol. Reo. 42, 707-724. Cochrane, V. W. (1958). “Physiology of Fungi,” p. 308. Wilry, New York. Cocito, C. (1979). Microhiol. Ruo. 43, 145. Cohen, S . , and Shapiro, J. A. (1980). Sci. A ~ R 242, . 40.

104

VEDPAL SINGH MALIK

Collie, J. N. (1893). J . Chetn. Soc. 329. Collie, J. N. (1907). J. Chem. SOC. 1806. Chrcoran, J . W. (1974). I n “Genetics ofIndustrial Microorganisins” (2. Vanek, Z. Hostalek, and J. Cudlin, etls.) Vol. 2, pp. 339351. Academia, Prague. Crawford. I. P., and Stauffer, C . V. (1980). Annu. Rev. Biochem. 49, 163. CundlLT, E . (1978). Suture ( . h 7 d 0 7 1 ) 272, 7%. Chidlitf, E., and Tlio~npson,J. (1979). Nature ( L o d o n ) 278, 859, II’Amato, R. F., and Pisano, M . A. (1976). Antonic oun Lrezweithoek J . A4ic~obiol.Serol. 42, 299308, Davis, R . D. (1980). Science 209, 78. Davies, J , , and Srnith, D. I. (1978). Anriu Reo. ~Microl>bl.32, 468. Davics, J . , Houk, C., Yagisawa, M., and White, T. J . (1979). I n “Genetics of Industrial Microorganisms” (0. K . Sehek and A. I. Laskin, eds.). Airier. Soc. Microbial., Washington, D.C. Deniain, A. L. (1963). Clin. Med. 70, 2045-2051. Dnmain, A. L. (1966). Ado. Appl. Micwbiol. 8, 1. Deinaiii, A. L. (1968). L~oydirr31, 395418. Deinain, A . L. (1972). J . A p p l . C h e m Biotechttol. 22, 345. Demain, A. L. (1973). Ado. A p p l . Microbiol. 16, 177. Deinain, A. I,. (1974a). Ann. N . Y . Acnd. Sci. 235, 601. Demain, A. L. (1974b). Llt~ydin37, 147. Deinain, A. L. (1980). Seurch 11, 148. Deinain, A. L., antl Rirnhaum, J. (1968). Crcrr. Top. Microbiol. l m n n n o l . 46, 1. Demain, A. I , . , and Inamine, E. (1970). Bacteriol. Reo. 34, I . Demairi, A. L., and Kennel, Y. M. (1978). J. Ferment. l’echnol. 56, 323. IIt.inain, A. L., and Masurekar, P. (1974). J . Gen. Mitxohiol. 82, 143. Deniain, A . L., and Piret, J. (1979). In M . Luckner and K. Schrieber, eds.), pp. 183-188. “Regulation of Secondary Product and Plant Hormone Metabolism. Pergamon, Oxford. Demain, A. L., Newkirk, 1. F., and IIendlin, D. (1963). J. Bucteriol. 85, 339. DeMoss, H. D. (1967). In “Antibiotics. Violaccin” (D. Gottlieb and P. D. Shaw, eds.), Vol. 2, pp. 77-81. Springer-Verlag, Berlin arid New York. Ikshpaiidr, V. N . (1968). H i n d Antibiot. Bull. 11, 106-112. Dcuel, T. F., Ginsberg, A., Yeh, J., and Shelton, E., and Stadtman, E. R. (1970). J . B i d . Chern. 245, 5195. Diaz, J. C., and Clowes, R. C. (1980). J. Bacteriol. 141, 1015. Dimroth, P., Ringelmann, E., and Lynen, F. (1976). hhr. J . Biochern. 68, 591. Dowding, J . E. (19794. J. Gen. Micwbiol. 115, 385. Dnwding, J. E. (197911). F E M S M i c w b . Lett. 6, 9 5 Doy, C. I I . (1968). Reo. Pure A p p l . Chem. 18, 41. Drew, S. W., and Demain, A. L. (1975a). Antirnicroh. Agents Chemother. 8,s-10. Drew, S. W., and Dernain, A . L. (197%). Eur. J . Appl. A!icrol?iol. 2, 121. Drew, S. W.. and Demain, A. L. (1975~).J. Antibiut. 28, 889-895. Drew, S. W., antl Demain, A. L. (1977). Annu. Rev. it4icrobiol. 31, 342356. Dubois, E. M . , Grenson, A., and Wiatne, J. M . (1974). Eur. J . Biockein. 48, 603-616. Dulaney, E. 1,. (1948). J . Bucteriol. 56, 305313. Dulancy, E. L., and Dulaiiey, D. D. (1967). Truns. N . Y. A m d . Sci. 29, 782. Eble, T. (1978). J . Chroinutogr. L i b . 15, 231-271. Egorov, N . S . , Toropova, E. G . , and Suchkova, L. A. (1971). MikrolJiologiyu 40, 475. Ehrensvard, G . (1955). E x p . Cell Res. Suppl. 3, 102. Elander, K. P.. Mabe, J. A.. and Gorman, M. (1970). A p p l . Microhiol. 19, 721.

GENETICS AND BIOCHEMISTRY OF SECONDARY METABOLISM

105

Elander, R. P., Mabe, J. A., Hamill, R . L., and Gorman, M. (1971). Folia Microbiol. 16, 156-165. Elson, E. W., and Oliver, R. S . (1978). 1. Antibiot. 31, 586. Elstner, E. F., and Suhadolnik, R. J. (1971). J. Biol. Chem. 246, 6973. Elstner, E. F., and Suhadolnik, R. J. (1975). Methods Enzymol. 43, 515-520. Englesherg, E., and Wilcox, G. (1976). Annu. Rev. Genet. 8, 219-242. Ensing, J. C. (1978). Annu. Reu. Microbiol. 32, 185. Fadeeva, L. E., and Malkov, M. A. (1978). Aritibiotiki 23, 1061. Farley, J. R., Mayer, S., Chardler, C. J., and Segel, I. H . (1979). J. Bacteriol. 137, 350. Fisher, P. B. (1980). Process. Biochem. 15, 2. Flickinger, M .C., and Perlman, D. (1979). Antimicrob. Agents Chemother. 15, 282. Floss, H. G., Robbers, 1. F., and Heinstein, P. F. (1974). Rec. Adu. Phytochem. 8, 141. Fontana, R . , Canepari, P., Satta, C . , and Coyette, J. (1980). Nature (London) 287, 70-72. Foster, J. W. (1949).“Chemical Activities of Fungi,” pp. 164-169. Academic Press, New York. Foster, J. W., and Waksman, S . A. (1939). J . Bacteriol. 37, 599. Francis, M.,Cella, R., and Vining, L. C . (1975). Can. 1. Microbiol. 21, 1151. Francis, M. M., Vining, L. C., and Westlake, D. W. S . (197th). J. Bacteriol. 134, 10-16. Francis, M. M., Vining, L. C., and Westlake, D. W. S. (l978b). J. Bacteriol. 134, 10. Freeman, R. F . , and Hopwood, D. A. (1978). J. Gen. Microhiol. 106, 377. Freeman, R. F., Bibb, M. J., and Hopwood, D. A. (1977). 1. Gen. Microbiol. 98, 453. Freese, E., Heinze, J. E., and Galliers, E. M. (1979). J. Gen. Microbiol. 115, 193. Friebel, T. E., and Demain, A. L. (1977a). J. Bacteriol. 130, 1010-1016. Friebel, T. E., and Demain, A. L. (1977b). FEMS Mi~robiol.Lett. 1, 215-218. Friedrich, C. G., and Demain, A. L. (1977). 1.Antibiot. 30, 760-761. Friend, E. J., Warren, M . , and Hopwood, D. A. (1978). J. Gen. Microbiol. 106, 201-206. Froyshov, 0. (1974). FEBS Lett. 44, 75. Froyshov, 0. (1977). FEBS Lett. 81, 315318. Froyshov, O., and Laland, S. G. (1974). Eur. 1.Biochem. 46, 235. Fujikawa, K., Suzuki, T., and Kurahashi, K. (1968). Biochetn Biophys. Acta 161, 232. Fujiwara, T., Tanimoto, T., Matsumoto, K . , and Kondo, E. (1978). 1.Antibiot. 31, 966-969. Funk, A , , and Divekar, P. V. (1959). Can. J . MicrohioZ. 5, 317321. Fynn, G. H., and Davison, A. C . (1976). 1. Gen. Microbiol. 94, 68-74. Gallo, B. J. (1979). Int. Symp. Genet. Ind. Microorg. 3rd, Madison, Wisconsin 1978 Abstr. No. 35. Gallo, M., and Katz, E. (1972). J. Bacteriol. 109, 659467. Garcia, E., Rancruft, S. Rhee, S. G., and Kustu, S. (1977). Proc. Natl. Acad. Sci. U.S.A. 74, 1662. Gatenbeck, S., and Hermodsson, S. (1965). Acta Chem. Scand. 19, 65. Gatenbeck, S., and Sjoland, S. (1964). Biochim. Biophys. Acta 93, 246. Gaworowska, M. J., Emilianowicz, C. S., Switalski, L., and Raczynska, B. K. (1975).Antimicrob. Agents Chemother. 8, 526. Gersch, D. (1980). Process. Biochem 15, 21. Gerscli, D., Romer, W., Bocker, H., and Thrum, J . (1978). FEMS Microbiol. Lett. 3, 39. Gersch, D., Skurk, A . , and Romer, W. (1979). Arch. Microbiol. 121, 91. Ghisalha, O., and Nuesch, J. (1978a). J. Antibiot. 31, 215. Ghisalba, O., and Nuesch, J. (1978b). 1. Antibiot. 31, 202. Gibson, F., and Pittard, J. (1968). Bacterial. Reu. 32, 465. Gifiorn, F . , and Goltschalk, G. (1975). J. Bacteriol. 124, 1052. Ginther, C. L. (1979). Antimicrob. Agents Cheniother. 15, 522. Godfrey, J. C., and Price, K. E. (1972). Adu. Appl. Microbiol. 15, 231.

106

VEDPAL SINGH MALIK

Godfrey, 0.(1973). Antitriimoh. Agents Cheinothw. 4, 73. Goldberg, N . D., and IIaddox, M. K. (1977). Annu. Reo. Biocheiti. 46, 823396. Goldhcrger, R. F., etl. (1979). “Biological Regulation and Development,” Vol. 1. Plenum, New York. Gordee, E., and Day, L. E. (1972). Antiinicwh. Agents Chemother. 1, 315. Gordee, E. Z., and Day, L. E. (1972). Antiinicrob. Agents Cheinother. 1, 315. Gorisch, 11.. and Ligens, F. (1972). Arch. h4ikrobiol. 82, 147. Gorisch, H . , a i d Lingelis, F. (1974). Biochemistry 13, 3790. Gorman, M., and Huber, F. M. (1979). Annu. Rep. Ferment. Proc. 2, 203. Gottlieh, S. (1976). J. Antikiot. 29, 987. Goulden, S. A., and Chattaway, F. W. (1968). Biocheiii. J. 110, 550. Gouldcn, S. A . , and Chattaway, F. W. (1969). /. Cen. Mic7ubio1. 59, 111. Gourevitch, A., Pursiano, T. A., and Lein, 1. A. (1961). Arch. Biochetn. Biophys. 93, 283. Grafe, U., Bocker, H., Reinhardt, G., Tkocz, I I . , and Thrum, H. (1974). Z. M g . Mikrohiol. 14, 181. Grafe, U . , Bocker, H . , and Thrum, H. (1977). 2. Allg. Mikrobiol. 17, 201. Grafe, Y.. Bocker, H . , Reinhardt, G . , and Thrum, H. (1974). Z. Allg. Mikrobiol. 14, 659. Graham, M . Y., and Weisbluirr, B. (1979). J . Bucteriol. 137, 1464. Greer, H . , and Fink, C . R. (1979). Proc. N a t l . Acad. Sci. U . S . A . 76, 4006. Gregory, K. F., and Huang, J . C . C. (1964). J. Bacterial. 37, 1287. Grnotwasaink, 1. W., and Gaucher, G. M. (1980). J. Bacteriol. 141, 443. Haavik, H . I. (1974). J . Cen. Microbiol. 84, 321-326. Haavik, H.1. (1979). Folia Mitrobiol. 24, 365. Hachimori, A . , h4atsunanga, A , , Shirnizu, M., Samejirna, T., and Nosoh, Y. (1974). Biochim. Biophys. Acta 350, 461. Hayakawa, T., Tanaka, T., Sakaguchi, K., Otake, N., and Yonehara, H . (1979). J . Gen. Appl. Microhiol. 25, 255. Helser, T. E., Davies, J , E . , and Dahlhcrg, J. E. (1971). Nuture (London)New B i d . 233, 12. Herrdlin, D. (1949). Arch. Biochem. Bioph!ls. 24, 435. IIinnen, A,, and Nuesch, J. (1975). Antimicrub. Agents Chemother. 9, 824. Hitchcock, M. J. M . , and Katz, E. (1978). Antimimob. Agents Chernother. 13, 104-114. Hodgson, B. (1970). J . Thew. B i d . 30, 111-119. Hodgson, D. A . (1980). Ph.D. Thesis, University of East Anglia, England. Iioeksema, H., and Krueger, W. C. (1976). J. Antibiot. 29, 704-709. Hoeksema, H., and Smith, C. G. (1961). Prog. I t i d . Microhiol. 3, 91-139. Hoeksema, H . , Mizsak, S . , and Baczynsky, J. (1979). J. Aiztihiot. 22, 773-776. Holtje, J. V. (1978). Eur. J , Biochein. 86, 345. Holtje, J. V. (1979). Antimimob. Agents Cheniotht-r. 15, 177. Holzer, H . , and IIeinrich, P. C . (1980). Annu. Rev. Biochem. 49, 63. Hook, D. J , , Chang, L. T., Elander, R. P., and Morin, R. B. (1979). Biochem. Biophys. Res. Conmun. 87, 258. Hopwood, U. A. (1978). Annu. Rel;. Microbiol. 32, 373392. Hopwood, D. A. (1980). Int. S y n p . Aclinomycete Biol. Hopwood, D. A. (1980b). I n “Antibiotics of the Future” (L. Ninet, ed.). Academic Press, New York. IIopwood, D. A., and Chater, K. F. (1980). Philos. Trunu. R . Sac. London Ser. B 290, 313. Hopwood, D. A , , and Merrick, M. J. (1977). Bacteriol. Reo. 41, 595-635. Horinouchi, S., and Weishlum, B. (1980). Proc. Natl. Acad. Sci. U . S . A . 92. Horner, W. H . (1964). Proc. Natl. Acad. Sci. U . S . A . 67, 305.

GENETICS AND BIOCHEMISTRY OF SECONDARY METABOLISM

107

Hostalek, Z. (1964a). Folia Microbiol. 9, 78. Hostalek, 2. (1964b). Folio Microhiol. 9, 89. Hostalek, Z. (1Wc). Folia M i ~ r o b i o l .9, %. Hostalek, Z. (1979). Folia Mimobiol. 25, 445. Hostalek, Z., Tinterova, M . , Jechova, V., Blumauerova, M., Suchy, S., and Vanek, Z. (1969). Biotechnol. Bioeng. 11, 539. Hostalek, Z . , Tobek, I., Bobyk, M. A., and Kulayev, I. S. (1976). Folia Microhiol. 21, 131. Hostalek, Z., Blumauerova, M . , and Vanek, Z. (1979). Econ. Microbiol. 3, 293. Hotta, K., Okami, Y., and Umezawa, H. (1977). J . Antibiot. 30, 1146. Howells, J. D., Anderson, L. E., Coffey, G. L., Senos, 6. D., Underhil, M. A., Vogler, D. L., and Erlich, J. (19’72). Antirnicrob. Agents Chemother. 2, 79. Huang, M . , and Gibson, F. (1970). J. Bacteriol. 102, 767. Hughes, P. F., Troost, T., and Katz, E. (1977). Abstr. Annu. Meet. Am. Sac. Mi~robiol.p. 253. Hurley, L. J., and Bialek, D. (1974). J . Antihiot. 27, 49. Hurst, A. (1966). J . Cen. Microbiol. 45, 503. Hutchison, K. W., and Hanson, R. S. (1974). J. Bacteriol. 119, 70-75. Hutter, R . , Kieser, T., Crameri, R . , and Hintermann, G. (1979). Proc. Int. S!ynp. Actinoniycete B i d . Cologne. Imsande, J. (1978). Microbiol. Rev. 42, 67-83. Inamine, E., Lago, B. D., and Demain, A. L. (1969). In “Fermentation Advances” (D. Perlman, ed.), pp. 199-221. Academic Press Inc., New York. Ishihara, H . , Endo, Y . , Abe, S., and Shimura, K. (1975). FEBS Lett. 50, 4346. Ito, M . , Aida, J . , and Uemura, T. (1969). Agric. B i d . Chem. 33, 262-269. Ito, M., Aida, K., and Vermura, T. (1970). Proc. Int. Congr. Chemother. 6th p. 1128. Janglova, Z., Suchy, J . , and Vanek, Z. (1969). Folia Microbiot. 14, 208. Jarvis, F. G., and Johnson, M. J. (1947). J . Am. Chern. Soc. 69, 3010. Jensen, R. A,, and Nasser, D. S. (1968). J. Bacteriol. 95, 188. Johnson, M. J. (1952). Bull. WHO 6, 99-121. Johnston, H. M., Barnes, W. M . , Chumley, F. G., Bossi, L., andRoth, J . R. (1980). Proc. h’atl. Acad. Sci. U . S . A . 77, 508. Jones, A , , and Vining, L. D. (1976). Can. J . Mimohiol. 22, 237-244. Jones, A., and Westlake, D. W. S. (1974). Can. J . Microbial. 20, 1599. Jones, A., Francis, M., Vining, L. C., and Westlake, D. W. S. (1978). Can. J . Microbiol. 24, 238. Jones, G. H . (1975). Biochem. Biophys. Res. Commun. 63, 469. Jones, G. H. (1979). Arch. Biochem. Biophys. 198, 195. Jones, G. H., and Weissbach, J. (1970). Arch. Biochem. Biophys. 137, 558-573. Kahan, J. S., Kahan, F. M . , Goegelman, R., Currie, S. A., Jackson, M . , Stapley, E. O., Miller, T. W. Hendlin, D., Mochales, S., Hernandez, S., Woodruff, H. B . , and Birnbaum, J . (1979). J . Antibiot. 32, 1. Kalakoutskii, L. V., and Agre, N . S . (1976). Bacteriol. Rev. 40, 469. Kane, J., and Jensen, R. A. (1970). J . Biol. Chem. 245, 2384. Kane, J. F. (1975). Arch. Biochem. Biophys. 170, 452. Kane, J. F., Holmes, H. W., and Jenson, R. H. (1972). J . B i d . Chern. 247, 1587. Katz, E. (1967). In “Antibiotics” (D. Gottlieb and P. D. Shaw, eds.), Springer-Verlag, Vol. 2. Berlin and New York. Katz, E. (1968). Lloydia 31, 364. Katz, E., and Demain, A. L. (1977). Bacteriol. REV. 41, 449474. Katz, E., and Weissbach, H. (1963). J . Biol. Chem. 238, 666-675.

108

VEDPAL SINGH MALIK

Kauffman, G. B. (1979). J. Chenz. Ecluc. 56, 454. Kawaguchi, A,, Tomodo, H., Okudo, S . , Awaya, J . , and Omura, S . (1979). Arch. Biochem. Biopluls. 197, 30. Keilman, G. R . , Burtis, K., Tanimoto, B., and Doi, R. 11. (1976). J. Bacteriol. 128, 8 0 4 5 . Keller, E. B., and Calvo, J . M. (1979). Proc. Natl. Acud. Sci. U . S . A . 76, 6186. Kennel, Y. M., and Deinain, A. L. (1978). E x p . Mycd. 2, 234-238. Khokhlov, A. S., and Tovarova, I. I. (1972). Postepy Hig. Med. Dosw. 26, 469491. Khokhlov, A. S., and Tovarova, I. I. (1979). In “Regulation of Secondary Product and Plant Hormone Metabolism” (M. Luckner and K. Schreiber, eds.), pp. 133-145. Pergainon, Oxford. Khokhlov, A. S . , Anisova, L. N., Tovarova, I. I . , Kleiner, E. M., Kovalenka, I. V., Krasilnikova, 0. I., Kornitskaja, E. Y., and Pliner, S.A. (1973). Z. Allg. Mikrobiol. 13,647455. Kibby, J . , McDonald, L. A , , and Richards, R. (1980). J . Chem. Soc. Cherra. Cornin. 768. Kida, M., Asako, T., Yoneda, M . , and Mitsuhashi, S. (1975). In “Microhiol Drug Resistance” (S. Mitsuhashi and H. Hasimoto, eds.). Univ. Park Prcss, Baltimore, Maryland. Kieser, T., Hinterinan, G., Crameri, R., and Hutter, R. (1979). Proc. Znt. Symp. Actinoitiycefe HiO/.

C<JlUgnL..

Kimura, A. (1967). Agric. B i d . Chrm. 31, 845-852. Kirby, J. P. (19880). Processs.Bioclicm. 15(7), 14. l . 3, 283. Kirby, R . (1978). F E M S M i ~ ~ o h i oLett. Kirsch, E. J. (1967). I n “Antibiotics” (D. Gottlieh and P. U . Shaw, eds.). Vol. 2. SpringerVerlag, Berlin and New York. Klein, H. L., and Welch, S. K. (1980). Nucleic Acids Hes. 8, 4651. Klciner, E. M., Pliner, S. A., Soifer, V. S . , Onoprienko, V. V., Balashova, T. A . , Hozynov, B. V., and Khokhlov, A. S. (1976). Bioorg. Khirn. 2, 1142-1147. Kleinkauf, H . , and Gcvers, W. (1969). Cold Spring Harbw S!/irap. Quant. B i d . 34, 805-813. Kleinkauf: H . , and Koischwitz, H. (1974). I n “Liptnann Symposiun: Energy, Biosynthesis and Regulation in Molecular Biology,” pp. 336344. De Gruyter, New York. o l 600. . Klucpfel, D., Laiicini, G. C., and Sartori, G . (1965). Appl. M i ~ ~ ~ b i13, Kniep, B., and Grisebach, H. (1976). FEBS Lett. 65, 44. Kniep, B., and Grisehach, H. (1980). .I. Antihiot. 33, 416. Koch, G . , and Richter, D., eds. (1979). “Regulation of Macromolecular Synthesis by Low Molecular Wright Mediators.” Academic Press, New York. Kogt*kai-,R . C . , and Deshpande, V. N . (1979). Biochinz. Biophys. Acta, 133, 37. Kojima, M., Yainada, Y . , and Vrnezawa, 1%.(1969). Agric. B i d . Cherrt. 33, 1181. Komatsn, K., and Kodaira. R. (1977). J. Antihiot.‘30, 226. Kominek, L. A. (1972). Antiiiiicrob. Agents C h i o t h e r . 1, 123. Kominrk, L. A. (1975). Antimicrob. Agerrt.9 Cheinother. 7, 861. Kralovcova, E., Blumauerova, M., and Vanek, Z. (1977). Folio Microbial. 22, 182. Krassihiikov, N. A. (1958). Acutl. Sci. USSR (Moscow) p. 365. Krrhs, G., a d Beavo, J. A. (1979). Annu. net.. Biocheiu. 48, 923. Krupinsky, V. M . , Robbers, J. E . , and Floss, H. G. (1976). J . Bacteriol. 125, 158. Kuruhashi, K . , Yainada, M., Mori, K., Fujikawa, K., Kainbe, M . . Imae, E., Takahashi, II., and Sakamoto, Y. (1969). Cold Spring Harhor S!ynap. Quunt. B i d . 34, 815. Kurylo-Borowsky, Z. (1967). Z I L “Antibiotics” (D. Gottlieh and P. D. Shaw, eds.), Vol. 2, pp. 342352. Springer-Verlag, Berlin and New York. Kurylowicz, W., and Kowszyk-Gindifer, Z. (1979). Ifind. Antibiot. Bull. 21, 115. Lagosky, P. A., a i d Chany, F. N. (1980). J . Bacteriol. 144, 499. Laland, S. G., and Zirnincr, T. L. (1973). I.:,wuys Biochrin. 9, 31-57. Lancini, 6. C., and White, R. C. (1973). Process. Biochcm. 8, 14-16.

GENETICS AND BIOCHEMISTRY OF SECONDARY METABOLISM

109

Laskowski, M., and Kato, I. (1980). Annu. Reo. Biochem. 49, 593. Lawrence, J., Cox, G. B., and Gibson, F. (1974). J . Bacteriol. 118, 41. Lee, S. G., Littau, V., and Lipmann, F. (1975). /. Cell B i d . 66, 238. Lemke, P. A , , and Nash, C. H . (1972). Can. J . Microbiol. 18, 255. Liddel, J. M., Shaw, W. V . , and Swan, I. D. A. (1978). J . Mot. Biol. 124, 285. Lipmann, F. (1971). Science 173, 875-884. Liras, P., Villanueva, J. R., and Martin, J. F. (1977). J . Gen. Microhiol. 102, 269. Lisivinova, S. N., Dmitrieva, S. V., Zaslavskaya, P. L., and Orlova, N . V. (1979). Antibiotiki 24, 888. Liu, C . M., McDaniel, L. E., and SchlafFner, C. P. (1972). J . Antibiot. 25, 116. Liu, C. M., McDaniel, L. E., and SchlafFner, C. P. (1975). Antimicrob. Agents Chemother. 7, 196-202. Liu, C. M., Hermann, T., and Miller, P. A. (1977). J . Antihiot. 30, 244-251. Liu, C., Hermann, T. E., and Miller, P. A. (1979). J . Antibiot. 32, 1025. Lockwood, L. B., and Nelson, G. E. N . (1946). Arch. Biochem. 10, 365. Lomovskaya, N. D., Chater, K . F., and Mkrtumian, N. L., (1980). Microbiol. Rev., 44, 206. Longley, R. P., Halliwell, J. E., Campbell, J. J. R., and Ingledew, W. M. (1972). Can. J . Microbiol. 18, 1357. Losick, R. (1981). In “Molecular Cloning and Gene Regulation in Bacilli.” Academic Press, New York. In press. Lowe, D. A , , and Westlake, D. W. S. (1971). Can. J . Biochem. 49, 448. Lowe, D. A , , and Westlake, D. W. S. (1972). Can. J . Biochern. 50, 1064. Luckner, M . , Nover, L., and Bohm, U . (1977). “Secondary Metabolism and Cell Differentiation.” Springer-Verlag, Berlin and New York. Luengo, J. M.,Revilla, G., Villanueva, J . R., and Martin, J. F. (1979). J . Gen. Microbid. 115, 207. Luengo, J. M., Revilla, G., Lopez, M. J., Vdlanueva, J. R., and Martin, J . F. (1980). J. Bacteriol 144, 869. Lurie, L. M., Verkhotseva, T. P., and Levitov, M . M. (1975). Antibiotiki (Moscow)20, 291. Magasanik, B., Prival, M., Brenchley, J . , Tyler, B., Deleo, A , , Streicher, S., Bender, R., and Paris, C. (1974). Cum. Top. Cell Reg. 8, 118. Maggon, K. K . , Gupta, S. K., and Vankitasubramanian, T. A. (1977). Bacteriol. Reu. 41, 822-855. Magni, G . , Pallotta, G . , Natdhni, P., Ruggieri, S., Santarelh, I., and Vita, A. (1978). J . Biol. Chem. 253, 2501. McCray, J. W., and Hermann, K. M. (1976). J . Bacteriol. 125, 608. McGuire, J. C . , Glotfelty, G., and White, R. J. (1980). FEMS Mimobiol. Lett. 9, 141. Maier, W., Erge, D., and Groger, D. (1980). Planta Mecl. 40, 104. Majumdar, M. K., and Majumdar, S. K. (1970). Biochem. J . 120, 271-278. Majumdar, M . K., and Majumdar, S. K. (1971). Fotia Microbiol. 16, 285-292. Majumdar, M. K., and Majumdar, S. K. (1972). Biochem. J . 122, 397-404. Matins, J. F., Normansell, I. D., and Holt, G . (1979). 1.Gen. Microbiol. 112, 113. Malik, V. S. (1972). Arlzj. A p p l . Microbiol. 15, 297-336. Malik, V. S. (1977). J . Antibiot. 30, 897-899. Malik, V. S . (197%). Ado. Genet. 20, 37. Malik, V. S. (1979h). Ado. Appl. Microbid. 25, 75. Malik, V. S. (1980a). Trends Biochem. Sci. March, p. 82. Malik, V. S. (1980b).Ado. Appl. Microbiol. 27, 1. Malik, V. S . (1980~).Biotechnol. Lett. 2, 455. Malik, V. S., and Reusser, F. (1979). Plusinid 2, 627.

110

VEDPAL SINGH MALIK

Malik, V. S., and Vining, L. C. (1970). Can. J . Microbiol. 16, 173. Malik, V. S . , and Vining, L. C. (1971). Can. J . Microbiol. 17, 1287-1290. Malik, V. S., and Vining, L. C. (1972a). Can. J. Microbiol. 18, 137. Malik, V. S., and Vining, L. C. (1972b). Can. J. Microhiol. 18, 583. Marahiel, M. A,, Lurtz, R., and Kleinkauf, H. (1980). Sixth Znternatiorinl Ferfnetitation Symposium. F.4.3.5 P. London, Ontario, Canada. Margalith, P . , and Pagani, H. (1961). Appl. Microbiol. 9, 325. Marshall, R. Redfield, B., Katz, E . , and Weissbach, H . (1968). Arch Biochem. Biophys. 123, 317. Martin, J. F. (1976). In “Microbiology-1976.” (D. Schlessinger, ed.), pp. 548-552. Amer. Soc. for Microbiology, Washington, D.C. Martin, J. F. (1977a). A h . Biochem. Eng. 6, 105. Martin, J. F. (1977b). Annu. Rev. Mic-robiol. 31, 13. Martin, J. F. (1978). In “Antibiotics and Other Secondary Metabolites. Biosynthesis and Production” (R. Hutter, T. Leisinger, J. Nuesch, and W. Wehrli, eds.), pp. 1 9 3 7 . Academic Press, New York. Martin, J. F . , and Demain, A. L. (1976). Biochetn. Biophys. Res. Cornmun. 71, 1103-1109. Martin, J. F., and Dernain, A. L. (1977a). Can. J . Microbiol. 23, 1334. Martin, J. F., and Dernain, A. L. (1977b). J . Bacteriol. 132, 590. Martin, J. F., and Demain, A. L. (1978). In “The Filamentous Fungi” (J. E. Smith and D. R. Berry, eds.), pp. 426450. Arnold, London. Martin, J. F., and McDaniel, L. E. (1974). Deu. lnd. Miaobiol. 15, 324337. Martin, J. F., and McDaniel, L. E. (1975). Bintechnol. Bioeng. 17, 925-938. Martin, J. F., Liras, P . , and Deinain, A. L. (1978). Biochem. Biophys. Res. Conmiun. 83, 822. Martin, J . F., Gil, J. A., Naharro, G . Liras, P., and Villanueva, J . R . (1979a). In “Genetics of Industrial Microorganisms” (0.K. Sebek and A. I. Laskin, eds.), pp. 205-209. Amer. Soc. for Microbiology, Washington, D.C. Martin, J . F . , Naharro, G., Liras, P., and Villanueva, J. R. (1979b). J. Antibiot. 32, 600406. Martinelli, E . , Antonini, P., Cricchio, R . , Lancini, C . , and White, R. J. (1978).J. Antibiot. 31, 949-9,51.

Martinez-Molina, E., and Olivares, J. (1979). Can. J. Miaobiol. 25, 1108. Masamune, S., Bates, G. S., and Corcoran, J. W. (1977). Angew. Cheni. 16, 585406. Masurekar, P. S., and Dernain, A. L. (1972). Can. J. Microbiol. 18, 1045-1048. Masurekar, P. S., and Deinain, A. L. (1974a). A p p l . Mic-robiol. 28, 265. Masurekar, P . S., and Demain, A. L. (1974b). Antinticrob. Agents Chen~other.6 , 366. Matern, U.,Brillinger, G. U., and Pape, H . (1973). Arch. Mikrobiol. 88, 3 7 4 8 . Matteo, C. C., Glade, M., Tanaka, A , , Piret, J., and Deniain, A. L. (1975). Biotechnol. Bioeng. 17, 129-143. Maxon, W. D., and Chen, J. W. (1966). J . Fw-ment. Technol. 44, 255-263. Mehta, R. J., Nash, C. H . , Speth, J. L., and Oh, Y. K. (1980). Deu. lnd. Microhiol. 21, 227. Merrick, M. J. (1976). J. Gen. Miaobiol. 96, 299. Mertz, F. P., and Doolin, L. E. (1973). Can. J . Microbiol. 19, 263. Michalic, J., Czerska, W. E . , Switalski, L., and Bojanowska, K. R. (1975). Antimicrob. Agents Chetitother. 8, 526. Micheli, M. R. (1980). Microbiologicn 3, 4 3 4 8 . Mikulik, K., Karnetova, J., Quyen, N., Blurnauerova, M., Komersova, I . , and Vanek, 2. (1971). J. Antihiot. 24, 801409. Miller, A. L., and Walker, J. B. (1969). J . Bactwiol. 99, 401. Miller, A. L., and Walker, J. B. (1970~).J. Bucteriol. 104, 8. Miller, A. L., and Walker, J. B. (1970b). J . Bacteriol. 104, 8.

GENETICS AND BIOCHEMISTRY OF SECONDARY METABOLISM

111

Miller, J. H., and Reznikoff, W. S. (1978). “The Operon.” Cold Spring Harbor Lab., Cold Spring Harbor, New York. Miyamura, S., Koizumi, K., and Nakagawa, Y. (1979). J . Antibiot. 32, 1217. Muth, W. L., and Nash, C. H. (1975). A n t i r n i ~ ~ oAgents b. Chernofhw. 8,321. Nakano, H., Tomita, F., and Suzuki, T. (1974). Agric. Biol. Chem. 38, 2505. Nakano, H., Tomita, F., Yamaguchi, K., Nagashima, K., and Suzuki, T. (1977). Biotechnol. Bioeng. 29, 1009. Nakano, N. M., Ozawa, K., and Ogawara, H. (1980a). J . BactLwiol. 143, 1501. Nakano, N. M., Ozawak, K., and Ogawara, H. (1980b). FEMS Microbiol. Lett. 9, 111. Nakao, Y . , Suzuki, M., Kuno, M . , and Maejinia, K. (1973). Agric. Biol. Chem. 37, 1223. Nakatsukasa, W. M., and Mabe, J. A. (1978). J. Antibiot. 31, 805. Nara, T. (1979). Annu. Rep. Fernient. Proc. 2, 223. Nester, E. W., and Montoya, A. (1976). J . Boctwiol. 126, 699. Nester, E. W., Dale, B., Montoya, and Vold, B. (1974). Biochiin. Biophiys. Acta 361, 59. Nierlieh, D. P. (1978). Annu. Rev. Microbiol. 32, 393432. Nomi, R., and Nimi, 0. (1969). Agric. Biol. Chern. 33, 1459. Nomi, R., Nimi, O., and Kado, T. (1968). Agric. B i d . Chern. 32, 1256. Nyri, L., Lengyl, Z. L., and Erdelyi, A. (1963). J. Antihiot. Ser. A 16, 80. Ochi, K., and Katz, K. (1978). J. Antihiot. 31, 1143. Ogawara, H., and Nozaki, S. (1977). J. Antibiot. 30, 337. Ogawara, H., and Horikawa, S. (198Oa). Antimicroh. Agents Chernofher. 17, 1. Ogawara, H., and Horikawa, S. (1980b). J. Antibiot. 32, 1328-1335. Ohinan, D. E., Cryz, S. J , , and Iglewski, B. H. (1980). J . Bactwiol. 142, 836. Okazaki, H . , Oho, H . , Yainada, K., Beppu, T., and Ariina, K. (1973). Agric. B i d . Che7n. 37, 2319. Okazaki, H . , Bcppu, T., and Ariina, K . (1974). Agric. B i d . Cheni. 38, 1455-1461. Omura, S. Tanaka, H., Okada, Y . , and Marumo, H. (1976). J . Chem. Soc. C h e m Connn. 320-321. Omura, S . , Iwasaki, H., and Kitano, C. (1979). J . Antihiot. 32, 1058. Ortman, R . , Matern, Y . , Grisebach, H., Stadler, P., Siniiwell, P. V., and Paulsen, H. Dur. J. Hiochem. 43, 265. Otsuka, I I . , Quigley, F. R., Groger, D., Anderson, J. A., and Floss, H. G . (1980). Planfa A4etl. 40, 109. Oxford, A. E., Raistrick, H., and Simonart, P. (1935). Biochem. J . 29, 1102. Papc, H . , and Brillinger, G . U. (1973). Arch. Alikrohiol. 88, 25. Pass, L., and Raczynska-Bojanowska, K. (l969a). Acta Biochiin. Pol. 15, 355. Pass, L., and Raczynska-Bojanowska, K. (196%). Acta Biochini. Pol. 16, 297311. Pass, L., Styczynska, D., and Bojanowska, R. K. (1974). Acta. Biochiin. Pol. 21, 213. Pastan, I., and Adhya, S. (1976). Bacterial. Rev. 40, 527-551. Patel, N . , Pierson, D. L., and Jensen, R. A. (1974). ,/. B i d . Che7n. 252, 5839. Paulus, H. (1967). In “Antibiotics” (U. Gottlieb and P. D. Shaw, eds.), Vol. 2, pp, 254-267. Springer-Verlag, Berlin and New York. Peberdy, J. F. (1980). Enzyme Microh. Technol. 2, 23. Pensikova, G. A., and Levitov, M . M . (J970). Biologi!yu 39, 337. Petroski, R. J., and Kelleher, W. J. (1977). FEBS Lett. 83, 55-57. Piggot, P. J.. and Coote, J. 6. (1976). Bactwiol.Eel;. 40, 908-962. Pirt, S. J. (1976). Deo. Intl. Mic~ohiol.17, 1. Pirt, S. J . , and Righelato, R. C. (1967). A p p l . hlic~ohiol.15, 1284. Piwowarski, J. M . , and Shaw, P. I).(1979). Antimicrob. Agents Chetnothcr. 16, 176. Ph, L. C . (1971). Riochim. Biophys. Actu 242, ,541,

I12

VEDPAL SINGH MALIK

Pogell, B. M. (1975). Methods Enzymol. 43, 508. Polsinelli, kf., Albertinc, A,, Cassani, G ., and Ciferri. 0.(19&5).J . Cen. Mimobiol. 39, 239. Prusiner, S . , and Stadtman, E. R . (1973).“The Enzymes of Glutaminc Metabolism.” Academic Press, New York. Puc, A , , and Socic, H . (1977). Eur. J . Appl. Microhiol. 4, 283. Purich. D. L., and Frotnnr, H. J. (1973).J . R i d . Chein 248, 461. Queener. S. W., Sehek, 0. K., and Vezina, C. (1978). Annu. Heo. Alicrobiol. 32, 593. Raczynska, K. B., Ruczdjz, Z.. Rafalski, A,. and Sawnor, D. K. (1973). Antimi~roh.Agents Chcinotlrer. 3, 162. Rafalski, A . , Raczynska, K. R. (1972). Acta Biochini. Pol. 19, 71. Rafalski, A,, and Raczynska, K. B. (1973). Acta Microhid. Pol. 22, 87. Ragan, C. & I . . and Vining, L. C. (1978). Can. J . Microhiol. 24, 1012. Raistrick, H. (1931). Philos. Trans. R. Sor. London S w B 220, 1. Kaistrick, H . , and Clark, A. B. (1919). BZ(~cheiii.J . 13, 329. Ramsev, H . I I . , Hussain, S. M., and Williams, R. P. (1973). Abstr. Annu. Meet. Am. Soc. Microbial. P239, p. 180. Redshaw, P. A., McCann, P . A,, Pentdla, M. A,, and Pogel, B. M . (1979). / . Hacteriol. 137, 891. Richinonrl, M . H . , and Sykes, H. B. (1973). Ada. hfiflfJhiol. Physiol. 9. Riley, XI., antl Anilionis, A . (1978). Annu. Reo. Microbiol. 32, 519-560. Kolhers, J. E . , Robertson, L. W., Hornemarrn, K . M.,Jindra, A , , and Floss, H. C. (1972).J . nnctmioi.112, 791. Roberts, J. W. (1976). In “RNA Polymerase” (R. Losick, ed.), pp. 247-271. Chamberlin, New York. Roeder, 6. S.. and Fink, G. R. (1980). Cell 21, 239. Koetler, 6. S., Farabaugh, P. J . , Chaleff, D. T., and Fink, G . R. (1980). Science 209, 1375. Rogers, T. O., and Birnbaum, J. (1974). Antimicrob. Agents Chemotlzer. 5, 121-132. Rogolskv, M.,antl Nakamura, H. T. (1974).J . Ractcriol. 119, 57-61, Roland, I . , Froyshuv, O., Laland, S. G. (1977). F E B S Lett. 84, 22-24. Rnlinson, G. N. (1979). J . Antimicroh. Chenmther. 5(1), 7-14. Roirrano, A. H.,and Margiotta, E. (1980). In Second Confrrence on Genetics and Molecular Biolog!y of Industrial 1Microcwgunisni.s. Bloomington, Indiana. Rose, A. H. (1980). “Economic Microbiology.” Academic Press, New York. Rosenberg, M.,and Court, D. (1980). Annu. Reo. Genet. 13, 319. Rossi, A,, and Chrcoran, J. W. (1973). Biocheni. Biophys. Res. Coininun. 50, 597. Roszkowski, J. (1972).Acta Microbiol. Pol. Ser. B . 4(1), 9-22. Ruczaj, Z . , Ostrowska, K. B., Sawmor, K. D., arid Raczynska, B. K. (1972). Acta M i ~ ~ ~ b i o l . Pol. 4, 201. Rudd, H . A. M.,and Hopwood, D. A. (1979). J . Gen. Microbiol. 114, 3543. Hudd, B. A. M . , and Hopwood, D. A. (1980).J . Gen. Microbiol. 119, 333. Ruhland, W. (1958).“Encychpedid of Plant Physiology,” Vol. X. Springer-Verlag, Berlin and New York. Sadoff, H . L. (1972). I n “Spores V” (H. 0. Campbell, ed.), pp. 157-166. Ainer. Soc. for hlicrohinlogy, Washington, U.C. Saltero, F. V., and Johnson, M . J. (1953). A p p l . Microbiol. 1, 52-57. Sanchez, S., Paniagua, L.. Mateos, R. C., Lara F., and Mora, J. (1980). Int. F m i e n t . Synip., 6th Tmonto. Sankaran, L., and Pogell, B. M. (1973). Nature (London) New B i d . 245, 257-260. Sankaran. I,., and Pugell, €3. M .(1975). Antimicroh. Agents Cheniother. 8, 721.

GENETICS AND BIOCHEMISTRY OF SECONDARY METABOLISM

113

Sarkar, N . , and Pauhs, H. (1972). Nafure (London) New B i d . 239, 228-230. Sarkar, N . , Langley, D., and Paulus, H. (1977). Proc. W a d . Acad. Sci. U.S.A. 74, 1478. Satoh, A,, Ogawa, H . , and Satomura, Y. (1976). Agric. B i d . Chein. 40, 191-196. Sawada, Y., Baldwin, J. E., Singh, P. D., Solomon, N . A., and Demain, A. L. (1980a). Antiinieroh. Agents Chemother. 18, 465. Sawada, Y., Hunt, N. A . , and Demain, A. L. (l980b). J . Antibiot. 32, 1303. Sawada, Y . , Solomon, N . A,, and Demain, A. L. (1980~).Biotechnol. Lett. 2, 43. Sawai, T., Takahashi, I., and Yamagishi, S . (1980). Antimimob. Agents Chemother. 13, 910999. Sawula, R. W., and Crawford, I. P. (1973).J. B i d . Chem. 248, 3573. Schaeffer, P. (1969). Bacteriol. Reu. 33, 48-71. Scherer, S., and Davis, R. W. (1980). Science 209, 1380. Schrempf, H . , and Goebel, W. (1979). In “Plasmids of Medical Environmental and Commercial Importance” (K. N . Timmis and A. Puhler, eds.). Elsevier, Amsterdam. Schwartz, J. L., and Schwartz, S. P. (1979). Antimic~ob.Agents Chemother-. 5, 123. Seddon, B., and Fynn, G. H . (1973). J. Gen. Mimobiol. 74, 305. Secldon, B., and Mandis, S. (1978). Biochem. Soc. Trans. 6, 412. Sermonti, G., Petris, A , , Micheli, M., and Lanfaloni, L. (1977).J . Gen. Microbiol. 100, 347. Sermonti, G . , Petris, A , , Micheli, M., and Lanfaloni, L. (1978). Mol. Gen. Genet. 164, 99. Sermonti, G., Lanfaloni, L., and Micheli, M. R. (1980). Mol. Gen. Genet. 177, 453. Setlow, P. (1973). Riochem. Biophys. Res. Coininun. 52, 365372. Shaw, P. D., and Piwowarski, J. (1977).J . Anfibiut. 30, 404. Shaw, W. V. (1979). Ann. N . Y . h a d . Sci. 182, 234. Shaw, W. V., and Hopwood, D. A. (1976).J . Gen. Microbiol. 94, 159-166. Shaw, W. V., Packman, L. C., Burleigh, B. D., Dell, A . , Morris, H. R., and Hartley, B. S. (1979). Nature (London) 282, 870. Silaeva, S. A , , Glazer, V. M . , Shestakov, S. V., and Prokofiev, M. A. (1965). Biokhirniya 30, 947-955. Sims, A. P., Toone, J., and Box, V. (1974). (1974). J . Gen. Microbiol. 80, 485. Simuth, J . , Hudec, J . , Chau, H. T., Danyi, O., andzelinka, J . (1979).J . Antibiot. 32, 53-58. Sjoland, S., and Gatenbeck, S. (1966). Acta Chem. Scand. 20, 1053. Smith, C. G., and Hinman, J . W. (1963). Prog. Ind. Microbiol. 4, 137-163. Smith, R. L., Bungay, H. R., and Pittenger, R. C. (1962). Appl. Mierobiol. 10, 293. Snoke, J. E . , and Cornell, N . (1965). J. Bacteriol. 89, 415. Soltero, F. V., and Johnson, M. J. (1953). Appl. Microhiol. 1, 5257. Soltero, F. V., arid Johnson, M. J. (1954). Appl. Microbiol. 2, 41. Spalla, C., Filippini, S., and Grein, D. (1978). Folia Mimobiol. 23, 505. Spizek, J., Malek, I., Suchy, J., Voridracek, M., and Vanek, Z. (1965). Folia Microbiol. 10, 263-266. Start, W. M., Wilgus, R. M., and Dubus, R. (1980). Deu. 2nd. Microbiol. 21, 77-89. Stephens, J. C . , Artz, S. W . , and Ames, B. N. (1975). Proc. Natl. Acad. Sci. U . S . A . 72, 4389-93. Stewart, W. W. (1971). Nature (London) 229, 174. Stinchcomh, D. T., Thomas, M., Kelley, J., Selker, J., and Davis, R. W. (1980). Proc. Natl. Acad. Sci. U.S.A. 77, 4559. Stirling, I., and Elson, S. W. (1979).J . Antibiot. 32, 1125. Stoops, J. K., and Wakil, S. J. (1980). Proc. Natl. Acad. Scd. U.S.A. 77, 4544. Streicher, S. L., and Tyler, B. (1980). Proc. Natl. Acad. Sci. U.S.A. 78, 229. Struhl, K . , and Davis, R. W. (1980). J . Mol. Biol. 136, 1309.

114

VEDPAL SINGH MALIK

Struhl, K., Davis, R. W., and Fink, G. R. (l979a). Nature (London)279, 78. Struhl, K., Stinchcomb, D. T., Scherer, S., and Davis, R. W. (1979b). Proc. Natl. Accitl. Sci. U.S.A. 76, 1035. Struhl, K., Stinchcomb, D. T., and Davis, R. W. (1980). J . hlol. Biol. 136, 291. St. John, T. P., arid Davis, R. W. (1979). Cell 16, 443. Suarez, J . E., and Chatcr, K. F. (1980). Nature {London) 286, 527. Summers, A. 0. (1978). Annu. Reo. Microhiol. 32, 637. Suter, M . , Hutter, R., and Leisinger, T. (1978). I n “Genetics of Actinomycetales” (E. Freerksen, I. Tarnok, and J. E. Thumin. ed Fischer-Verlag, Stuttgart. Snznki. M . , Kuno, M., Maejima. K., and Nakao, Y. (1974). Agric. Riol. Chem. 38, 667. Swain, T. (1977). Annu. Ruu. Plant Pluysiol. 28, 479. Switzrr, R. L. (1977). Annu. Reo. Microbiol. 31, 135. Szeszak, F., and Szabo, G. (1973). Acttr B i d . Acad. Sci. Ifung. 24, 11-17. Tabcr. W. A., arid Vining, L. C. (1963). Can. J. Micrubiol. 9, 1. Tadrew, P. L., and Johnson, M. J . (1958). J . Bactwiol. 76, 400. Takeda Chemical lntlnstries Ltd. Japan (1975). Patcnt JA-110723. Takeda Chemical Industries, Ltd. Japan (1975). Patent J.S. 109680. ’rakeda, K., Aihara, K . , Furumai, T., and Ito, Y. (1979). /. Antibiot. 32, 18-28. Takeda, K., Okuno, S., Ohashi, Y., and Fufiimai, T. (197y78a). J . Antihiot. 31, 1031-1038. Takeda, K., Okuno, S., Ohashi, Y., and Fufumai, T. (197813). J . Antibiot. 31, 1039-1045. J . Antibiot. 31, 1023-1030. Takeda, K., Okuno, S., Ohashi, Y . , and Fufumai, T. (1978~). Tamnra, A., ’I‘akeda, I., Naruto, S . , and Yoshimura, Y. (1971). J . Antihiot. 24, 270. Tatum, E. L., and Gross, S. R. (1954). Proc. Natl. A c n d Sci. U . S . A . 40, 271. Tereshin, I . M. (1976). Poyene Antibiotics, Present and future. Tokyo, University of’rokyo. 144 PP. Thomas, D. Y . , and James, A. P. (1980).J . Bactevioiol. 143, 1179. Thompson, J . M., Ward, J. M., and Hopwood, 1). A. (1980). Nature (London) 286, 525. Tomasz, A. (1979). Annu. Reo. Mic-robiol. 33, 113. Toropova, E. G., Egorov, N. S., and Suchkova, L. A. (1973). Antibiotiki (Moscow) 18, 587. Treichler, H. J., Licrsch, M., Nuesch, J . , and Dobeli, 14. (1979). In “Genetics of Industrial Microorganisms” (0.K . Sebek and A. L. Laskin, eds.), pp. 97-104. Amer. Soc. for Microbiology, Washington. D.C. Tronick, S. R . , Ciardi, J. E., and Stadtman, E. R. (1973). I . Bacteriol. 115, 858. Trowsdale, J.. Shiflett, M., and Hoch, J. A. (1878). Nature (London) 272, 179-181. Tsuji, N., Kohayaski, M . , Terui, Y . , and Tori, K. (1976). Tetrahedron 32, 2207. Turnbough, C. L., j r . , and Switzer, R. L. (1975). J . Bncteriol. 121, 115. Turner, W. B. (1971). “Fungal Metabolites.” Academic Press, New York. Turner, W. B. (1973). I n “Actinomycetalcs: Characteristics and Practical Importance.” The Society for Applied Bacteriology Symp. Ser No. 2.(G. Sykes and F. A. Kinner, eds.), Academic Press, New York. Tylcr, B. (1978). Annu. Rev. Microbiol. 47, 1127-1162. Udvardy, E. N. (1980). Process. Biochem. 15, 5. Umbarger, H. E. (1978). Arrnu. Rev. Biochem. 47, 533. Umezawa, H. (1967). “Index of Antibiotics from Actinornycetes.”Univ. of Tokyo Press, Tokyo. Unowsky, J., and Hoppe, D. C. (1978). J . Antibiot. 31, 662. Vanek, Z.(1958). Folia B i d . 4, 100. Vanek, Z., and Hostalek, 2. (1972). Postep. H i g . M e d . Dows w . 26, 445. Vanek, Z., and Mukulik, K. (1978). Folia Miwobiol. 23, 309. Vanek, Z., Cudlin, J, Blumauerova, M., and Hostalek, Z. (1971). F d i u Microbiol. 16, 225. Vdsdntha, N . , and Freese, E. (1980). 1. Bacteriol. 144, 1119.

GENETICS AND BIOCHEMISTRY OF SECONDARY METABOLISM

115

Vining, L. C . , and Taber, W. A. (1979). Econ. Microbial. 3, 389. Vining, L. C. (1979). Adu. A p p l . Microbiol. 25, 147. Vining, L. C., and Wright, J. C. (1977). I n “Biosynthesis, A Specialist Periodical Report” Bd. 5, S. 240. Chemical Society, London. Vining, L. C . , Malik, V. S., and Westlake, D. W. S. (1968). Lhydia 31, 355. Vitkovic, L., and Read, E. (1880). A m . Soc. Microbiol. Annu. Meet., Miami. Vitkovic, L., and Sadoff, H . L. (1977a). J. Bacteriol. 131, 891. Vitkovic, L., and Sadoff, H . L. (1977b). J. Bacteriol. 131, 897. Wagman, G . H., and Weinstein, M. J. (1980). Annu. Rev. Microbial. 34, 537. Waksman, S. A. (1943). J. Bacteriol. 46, 299. Waksman, S. A. (1956). G . Microbiol. 2, 1. Walker, J. B. (1964). J. Biol. Chein. 239, 578-581. Walker, J. B. (1974). J. Biol. Chem. 249, 2397. Walker, J. B. (1975). Methods Enzymol. 43, 451. Walker, J. B. (1978). In “Cyclitols and phosphonositides” (W. Wells and F. Eisenberg, Jr., eds.), pp. 423438. Academic Press, New York. . 105-113. Walker, J. B. (1980). Deo. Ind. M i ~ r o b i o l21, Walker, J. B., and Hnilica, V. S. (1964). Biochim. Biophys. Acta 89, 473. Walker, J. B., and Walker, M. S. (1967). Biochemistry 6, 38213829. Walker, M. S., and Walker, J. B. (1971). J. Biol. Chem. 246, 7034. Walker, M . S., and Walker, J. B. (1964). Biochiin. Biophys. Actu 193, 201. Wallace, R. J., Vance, P., Weissfeld, A., and Martin, R. R. (1978). Antimimob. Agents Chernother. 14, 704-709. Watanabe, S., and Tanaka, K. (1976). Biochem. Biophys. Res. Cornmun. 72, 522. Wedler, F. C., and Hoffman, F. M . (1974). Biochemistry 13, 3207. Weinberg, D. (1978). FoZia Microbiol. 23,496. Weinberg, E. D. (1970). A h . Microbiol. Ph!ysiol. 4, 1. Weinberg, E. D. (1971). Perspect. Biol. Med. 14, 565. Weinberg, E. D. (1974). Deu. Ind. Microbiol. 15, 70. Weinberg, E. D., and Tonnis, S. M. (1967). Can. J . Microbiol. 13, 614-615. Weisblum, B., and Graham, M. (1979). J. Bacteriol. 137, 635. Weisblum, G., Holder, S. B., and Halling, S. M. (1979). J. Bacteriol. 138, 990. Whitaker, A. (1980). Process. Biochem. 15, 10. Wickner, W. (1980). Science 210, 861. Wieland, 0. H., Hartmann, V., and Siess, E. A. (1972). FEBS Lett. 27, 240. Wilkerson, S. G., Nash, C. H . , and Queener, S. W. (1978). Int. Symp. Genet. Ind. Micromg,, 3rd, Madison (Abstr.). Wilson, D. B. (1978). Annu. Reu. Biochem. 47, 933. Woodruff, H. B. (1966). Soc. Gen. Microbiol. Symp. 16, 22. Woodruff, H. B. (1980). Science 210, 311. Woyczikowska, B., Pass, L., Girdwoyn, M., and Raczynska, K. G. (1973). Actu Biochirn. Pol. 20, 285. Wright, M. H., and Hopwood, D. A. (1977). J. Gen. Microbiol. 102, 417. Yanagimoto, M . , and Terui, G . (197la). J . Ferment. Technol. 49, 604. Yanagimoto, M., and Terui, G . (1971b). J. Ferment. Technol. 49, 611. Zaidenzaig, Y., Fitton, J. E., Packman, L. C., and Shaw, W. V. (1979). Eur. J. Biochem. 100, 609. Zalkin, 11. (1973). Adu. Enzymol. 38, 1. Zeeck, A., Zahner, H. , and Mardin, M. (1974). Justus Liegigs Ann. Chem. 1100-1125.

This Page Intentionally Left Blank

Partition Affinity Ligand Assay (PALA): Applications in the Analysis of Haptens, Macromolecules, and Cells B o MATTIASSON, MATTS RAMSTOW,

AND

TORBJORN G. I. LING

Department of Ptcre and Applied Biochemistry, Chemical Center, University of Lund, Luncl, Sweden I. Introduction . . . . . . . .

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

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

117

iia 118 120 121 122 123

Use of Hydrophobicity in Alteration of Partition . . . . . . . . . . . . Biospecific Interactions-Alfinity Partition . . . . . . . . . . . . . . . . . Separator Molecules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Systems Studied . . . . . . . . . . . . A. Assay of Digoxin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Assay of Triiodotbyronine ( C. Interaction hetween Hydro D. Assay of Concanavalin A , . XII. Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Analysis of Staphylococcus niireus . . . . . . . . . . . . . . . . . . . . B. Assay of Saccharoinyces cweoisiae . C. Assay of Streptococcus B, . . . . . . . . . .............. XIII. Discussion . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

VIII. IX. X. XI.

124 126 126 127 127 129 129 129 133 134 138 141 145 146

I. Introduction For many years mostly old, conventional methods have been used for identifying and quantifying microorganisms (Rytel, 1979a). However, during the last decade research in the field of analytical microbiology has been focused more on the development of quick and sensitive assays (Johnston and Newson, 1977; Rytel, 1979b). These methods have been based either on the metabolism of the living organism, the surface properties of intact cells, or even loose fragments of the cell surface. Cell metabolism has usually been followed by gas chromatographic investigation of metabolites (Phillips et al., 1977; Coloe, 1977) or by measurements of conductivity (Kahn et al., 1977). Cell surface properties have been used for immunological quantification of intact cells (Kronvall, 1973; Edwards and Larsson, 1974) as well as of loose fragments of structures (Pickering, 1976; Coonrod and Rytel, 1973). A wide variety of immunological methods have been developed for quantifying microorganisms (Rytel, 1979b). Most of these are based upon equilibrium oonditions when binding of the antibody to the cell takes place. 117 A U V A N C E S IN APPLIED MICROBIOLOGY. VOLUME 28 Copyright @ 1982 by Acadrmic Pre\i, Inc. All rights of reprorlnrtion in airy form reserved. ISBN 0-12-002628-7

118

BO MATTIASSON E T AL.

This chapter concerns a newcomer to the aforementioned methods. The method-partition affinity ligand assay (PALA)-is normally used as a nonequilibrium method, i.e., the binding reaction does not have to reach equilibrium. Furthermore, it might be possible to quantify whole cells, cell fragments, and soluble structures in one and the same assay.

II. Phase Partitioning

If aqueous solutions of different water soluble polymers are mixed with each other, the mixture will often be turbid, and when left for a while, phase separation will occur. This phenomenon has long been known (Beijernek, 1896; Albertsson, 1958) and reportedly occurs in mixtures of polymers not too closely related. In the last 10-20 years, such aqueous two-phase systems have been used in biochemical preparative work and in the investigation of surface properties of cell subpopulations. Thus, it is also known that biomolecules separate in different ways in such phase systems. These differences have been utilized in the purification of a number of different macromolecules, such as proteins and nucleic acids (Albertsson, 1971). It is also possible to separate cells according to their surface properties (Albertsson, 1977a). Thus, young and old red blood cells can be separated in aqueous two-phase systems (Walter and Selby, 1966), and similar results have been obtained when studying the surface properties of different mutants of Salmonella typhimurium (Stendahl et al., 1977). As discussed later in this chapter, the systems hitherto used in preparative work usually separate rather slowly into distinct phases. If an analytical procedure is to be based on phase separation, it must be accurate, reproducible, and preferably also quick. Another desirable characteristic of such a phase system is that the phase components should not negatively influence the molecular interactions between cell surface structures and antibodies. Many mild two-phase systems permitting biospecific binding are available (Winlund and Chamberlain, 1971). Also, enzyme-catalyzed reactions in polymer solutions have been described (Laurent, 1971; Mattiasson et al., 1974; IIGgerdahl et al., 1981). Furthermore, such phase systems have proved to be an attractive alternative as a fermentation medium, especially for the growth of cells with poor growth properties and for those producing toxins (Puziss and Heden, 1965). It therefore appeared reasonable to expect that the phase components would not have a negative influence on binding assays.

Ill. The Principleof PartitionAffinity Ligand Assay (PAM) A requirement for a binding assay in aqueous two-phase systems is that the reactants in the binding reaction have markedly different partition coefi-

119

PARTITION AFFINITY LIGAND ASSAY

cients (Mattiasson, 1980a; Mattiasson and Ling, 1980). Otherwise the separation may not be complete enough for analytical purposes. In the absence of such an asymmetric phase distribution, one of the reactants must be modified in order to achieve the desired separation. As pointed out previously, the separation must be quick and as complete as possible. The advent of modified “separator molecules” has made it possible to design binding assays also with reactant pairs that spontaneously move to the same phase (Mattiasson and Ling, 1980). Furthermore, the PALA principle includes incubation of the reaction mixture and subsequent separation with an added phase system, both steps being carried out in a single test tube. No separate washing steps, centrifugations, etc. are needed. In a direct binding assay, the binding between two different molecules is utilized to quantify one of them. One of the molecular species is labeled, usually with a radioactive isotope or with an enzyme. After binding has taken place, a well-mixed phase system is added and the reaction mixture is separated into two phases and the distribution of the label is measured. Furthermore, the two end points of the dose-response curve can be determined by measuring one sample containing labeled ligand and no antibody and one with labeled ligand and excess antibody. A binding assay is best when the two reactants distribute almost completely to different phases. Native molecules rarely do so. In practice, however, it is usually sufficient if only one of the reactants is clearly separated off in the phase system. In competitive binding assays, a native ligand to be quantified competes with a fixed amount of labeled ligand for the binding sites on the structure

t

I]

FIG. 1. Schematic diagram of a competitive binding assay between a limiting amount of laheled protein (or ligand), I, , and modified, 0 , and inirnodified, 0 , cells, respectively. (a)The free laheled protein partitions to the bottom phase. (b) The cells with covalently attached PEG-molecules, the separator cells, partition to the top phase. The labeled protein bound to the cells is thereby transported to the top phase. (c) Unmodified cells partition to the bottom phase. Sirice competition takes place in the incubation mixture, a shift in distribution of the labeled proteins occurs 011 addition of unmodified cells.

120

BO MATTIASSON ET AL.

with binding properties. The labeled ligand and the structure with binding properties must have quite different partition coefficients to secure a sufficiently wide range in the partitioning of the label. Proper partition of the labeled ligand can often be achieved by choosing an appropriate phase system, whereas the binding structure often has to be modified to secure such partitioning. The partitioning of the native ligand, when it is different from that of the labeled one, is often less important. A schematic diagram ofa competitive binding assay is given in Fig. 1. The labeled ligand is found almost exclusively in the bottom phase (Fig. la). When the modified binding protein, which on partition travels in the opposite direction, is added to the system, the complex-bound labeled ligand ascends to the top phase (Fig. 111).The presence of native proteins competing with the modified proteins for the binding sites thereby reduces the amount of labeled ligand in the top phase (Fig. lc).

IV. Phase Systems It has been shown that sufficiently concentrated aqueous solutions of some water-soluble polymers forming phase systems can be used for selective distribution of inacromolecules (Albertsson, 1971). Such phase systems have a top phase rich in one of the polymers and a bottom phase rich in the other. Compared to phase systems generally used in chemistry, i.e., those containing organic solvents, these water-rich systems have been shown to be better for separating biological material. Aqueous polymer phase systems are very mild and can be made to resemble physiological conditions more closely by the addition of different low-molecular-weight substances, such as salts and sugars. When partitioning complex and fragile structures such as cells and cell organelles, no destructive effects are demonstrable, but a stabilization of the partitioned material is observed. Soluble substances are distributed mainly between the two bulk phases of an aqueous two-phase system, whereas partitioning of particles, such as cell organelles and cells, often results in a partition both to the interface and one of the bulk phases. The distribution of biotnolecules in two-phase systems is characterized by the partition coefficient, Kpart, defined as the ratio of the concentrations in the top and bottom phase, respectively: Kwrt = Ctop/ChuLLom

( 1)

The distribution of a substance in a phase system is dependent on several parameters, such as the properties of the phase constituents and of the substance to be partitioned. A typical phase diagram of a two-phase system is shown in Fig. 2. The curved line in this diagram is called the binodial curve. Mixtures of polymers

PARTITION AFFINITY LIGAND ASSAY

121

FIG.2. Mixtures of two water-soluble polyiners, P and Q, respectively, represented by points above the binodial, such 3s point D, give hvo liquid phases, whereas mixtures below the binodial, such as point C , result in a homogeneous solution. The two binotlials shown differ in molecular weight in such a way that MW(2) > MW(1).

Polymer P (%)

in concentrations above the binodial curve, e.g., point D, result in immiscible phase systems, whereas polymer mixtures below the binodial curve, e.g., point C, give a homogeneous solution. Differences in molecular weight of the phase components can markedly influence the separating capacity of the phase system. Figure 2 shows the difference between the binodials for systems of the same type of polymers but with different molecular weights. It is seen that a lower concentration is sufficient for phase separation when using polymers of a higher molecular weight. The time required for phase separation varies from phase system to phase system. The phase systems used for analysis with the PALA method should give quick separation. This review concerns the poly(ethy1ene glycol)-salt system in which separation takes 5-15 minutes, and the poly(ethy1ene glycol)-dextran system in which separation takes 5-60 minutes. Depending on which one of the phases is to be used for the readout from the analytical procedure, it is possible to obtain the phase used in a clear state before the other phase has settled. This is secured by varying the ratio between the volumes of the two phases since the smaller phase is clarified more quickly than the larger one.

V. Modification of Partition Biological macromolecules, cell organelles, and cells distribute in aqueous polymer phase systems according to their surface properties. This distribution can often be changed by manipulation of certain parameters of the phase system. This section concerns methods for markedly increasing the effectiveness of the separation by addition of different low-molecular-weight sub-

BO MATTIASSON ET AL.

122

stances, polymers with covalently attached ligands (for affinity partitioning), and finally, the heavily modified separator molecules. The partition coeEcient of a soluble biomolecule expresses the partitioning in a phase system and can be ascribed to contributions by several factors: In K,,,,

= In

K,I

+ In K

h.,dPoPhobl(

+ In

KhYdruphlljr

+ In K,onf~,rmstlun + ...

(2)

where &I, Khydrophobic. Khydrophillc, and Kconformation denote partition coeficient factors due to electrical, hydrophobic, hydrophilic, and conformational effects, respectively (Albertsson, 1977b). In the case of partitioning cells and particles, the situation is more complicated because of the possibility of adsorption to the interface.

VI. Influence of Salt on Partition A phase system can be rendered isotonic and buffered in order to secure a more suitable environment for the substance to be partitioned. This can be done by the addition of salts, sugars, and various other organic and inorganic compounds. Such low-molecular-weight substances are generally distributed evenly in the phase system, i.e., the K,,,, is around 1. However, some of these low-molecular-weight substances do not obey this rule and an uneven distribution in the system results. If the ions of a salt are distributed unevenly in a system, there will be an electrical potential between the phases. This phenomenon can drastically alter the partition of macromolecules and cells (Albertsson, 1965). The partition coefficient of a protein can mathematically be described by the equation (Albertsson, 197713) In K:,,, =

KiBPt+ ( F Z / R T )A U

(3)

where AU is the interfacial potential, F is Faraday's constant, Z is the net charge of the protein, R is the gas constant, 1' is the absolute temperature, and K;,rt is the partition coefficient of the protein when the interfacial potential and/or the net charge of the protein is zero, i.e., at the isoelectric point of the protein. When introducing a salt with the ions Z + and Z-, respectively, which distribute unevenly in a phase system, an interfacial potential AU arises, which is given by the equation AU

=

[ R T / ( Z + + Z - ) F ] In ( K - / K + )

(4)

where K + and K - are the partition coeff'icients of the salt ions in the absence of interfacial potential. The interfacial potential, AU, increases with increasing K-IK' ratio, which means that a salt with two ions with a large difference

PARTITION AFFINITY LIGAND ASSAY

123

in affinity for the two phases will create a larger potential difference than a salt with ions with fairly similar affinity for the two phases. On addition of salt to such a system, 0.1 up to 0.5 M , the interfacial potential created by the net charge of the protein is masked, resulting in partition of the protein according to the interfacial potential created by the addition of the salt. The same principle may be applicable for the partition of cells and organelles carrying a large number of charges per particle. A pattern of the change occurring in the partition coefficients on addition of different salts to a phase system has been shown to exist. Thus, addition of potassium halogenides decreases the partition coefficient of a negatively charged protein in the following order (Albertsson, 1971): F- < C1- < Br- < I -

and the alkaline chlorides in the following order: Li+ < NH,' < Na+ = Cs+ < K'

When using ammonium and sodium ions, the difference in effect is the same whether the anion is chloride, nitrate, or sulfate, i.e., the difference in effect of the ions does not depend on the other ions present.

VII. Modification of Partition by Addition of Charged Polymers An alternative approach to increase both the selectivity and the sensitivity of phase separation is to add polymers bearing charged groups to the phase system. Charged polymers have been synthesized by substitution of the hydroxyl groups of poly(ethy1ene glycol) with positively and negatively charged groups, respectively (Johansson, 1970). In this way, bistrimethylamino-poly(ethy1ene glycol) (TMA-PEG) and poly(ethy1ene glyco1)sulfonate (PEG-S) have been prepared. These charged polymers preferentially pass to the PEG-rich top phase of a PEG-dextran system resulting in an uneven distribution of charges in the phase system. Even when the concentration of charged polymers is only 1% of the total amount of polymers in the system, it causes a drastic change in the partition. In such a system, positively charged structures will be attracted to the negatively charged PEG-S and therefore pass to the PEG-rich top phase, but when TMA-PEG is used instead, it will partition to the dextran-rich bottom phase because of the repulsive force between the charges. As for negatively charged structures, the situation is reversed. As seen in Fig. 3, rather low concentrations of charged polymers are sufficient to bring about a drastic alteration of the partition. A similar modification of the partition can be brought about by the addi-

124

BO MATTIASSON ET AL. 100

=

2 0

0

ap

111

t

2 E 50

x 0

b

P

B C

E 3 12.50

6.25 TMA-PEG (8)

0

6.25

12.50

PEG-S

(0

FIG. 3. Partition of two strains of Sabtwnella typhimuriutti 395 (open symbols) and M S (solid synibols) in the prcscnce of' different concentrations of TMA-PEG and S-PEG. 0, Top phase; 0, bottom phasc.. The cells were labeled with W r . (X-PEG concentrations are expressed as perPEG). (Reproduced with pcrinission from Stendahl et al., 1977.) crntage of X-PEG

+

tion of charged polymers, which causes the proteins to separate according to their isoelectric points. This is achieved by keeping the phase system at a constant charge and changing the pH of the system (Johansson et al., 1973).

VIII. Use of Hydrophobicity in Alteration of Partition When the electric effects of phase separation are inhibited by addition of an appropriate salt in the phase system, the value of In Kel in Eq. (2) be-

comes zero. It is then possible to let other factors such as hydrophobicity determine the partition pattern. The water-soluble polymers used in phase separation can be regarded as more or less hydrophobic (Albertsson, 1971). Poly(ethy1ene glycol) is more hydrophobic than dextran. This is clear from Fig. 4, where a hydrophilichydrophobic scale is given. More drastic effects of hydrophobic interactions on the partition pattern can be achieved by incorporation of small amounts of polymers modified with hydrophobic groups, such as fatty acids, or by inclusion, in the phase system, of detergents interacting with hydrophobic parts

PARTITION AFFINITY LIGAND ASSAY

Heptane

-

125

pdyprwykne glycol

-- Polyethylene glycol

Benzene -Ether

--

Acetone

--

H20

--

Salt + H20

-

--

Polyvinylalcohol

--

Methylcellulose

--

Hydroxypmpyldextran

--

Dextran

--

Carboxymethyl dextran Dextran sulfate

FIG.4. Hydrophobic ladder. To the left, a number of solvents have been selected from a spectrum of solvents with increasing hydrophobicity. Aqueous solutions of the polymers to the right are mutually immiscible, but because they all consist mainly of water they fall within a narrow part of the solvent spectrum to the left. (Reproduced with permission from Albertsson, 1971.)

0.05

0.10

Degree of substitution

(X)

FIG.5. The percentage of erythrocytes in the top phase as a function of the percentage of esterified PEG end groups. 0 , Dog erythrocytes; W, human erythrocytes. PEG was esterified with linolenic acid. (Reproduced with permission from Eriksson et al., 1976.)

126

BO MATTIASSON ET AL.

of a protein. (Johansson, 1976; Shanbhag and Axelsson, 1975; Westrin et al., 1976; Johansson and Westrin, 1978). The partition of albumin, known to interact with fatty acids, is strongly affected by the addition of palmitoyl-PEG into a PEG-dextran phase system in such a way that albumin partitions almost exclusively to the top phase of the system (Shanbhag and Johansson, 1974). Polymer systems containing saturated and unsaturated fatty acids coupled to one of the polymers have been used in the study of the surface properties of cells (Eriksson et al., 1976) and liposomes (Eriksson and Albertsson, 1978). The hydrophobic effect on the separation of particles is even more striking. Palmitoyl-PEG in concentrations down to 0.0001-0.001% have been shown to cause red blood cells to pass from the bottom to the top phase (Eriksson et al., 1976). An example of this effect is illustrated in Fig. 5.

IX. Biospecific Interactions-Affinity Partition When two interacting molecules are separated off in the form of a complex, the partition coeilicient is not identical with that of either of’ the molecules when separated alone. This observation has been utilized in the study of weak interactions between biomolecules (Backman and Johansson, 1976). The same type of biospecifc interaction has been used in a method similar to affinity chromatography. This method has been called affinity partition and is based on specific interactions between two molecules, one of which has been covalently attached to one of the phase polymers (Shanbhag and Johansson, 1974; Flanagan et al., 1976; Hubert et al., 1976). These ligand-containing polymers partition to the phase rich in the same unsubstituted polymer and thereby drag with them the molecules that interact with the ligand. This is also the case even if the interacting molecule has a high affinity for the other phase of the system.

X. Separator Molecules Polymer aqueous phase systems have proved useful for separating biomolecules, but only recently have they been applied for analytical purposes. A prerequisite for phase separation for analytical purposes is that the two reactants separate into different phases in the system. Sometimes this occurs spontaneously (Mattiasson and Eriksson, 1981; Ling and Mattiasson, 1981), and sometimes both reactants partition to one and the same phase. The desired separation can, however, be brought about by chemical modification of one of the reactants, i.e., converting it into a “separator molecule.” When the reactants separate into different phases, the complex

PARTITION AFFINITY LIGAND ASSAY

127

will presumably show a predilection for the phase to which one of the reactants separates spontaneously. If the molecular entities are similar in size and thus exert a pull of equal strength, the complex may be found at the interface. Depending on which of the phases the ligand prefers, there are two different possibilities for procuring the separator molecule. When working with systems utilizing differences in hydrophobicity such as PEG-MgSO, or PEG-dextran, the reactant to be converted to a separator molecule may either be made more hydrophobic and then be recovered from the PEG-rich top phase, or be made more hydrophilic and be recovered from the more hydrophilic bottom phase. As previously mentioned, separation may take place in phase systems based on properties other than hydrophilicityhydrophobicity. Such possibilities must be taken into account in the evaluation of the possibilities of setting up a PALA for a specific molecular species. Another factor to bear in mind when modifying molecules to change their properties is that too heavy derivation may be deleterious to the binding properties of the molecule. This may then lead to a separator molecule with good partition properties but poor binding properties. Such a situation is illustrated in Fig. 15 where the capacity of Staphylococcus aureus cells to transfer lZ5I-labeledIgG from the top to the bottom phase is plotted against the degree of derivation of IgG. Thus in order to get an efficient separator molecule, both the change in partition and decreased binding capacity must be considered.

XI. Systems Studied The PALA procedure has been used for assaying a number of biochemical species ranging from low-molecular-weight ligands via macromolecules to whole cells. There are no essential differences in the phase systems used for the different species analyzed.

A. ASSAY OF DICOXIN Digoxin was chosen as an example of a low-molecular-weight substance to be analyzed (Mattiasson, l980a). Digoxin is a cardioactive drug that is frequently used in the treatment of cardiac arrhythmia where therapeutic and toxic concentrations are very close. The molecule consists of a steroid unit and a chain consisting of a trisaccharide. In a two-phase system consisting of 7.5% (wiw) PEG-4000 and 22.5% (w/w) MgS04 -7HYOthe K,,,., for digoxin is 4.04. In an immunoassay of digoxin, the antibodies must partition to the bottom

128

BO MATTIASSON ET AL.

phase. This is also the case with native antibodies, and the preference for the bottom phase could be further enhanced by immobilizing the antibodies to very small, hydrophilic Sephadex particles. The assay was run as follows: 12sI-labeleddigoxiri and a serum sample containing digoxin were mixed in a test tube, and a limiting amount of antibodies was added. After incubation the phase system was added, the whole mixture was mixed thoroughly on a Vortex mixer for 10 seconds, and within few minutes separation occurred and a sample from one of the phases could be taken out for determination of its radioactivity. Some critical parameters of such an assay were examined in particular. The separation of free from bound digoxin was found to vary with the ratio bctween the volumes of the phases (Fig. 6). In assays where separation can be instantaneous and thus reproducible, it may be of interest to investigate the possibility of running iionequilibrium binding assays without loss of accuracy. When Sephadex-bound antidigoxin antibodies were used, binding between antigen and antibody required 15-20 minutes, whereas when free

I

1

I

1

2

3

Volume ratio

PEG

MgS04

FIG.6. Influeuct. of the volume of the top phase and the bottom phase on the separation. The amount of radioactivity in the top phase without antibodies in relation to the radioactivity in thr top phase with antibodies added, plotted as a fiinction of the composition of the phase system. (Reproduced with permission from Mattiasson, 1980a.)

PARTITION AFFINITY LIGAND ASSAY

129

antibodies were used, the corresponding time was 4 minutes. But generally speaking, shortening ofthe time allowed for binding to take place is attended by a decrease in sensitivity of the assay. The operational concentration range in the digoxin PALA was 1-8 nmolfliter. A correlation coeficient of 0.979 was found on comparison of the results of PALA with those of conventional radioimmunoassay (RIA). B. ASSAY OF TRIIODOTHYRONINE (T3) Triiodothyronine is one of the most frequently analyzed peptide hormones. In the phase system described in the previous section, it passes to the top phase with K,,,, = 10. The antibodies are recovered mainly from the bottom phase. Using an incubation time of 30 minutes before separation, the operational concentration range was 1.5-12 nmolfliter. Prolongation of the incubation to 12 hours reduced the lower limit to 0.5nmol/liter, and at the same time the reproducibility of the assay was substantially improved. A correlation coefficient of 0.981 was obtained for analysis of serum samples when comparing the PALA results with those of a conventional RIA (Mattiasson and Eriksson, 1981). C. INTERACTION BETWEEN HYDROPHOBIC MOLECULES AND ALBUMIN The interaction between defatted bovine serum albumin and a dye, Cibacron blue F3GA, was studied as a simple model system. In a phase system consisting of 13.5% (w/w) PEG-4000, 13.5% (w/w) MgSO, * 7H20, and 10 nmol/liter sodium phosphate, pH 7.00, K,,,, is 0.025for albumin and 11.4 for the dye. The quotient between the partition coefficients is 460. With the advantage of this quotient, a standard curve for albumin was constructed (Fig. 7). Since albumin can bind many different hydrophobic ligands, such as fatty acids, tryptophane, and several drugs, such ligands can be quantified in competitive binding assays, where they compete with, e.g., Cibacron blue FSGA, for the binding sites on albumin. Figure 8 shows the partitioning of the dye as a function of the amount of competing ligand added to a constant amount of albumin (Ling and Mattiasson, 1981).

D. ASSAY OF CONCANAVALIN A Concanavalin A (Con A) is a protein isolated from jack beans, Canavalia ensiLformis. It binds to methyl-a-D-mannopyranoside and a few other carbohydrates with similar structures. This lectin+arbohydrate interaction has

BO MATTIASSON ET AL. Kpart

10

5

2

1

0.5

0.2 I

I

1

I

1

I

I

0.1

0.2

0.5

1

2

5

10

mg

Albumin

FIG.7. A direct binding assay where the binding of a small ligand to a binding protein is studied. The partition coefficient for the dye, Cibachron blue FBGA, is determined for different amounts of added albumin. The phase system consisted of 13.5%(wiw) PEG-4000, 13.S% (wiw) MgSO, . 7H,O, and 10 mM sodiuin phosphate, pII 7.00.

I

,

1

5

10

I

I

I

15

20

25

Molar ratio

RB

FIG. 8. Competitive binding assay. Cibacron blue FBGA and a varying amount of SDS are mixed, and a constant amount of albumin is added. The phase system is the Same as that described in Fig. 7.

131

PARTITION AFFINITY LIGAND ASSAY

been used as a model system for enzyme immunoassay (Mattiasson and Ling, 1980).Con A was the binding protein and peroxidase was used as the labeled 1igand.This enzyme is a glycoprotein capable of interacting with Con A and it may be used as a naturally occurring ligand-label conjugate (Borrebaeck and Mattiasson, 1979). The enzyme has a partition coefficient of 0.063 in a phase system consisting of 13.5%(w/w) PEG-4000, 13.5%(w/w) MgSO, . 7H20, and 1Onmolflitersodium phosphate, pH 7.00. Native Con A has an even lower partition coefficient, Kpart= 0.031. PEG-modified Con A has a Kpartof 80 or more depending on the degree of modification, i. e., by modification K,,,, can be changed by a factor of 2500. As discussed earlier, when deriving reactants for a binding assay, the desired extreme partitioning is limited by the fact that high degrees of derivation reduce the capacity for biospecific binding (cf. Fig. 15; a similar pattern was also obtained for Con A). PEGmodified Con A used in this assay was shown to be capable of causing peroxidase to pass to the top phase. This transport could be disturbed by the presence of free carbohydrates in the binding assay. Figure 9 shows a calibration curve where free carbohydrates compete with the enzyme in the binding of PEG-modified Con A. The sensitivity is not in the same range as that for antigen-antibody reactions, but the affinity constant of Con A for carbohydrates is around lo4 liters/mol, which should be compared to 10' literdmol for antigen-antibody reactions. PALA can either be performed as described previously, or when enzyme labels are used, it can be run as a one-step procedure with all reactants, including phase system and reactants in the binding reaction and substrate for the enzyme, all mixed at the same time. Thus, binding, partitioning, and

FIG. 9. Calibration curve for methyl-aD-mannopyranoside obtained for the phase system described in Fig. 7. Carbohydrate, enzyme, and modified concanavalin A were incubated for 10 minutes in a total volume of 100 p1. The two-phase constituents were added to a final volume of 1 ml, and after being mixed on a Vortex mixer the phases were allowed to separate for 10 minutes. Then 200 pI were taken from each phase for determination of enzyme activity. Log K,,,, of the enzyme activity is plotted against the logarithm of the carbohydrate concentration. (Reproduced with permission from Mattiasson and Ling, 1980.)

a%

0.0

-

-0.5

-

I

0

10

l

l

25 50 100

I

l

l

250 5001000 pM

Methyl-aD- mannopyranoside

132

BO MATTIASSON ET AL.

the enzyme reaction take place simultaneously within the same vessel. Directly after mixing the mixture is poured into a spectrophotometer cuvette, and the cuvette is placed in a spectrDphotometer with facilities for registering the absorbance of one phase only. In the system studied here it is preferable to follow the events in the bottom phase. The course for this procedure is shown in Fig. 10. Thus, as seen in the figure, the total time needed for a readout of the assay was 10 minutes. In spectrophotometers equipped with cuvette-automatique several samples can be assayed simultaneously. Con A has also been used for quantification of a macromolecular carbohydrate, Dextran T40. In the competitive binding situation, dextran and peroxidase were competing for the sites on PEG-modified Con A. A calibration curve was obtained (Fig. 11). It is also possible to use an assay for quantifying native binding molecules in reactions where they also are used in a modified form as separator molecules. This was illustrated by the assay of native Con A in the presence of PEG-modified Con A and a common lipand, peroxidase. Since native and PEG-modified Con A have quite different partition coefficients, such quantitation can readily be performed. Figure 12 shows a calibration curve obtained. The examples so far discussed include antigenantibody interaction, lectin-carbohydrate interaction, as well as less well-defined hydrophobic interactions between ligands and serum albumin. The examples include quantification of haptens spontaneously present in either the bottom phase or in the top phase. Furthermore, it is clear from these data that macromolecules can be quantified with the use of the PALA technique.

320 0.2

-

I

FIG. 10. Course of' a system analogous to the one nsed in Fig. 9, where all re-

actants, including phase comporients and substrates, were mixed. Immediately afterwards the enzyme activity in the bottom phase was measured. The different conwntratians of methyl-a-D-mannopyranoside are indicated on the graphs (in Inn!). The initial period of decreasing ahsorhance is drie to phase separation. (Reproduced with permission from Mattiasson and Ling,

8o 0.1

4o

l6 L.

I

5 Time

,o 10

1980.) min

133

PARTITION AFFINITY LIGAND ASSAY

FIG.11. Calibration curve for determination of a macromolecular carbohydrate, Dextran T40, after competitive binding of dextran and peroxidase to the modified concanavalin A . Afterpartition had taken place, peroxidase activity in the top phase was assayed as a hnction of the dextran concentration. The enzyme scale is defined as: 070, enzyme without addition of modified concanavalin A (see Fig. la); 10010, enzyme and modified concanavalin A, but no dextran in the phase system (see Fig. lb). (Reproduced with permission &om Mattiasson and Ling, 1980.)

100 r

5 .-

80

CI

m 0

5 UI

6o 40 ““-0

0

10

20

1000 pM

40

Dextran T 4 0

XII. Cells The partition of cells in two-phase systems has been described earlier (Albertsson, 1971). In most reports, the cells partition to one of the phases, but in many cases a large fraction of the cells is enriched at the interface. Thus, when designing a phase system for analytical purposes it is important to secure efficient partition and if cells occur at the interface, to design the analysis in such a way as not to influence the results of the analyses. As has already been shown, the binding between lectins and antibodies to carbohydrates and antigens, respectively, can be utilized also in such rough separation systems as 13.5% (w/w) magnesium sulfate and 13.5% (w/w) PEG.

FIG. 12. Standard curve for determining native concariavalin A. The curve was obtained by adding a varying amount of native concanavalin A to the system consisting of 13.5% (w/w) PEG4000, 13.5% (wiw) MgSU4.7H2Oand 10 mM sodium phosphate, pH 7.00. Native and modified concanavalin A then compete for binding sites 011 peroxidase. Log K,,,, for peroxidase is plotted against the logaritlrm of concentration of native concanavalin A added. (Reproduced with permission &om Mattiasson and Ling, 1980.)

0.5

0.0

- 0.5 10

20

50

100

Native Concanavalin A

pM

134

BO MATTIASSON ET AL.

A, ANALYSISOF Staphylococcus aureus

As an initial model system, the interaction was studied between cells of Staphylococcus aureus strain Cowan I and human IgG (Mattiasson et al., 19Sla). In this case no real immunological reaction was used, but instead the biospecific interaction between protein A on the cell surface of the bacteria and the Fc region of IgG of subclasses I, 11, and IV was used. In the phase system used, PEG-MgSO, * 7 H 2 0 , the cells were recavered from the salt-rich bottom phase, i.e., the phase where most of the IgG molecules were found. To set up an assay, one of the entities had to be modified in order to secure an asymmetrical distribution. PEG modification of IgG according to procedures discussed earlier resulted in an antibody preparation recovered almost entirely from the top phase. The conditions for setting up an assay were thus established. Before being used in the assay, the PEG-derived antibodies were labeled with lZ5Iaccording to the lactoperoxidase method (Thorell and Johansson, 1971).

1 . Direct Binding Assay An analysis according to the procedure iIlustrated in Fig. 13 was set up. The amounts of bacterial cells relative to PEG-derived antibodies soon proved critical. A S . aureus cell reportedly possesses loG protein A molecules on its surface (Kronvall and Frommel, 1 9 7 0 t t h u s being able to bind an equal number of IgG molecules. Assuming that such a situation takes place, the cell will be covered by PEG-modified protein and thus partition to the top phase and the whole analytical procedure will fail. On the other hand, if the binding sites on the cell surface are in excess of the ligand, all

n

f

cil

FIG. 13. Schematic diagram of a direct binding assay between cells (or binding protein), , and laheled protein (or ligand),-, modified if necessary. (a)The free labeled protein with covalently attached PEG-molecules partitions to the bottarn phase. (h) The cells partition to thr top phase. (c) Labeled protein bound to the cells is found in the top phase.

135

PARTITION AFFINITY LIGAND ASSAY

1200

FIG. 14. Calibration curve for S. aureus obtained in a direct binding assay. The diagram shows the activity in the top phase as a function of the number of native S. aureus cells pres. ent in the sample. A constant amount, 1.2 X mol, of M-PEG-derived 1251-labeledIgG molecules was incubated for 30 minutes with a varying nuinher of native S. uureus cells. The incubatioii mixture was then allowed 30 minutes to separate in an aqueous two-phase system, as described in Fig. 7. A 200 p1 sample was taken out froin the top phase for measuring the activity. (Reproduced with perinission froin Mattiasson et u l . , 1981a.)

-

5

2

4

5

1100

8 5 .C

1000

.-2

2

8 900

it

I

I

lo6

Id

cell number

ligands (in this case the PEG-modified IgG) will bind to the cell surface and no discrimination between different amounts of cells will be possible. The calibration curve shown in Fig. 14 was obtained. It also turned out that the degree of PEG derivation of IgG molecules influenced not only the tendency of the modified antibody to persist in the PEG-rich top phase, but also, at higher degrees of substitution negatively influenced binding to protein A on the cell surface (Fig. 15). Taking these two limitations into account a direct binding assay was designed. It soon turned out that after binding to S. nureus, 1251-labeled PEG-modified IgG moved across the phase boundary to the bottom phase. As seen from the calibration curve, assays can be run only within a very narrow concentration range. Even if the assay did work, the maximum permissible ratio between the reactants would severely limit its value.

2. Competitive Binding Assnlj If a competitive binding assay is set up instead, the limits must be easier to set and its sensitivity may be higher. In this case native IgG molecules labeled with *“I and PEG-derived cells were used. The basic principle of this experimental set up was so that the labeled ligand should bind to the PEG-derived S. nureus cells and if a sample containing native S. nureus cells were added, competition for the labeled IgG molecules would take place.

136

BO MATTIASSON ET AL.

1

2

3

4

5

mg monomethoxy-PEG I 1 rng IgG

FIG. 15. Determination of the optimal degree of M-PEG-modification of IgG in a direct binding assay of S. u r w s The diagram shows the activity in the top phase as a function of mg M-PEG added per 1 mg IgG. The experimental conditions were as follows: A predetermined number of S. uureus cells were incuhated for 30 minutes with a predetermined amount of IgC molecules modified with a varying amount of M-PEG. The incubation mixture 12~I-labelcd was allowed 30 niiiiutes to separate i n an aqueous two-phase system, as described in Fig. 7. A 400-pl sample was taken from the top pliasr for measuring the activity. (Reproduced with permission from Mattiasson et n l . , llU31a.)

Addition of S. nureus would lead to a reduced number of counts in the top phase. The experimental situation is shown schematically in Fig. 1. First the amount of Iz5I-labeledIgG and PEG-derived staphylococci must be matched. This was done by plotting a titration curve when an increasing amount of labeled IgG was added to a constant number of cells. It is clear from Fig. 16 that a linear relationship was obtained with no tendency to inoles of '251-labeled IgG and saturation. In the subsequent assays 26 x 6 x lo7 PEG-derived cells were used. In the competitive assay the following reagents were mixed in the test tube: S . uurem and PEG-derived S . uureus cells, followed by addition of 12sI-labeledIgG. After a fixed period of time, usually 30 minutes, the phase system was added and separation took place. Figure 17 shows a calibration curve. This curve is far better than that obtained in the direct binding assay. The sensitivity achieved (at best lo5 cells) proved to be a kind of limit in the system studied. The IgG preparation used contained one '''1 per two IgG molecules. A value of 200 cpm requires the presence of approximately loll lZ5Iatoms. As mentioned earlier, each cell possesses approximately lo6bind-

I

I

I

20

I

I

40

I

I

60

I

80

Mol 1251-labeledIgG (

FIG. 16. Titration curve obtained by direct titration of a constant number (6 X 107) of M-PEG-modified S. attreits cells with 1251-laheledIgG. The diagram shows the activity in the top phase when increasing amounts of '"I-labeled IgG were incubated with the cells. After 30 minutes incubation the mixture was allowed to separate in an aqueous two-phase system consisting of 13.5%(w/w) PEG-4000, 13.5% (w/w) MgS0,~7M,O, and 10 mM sodium phosphate, pH 7.00. (Reproduced with permission from Mattiasson et d.,1981a.)

1000

-k &,

VI

900

c P

-

P

Q

5 .-

800

> r

2

Y

700

I

I

lo"

lo7

I

10'

Cell number

FIG. 17. Calibration curve for S. uureus cells in a competitive binding assay, using a constant amount of 1251-labeledIgG molecules. The diagram shows activity in the top phase as a function of the number of native S . aureus cells added to 6.3 X 10' M-PEG-modified S. aureus cells. mol of '251-labeledIgG for 30 minutes and thereafter The mixture was incubated with 27 x allowed to separate in an aqueous two-phase system consisting of PEG-6000 and Dextran T250. A 200-pl sample was taken from the top phase for measuring the activity. (Reproduced with permission from Mattiasson et at., 1981a.)

138

BO MATTIASSON ET AL.

ing sites for IgG. This then leads to a minimum cell count of about lo5, since this is the smallest number of cells to bind 10" labeled IgG molecules and thereby able to give 200 cpm. Further improvements of the method would be: (1) to use more heavily labeled antibodies, (2) to use an excess of labeled ligands to saturate the binding sites on the cells to be assayed, and (3) to use another type of label-preferably an enzyme. Point (I)will only marginally influence the analytical outcome. Thus, this was not tried further in these experiments. The two other points were tried on experiments with other systems, but the results of those investigations may nevertheless have implications bearing on the systems discussed here.

B. ASSAYOF Saccharomyces cwevisiae Utilizing a partition affinity ligand assay, the binding of lZ5I-labeledconcanavalin A to the surface of the yeast cell, Saccharomyces cereuisiae, was used in a study to quantify yeast cells (Mattiasson et al., 198lb). Concanavalin A is a lectin binding specifically to a-mannosides and a-glucosides. This lectin binds to the mannans present on the surface of the yeast cells. Concanavalin A binding to carbohydrates is characterized by rather low association constants, but owing to multipoint attachment, the binding is firm (Mattiasson, 1980b).

1 . Competitive Binding Assay For comparison with the previous assay, a competitive assay like that carried out with the S. aureus cells was performed. Figure 18 shows a calibration curve when 300,000 PEG-derived yeast cells were mixed with varying amounts of native yeast cells before the addition of lZ5I-labeledconcanavalin A. After 30 minutes, the phases separated and the cpm in the top phase was determined. The curve shows that 5 x 104-5 x lo5 cells can be detected. In this context it should be borne in mind that yeast cells are larger than bacteria and thus, at least theoretically, should possess more binding sites. '251-labeledCon A was titrated with varying amounts of PEG-modified yeast cells. Plotting of cpm in the bottom phase against the number of cells added gave curves exemplified in Fig. 19. The time dependence of binding was also studied, and at least 80% of the binding occurred within the first three minutes. One reason for counting the bottom phase and not the top phase in such titration is that when starting with small amounts of cells the cell surface will presumably be occupied to a large extent by Con A, and thus the tendency to pass to the top phase is reduced. In such a case, enrichment at the interface may be expected. Thus, under such circumstances, analyses of the bottom

-

1500

50

100

150

Cell number ( * lo3)

FIG. 18. Calibration curve obtained when a predetermined number of PEG-modified yeast cells (300,000)were mixed with a varying number of native yeast cells before iacubation with IZ5I-labeledconcanavalin A. Separation of free from bound ligand was achieved by partition in a phase system consisting of13.0% (wiw) PEG-4000, 13.0%(wiw) MgS04.7H,0 and 11.0%(wr'w) 0.1 M Tris-lHC1, 1 m M Ca2+, 1 m M M g 2 + , and 1 mM Mn'+, pH 7.00. Thirty minutes were allowed for phase separation after which a sample was taken from the bottom phase for measurement of the activity.

1500

E

-4 UY W

z P

2

lo00

c c 0

n W

r

1

2

3

4

Cell number ( x lo5)

FIG. 19. Titration cume obtained when a varying number of PEG-modifed yeast cells were added to a fixed amount of lZ5I-labeledconcanavalin A. The figure shows the activity found in the bottom phase versus the number of derived cells added. The effect of the incubation time was also studied. 0 , 3 minutes incubation; . , 30 minutes incubation.

140

BO MATTIASSON E T AL.

phase will give a fair measure of what has happened, whereas analysis of the top phase may only reflect a minor part of the binding that has taken place.

2 . A Two-step Competitiue Assay The assumption that exposure of the cells to an excess of '251-labeleclligarid would cause a heavier binding led us to design a two-step incubation experiment. First, the cells to be analyzed were incubated with the lZ51-labeled ligand, and after a definite period of time PEG-derived cells were added in excess in order to pick up unbound ligands. After a short time (usually 1 minute) the phase system was added. The lZ51-labeledligand bound to the PEG-derived cells was recovered from the top phase. This two-step procedure makes it possible to bind ligands more effectively to the native cells and to use derived cells in excess. In competitive assays, the relation between the native and the derived cells will influence the result of the analysis. Here, the large excess of PEG-modified cells in the second step guarantees an effective separation of bound froin free ligancl from the first incubation. Furthermore, to eliminate risks of exchange of ligands between the cell populations, the second step has to be quick. Figure 20 shows the results obtained, as counts in the top phase and the bottom phase when using 225,000 PEG-derived cells in the last step. Thus, by introducing a two-step technique, resolution can be improved by a factor of approximately 10. Still further improvement may be obtained by

000 c

FIG.20. Calibration

ln

I S Q

C U I V obtained ~

in a two-step competitive binding assay.

Native yeast cells were incubated with a 1~rt.(lcteriniiirdamount of '251-labeled wncanavalin A. Alter 30 minutes an exccss nnmher oi'PE6-derived yeast cells was addecl, followed, usually after 1 minute, by the addition of a phase system as describeti in Fig. 18. The diagram shows the activity found in the top (0)and bottom (W) phases, rcspectively, as a fiinctioii of the nuiiiber of native yeast cells.

600

400

200

12

24

36

40

60

72

Cell number ( 8 lo3)

PARTITION AFFINITY LIGAND ASSAY

141

optimizing the ratio of the reagents in the two-step incubation, but even then the sensitivity will still depend largely on the rate of decay of the radioactive isotope.

C. ASSAYOF Streptococcus B, To additionally improve the sensitivity of the assay the ligand must be labeled in such a way that the signal from the label can be amplified further. Such a label would be a marker enzyme. In an effort to quantify StreptococLXAS strain B cells in PALA, experiments were done with radioactive isotopes as well as with enzymes as markers (Ling et al., 1981).

mg monomethoxy-PEG / 1 ml cell suspension

FIG. 21. Analysis of the effect of M-PEG modification on the partitioning of Streptococcus cells, determined as the ability to transport '251-labeled antibodies to the top phase. 9OO-pl phase system consisting of 5%(wiw) PEG6000 and 7 . 5 8 (w/w) Dextran T250 was added to 100yl incubation mixture, 0.1 hl sodium phosphate, pH 7.00. The radioactivity in the top phase is plotted as a function of the amount of activated M-PEG added to a 1 ml suspension containing loRStreptococcus cells.

142

BO MATTIASSON ET AL.

1 . Competitive RIA The experimental set-up is illustrated in Fig. 1, i.e., a competitive situation between the native cells and a definite number of PEG-derived cells and a fixed, limiting number of labeled specific antistreptococci B antibodies. In the studies performed here, the phase system used consisted of poly(ethylene glycol)dextran. The degree of PEG substitution of the cells was investigated and the relation found is clear from Fig. 21. Quantification of the amount of cells necessary to transport the labeled antibodies from the bottom phase, across the phase boundary, and up to the top phase was determined. A calibration curve illustrating the counts in both the top phase and bottom phase is given in Fig. 22. With the use of the appropriate amounts, as indicated by the previous experiments, an assay of Streptococcus B gave the calibration curve shown in Fig. 23. Here the sensitivity was better than obtained with S . aureus.

14

6

61.25

125

250

500

1000

Number of PEG-derived cells ( =lo3)

FIG.22. Determination of the optimal amount of M-PEG-modified (10 mg per 1 ml cell suspension) Streptococcus cell suspension necessary to transport the labeled antihodies from the bottom phase, across the phase boundary, and up to the top phase. Various amounts of PEG-modified Streptococcus cells were incubated with a predetermined amount of '"Ilabeled antibodies and partitioned in a two-phase system consisting of PEG-6000 and Dextran T250 as described in Fig. 21. The figure shows the activity, i n a 200-4 sample, taken from the top (0)and bottom phases, respectively.

(m)

PARTITION AFFINITY LIGAND ASSAY

6oI 4Wl 0

]

25

,

,

,

50

125

250

143

Cell number ( .lo3)

FIG. 23. Calibration curve obtained for Streptococcus B, cells in a competitive binding assay using a constant amount of "51-lal~eledantibodies. The diagram shows the activity measured in the top phase after incubation, followed by partition in a phase system, as described in Fig. 21.

2 . Enzyme Iinmunoassny

A similar experiment was designed for enzyme immunoassay. When the antibodies were labeled by coupling horseradish peroxidase with the periodate method (Wilson and Nakane, 1978), an unexpected side-effect was observed. The enzyme-antibody corijugate was recovered from the top phase, whereas the unmodified streptococci were found in the bottom phase. This suggested the possibility of designing a direct binding assay. The cells and the labeled antibodies were mixed arid incubated for 60 minutes at room temperature before phase separation. The enzyme activity in the top phase was measured and a calibration curve as exemplified in Fig. 24 was obtained. In this context it must be stressed that the enzyme activity used was rather low and any calibration curve that can be obtained is due to the fact that the few enzyme molecules left in the top phase, when incubated in the proper substrate, convert many substrate molecules into products and thereby amplify the signal. In this case an isotope-based assay would not have been possible. This example thus lends further support to the contention that in

no MATTIASSON

144

ET AL.

0.50

0.45 h

f

2 4

- 0.40 1a r

’ z

\ \

0.35

C .-

z h

2 4

0.30

0.25

0.20

*t

0

’

2.5

I

50

I

I

12.5 25

1

I

1

50

125

250

Cell number ( “lo3)

FIG. 24. Direct biridiiig assay between antibodics against Strepfococcus B, cells, labeled with peroxiclase, a d nativc Streptococcus cells. The laheled antihodies partition to the top phase. whereas the Streptococcus cells are usually absorbed to the interface and in the bottom phase of a phase system consisting of 900 yl 5% (w/w) PEG-6000 and 7.5% (wiw) Dextran T250 added to 100 yL1 incubation mixture, 0.01 M Tris-HC1. and 0.09 A4 KCI, pH 7.00. After partitioning, a 2W-yI sample was taken from the top phase and mixed with 800 p1 reagent sulution to make a final concentration of 14 rnhl phenol, 0.8 mM 4-ainino-aritipyrine, 1 m M hydrogen peroxide, and 10 m M Tris-HCI, pH 7.00. The enzyme activity was measured in a spectrophotomrter at 510 niii.

many situations, enzyme immunoassays are preferable to radioimmunoassays. The sensitivity of the assay is now better than that of the competitive isotope-based assay. It is clear from the results presented previously that PALA is a powerful analytical tool in the analyses of haptens and macromolecules and perhaps especially in assays of microorganisms. It should also be stressed that an assay takes about 40-90 minutes from the time when the sample is mixed. It should be pointed out that when using enzyme-labeled reagents in the binding assay it may be possible, provided partitioning takes place in the presence of substrate for the marker enzyme, to make qualitative or even

PARTITION AFFINITY LIGAND ASSAY

145

semiquantitative measurements with the naked eye by reading the color intensity in both phases of the sample analyzed. By having two phases to compare and a constant total enzyme activity, the relative distribution between the phases can be read more accurately than by reading only one phase. This may facilitate development of PALA in such a way that it could be used also under field conditions. As discussed earlier in this chapter, a wide variety of methods for improving or modulating the partition pattern in aqueous two-phase systems are available, and it can already be predicted that some of these methods will help to improve the sensitivity of the analyses. In addition, the assays of haptens and some of the macromolecules clearly showed that addition of such a complexed and varying material as human serum did not influence the analytical outcome of the present method.

XIII. Discussion Much time and money has been spent in the development of new procedures in microbiological assays. As in the development of, e.g., enzymatic analysis, as well as radio- and enzyme immunoassays, new tests were developed first in the field of medicine. This is partly because a quick analysis of a specific sample may be of utmost importance in an acute clinical situation and also because much larger funds were available for medical research and service. This may have limited the techniques applied and definitely governed the selection of the systems analyzed. Microorganisms and viruses can, as mentioned earlier, be detected by the presence of: (1) whole living cells, (2) whole cells, (3) fragments of cells or free surface antigens, (4)antibodies against the bacterial cell antigens, and (5) bacterial exotoxins. Plate cultures are conventionally used for detecting living cells (Rytel, 1979a), but some assays based on metabolic activity have been developed (Rytel, 1979b). Fragments of cells, antigens, and antibodies are detected and quantified mainly by immunoassays. Most methods are designed to demonstrate the presence of one or at most two of the aforementioned items. For example, coagglutination requires multifunctional fragments; conventional RIA often requires soluble antigens or antibodies. The present method is not dependent on active metabolism in the cells, and it is possible to run assays of whole cells, fragments of cells, or free antigens. No comparative studies have been carried out so far to determine what the effect of fragmentation of the cells to be assayed has on the result of the analysis. However, the system has been shown to be useful in the analyses of many types of molecular species and particulate structures. The assay is very quick and sensitive and can presumably be improved. It is known from the literature that a wide variety of cells and cell frag-

146

BO MATTIASSON ET AL.

ments can be separated in aqueous two-phase systems. If this knowledge is applied to the use of separator molecules or second-separator cells in binding assays, a broad spectrum of simple and quick analytical systems useful for ready and accurate quantification of cells may be expected. ACKNOWLEDGMENTS

The authors thank Prof. P.-A. Albertsson and Dr. G. Johansson for valuable discussions. This project was supported by the National Swedish Board for Trchnical Development and Pharmacia Diagnostics AB.

REFERENCES Alhertsson, P . - k (1958). Biochim. Biophqs. Acta 27, 378395. Albertsson, P.-& (1965). Biochini. Biophys. Acta 103, 1-12. Albertsson, P.-A. (1971). “Partition of Cell Particles and Macromolecules,” 2nd ed. Almqvist & Wiksell, Stockholm. Albertsson, P.-A. (1977a). In “Cell Separation Methods” (H. Bloemendal, ed.), pp. 79-93. Elsevier, Amsterdam. Albertsson, P.-if, (1977b). Endeavour 1, 69-74. Albertsson, P.-A. (1978). J . Chrornatogr. 159, 111-122. Albertsson, P.-A., and Baltscheffsky, H. (1963). Biochein. Biuph!ys. Res. Coniitiun. 12, 14-20. Backman, L., and Johansson, G. (1976). FEBS Lett. 65, 3 9 3 3 . Beijeruek, M. N. (1896). Zentrulbl. Bacteriol. 2, 627. Borrehaeck, C., and Mattiasson, B. (1979). I n “Protides of Biological Fluids” (H. Peeters, ed.), 27th Colloqiurn, pp. 607-610. Pergamon, Oxford. Coloe, P. J. (1977). I n “Rapid Methods and Automation in Microbiology” (H. H. Johnston and S. W. R. Newson, eds.), p. 27. Research Studies Press, Forest Grove, Oregon. Coonrod, J . D., and Rytel, M. W. (1973). J . Lab. Clin. Mud. 81, 770-777. Edwards, E. A., and Larsson, G. L. (1974). A p p l . MiL-robiol.28, 972. Eriksson, E . , and Albertson, P.-A.- (1978). Biochim. Biophys. Acta 507, 425432. Eriksson, E . , Alhertsson, P.-A, and Johansson, G. (1976). Mol. Cell. Biochern. 10, 123-128. Flanagan, S. D., Rarondes, S. H., arid Taylor, P. (1976). J . B i d . Chem. 251, 858465. Higerdahl, B . , Mattiasson, B., and Albertsson, P.-A. (1981). BiotechnoZ. Lett. 3, 53-58. Hubert, P., Dellacherie, E., Neel, J., and Banlieu, E . E. (1976). FEBS Lett. 65, 169-174. Johansson, G . (1970). Biochim. Biophys. Acta 222, 381389. Johansson, G. (1976). Biochim. Biophys. Acta 451, 517-529. Johansson, G., and Westrin, H. (1978). Plant Sci. Lett. 13, 201-212. Johansson, G . , Hartman, A., and Alhertsson, P.-A, (1973). Eur. J . Biochern. 33, 379386. Johnston, H. H., and Newson, S. W. B., eds. (1977). In “Rapid Methods and Automation in Microbiology.” Research Studies Press, Forest Grove, Oregon. Kahn, W., Friedman, G., Rodriguez, W., Controni, G., and Ross, S. (1977). In “Rapid Methods and Automation in Microbiology” (H. H. Johnston and S. W. B. Newson, eds.), pp. 14-15. Research Studies Press, Forest Grove, Oregon. Kronvall, 6 . (1973). J . Med. Mimobiol. 6, 187-190. Kronvall, G., and Frommel, D. (1970). Zminunochemistry 7, 124-127.

PARTITION AFFINITY LIGAND ASSAY

147

Laurent, T. C. (1971). Eur. J. Biochem. 21, 498-506. Ling, T. 6. I., and Mattiasson, B. (1981). To be published. Ling, T. G. I., Ramstorp, M., and Mattiasson, B. (1981). Submitted. Mattiasson, B. (1980a). /. Immunol. Methods 35, 137-146. Mattiasson, B. (1980b). J. Appl. Biochem. In press. Mattiasson, B., and Eriksson, H. (1981). Submitted. Mattiasson, B., and Ling, T. G. 1. (1980). J. Zmnzunol. Methods 38, 217-223. Mattiasson, B., Johansson, A.-C., and Mosbach, K. (1974). Eur. J. Biochetn. 46, 341349. Mattiasson, B., Ling, T. G. I., and Ramstorp, M . (1981a). J. Irntnui~ul.Methods 41, 105-114. Mattiasson, B., Durholt, M., Nilsson, J . , and Ling, T. G. I. (1981b). I n “Lectins-Biology, Biochemistry, Clinical Biochemistry,” Vol. 2 (T. C. Bog-Hansen, ed.). Walter de Gruyter, Berlin. In press. Phillips, K . D., Tearle, P. Y . , and Wills, A. T. (1977). I n “Rapid Methods and Automation in Microbiology”(H. H. Johnston and S. W. B. Newson, eds.), p. 27. Research Studies Press, Forest Grove, Oregon. Pickering, L. K . (1976). J. Am. Metl. Assoc. 236, 1882. Puziss, M . , and Heden, C.-G. (1965). Biotech. Bioeng. 7, 355366. Rytel, M. W. (1979a). I n “Rapid Diagnosis in Infectious Disease” (M. W. Rytel, ed.), pp. 1-5. CRC, Boca Raton, Florida. Rytel, M. W . , ed. (197913). I n “Rapid Diagnosis in Infectious Disease” CRC, Boca Raton, Florida. Shanbhag, V. P., and Axelsson, C. 6. (1975). Eur. J. Biochern. 60, 17-22. Shanbhag, V. P., and Johansson, G . (1974). Biochirn. Biophys. Acta 61, 1141-1146. Stendahl, O., Edebo, L., Magnusson, K.-E., Tagesson, C., and Hjerten, S . (1977). Acta Pathol. Microhiol. Scand. Sect. B 85, 334340. Thorell, J. I., and Johansson, B. 6. (1971). Biochin. Biophys. Acta 251, 363369. Walter, H . (1977). In “Methods of Cell Separation” (N. Catsimooplas, ed.), Vol. 1, pp. 3 0 7 3 5 4 Plenum, New York. Walter, H . , and Selby, F. W. (1966). Biochini. Biophys. Acta 112, 146-153. Westrin, H., Albertsson, P.-A., and Johansson, G. (1976). Biochi~n.Biophys. Acta 436, 696706. Wilson, M. B., and Nakane, P. K. (1978). In “Irnrnunofluorescence and Related Staining Techniques” (W. Knapp, K. Holubar, and G . Wick, eds.), pp. 215-223. Elsevier, Amsterdam. Winlund, C., and Chamberlin, M. J., (1971). Anal. Biochem 41, 83-104.

This Page Intentionally Left Blank

Accumulation, Metabolism, and Effects of Organophosphorus Insecticides on Microorganisms RUP L A L ~ Ilepartment uf Zoology, University of Delhi, Delhi, India

I. Introduction . . . . .

.........

. . . . . . . . . . . . . . . 149

11. Entry of Organophosphorus Insecticides into

Microbial Environrnents 111. Accumulation . . . . . . . . . . . . . . ......... 1V. Metabolisiri . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Reactions Associated with Microbial Metabolism of Organophosphorus Insecticides in Microorganisms . . . . . . B. General Metabolism and thc Pathways of Metabolism . . . C. Enzymcs Associated with Microbial Metabolism of Organophosphorus Insecticidcs . . . . . . . . . . . . . . . . . . . . . . . V. Effects of Organophosphorus Insecticides on Microorganisms . A. General Effects on Species Popul and Microbial Activities . . . . . . . .............. B. Cytological and Biochemical Effe VI. Suinmary and Future Prospects . . . . . . . . . . . . . . . . . . . . . . . . . . Appendix ... .... References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

151 152 153

153 155 169

177 177 186 191 193 195

I. Introduction Effective pest control is essential for two of man's most urgent problems:

the provision of sufficient food for the ever-expanding human population and the maintenance of human health b y the control of vectors of serious human diseases. The greatest contribution toward the control of these pests has come from the use of pesticides. Among the pesticides, organochlorine insecticides enjoy an unique status because of their high insecticidal efficacy. However, because of their persistence in the environment, susceptibility to biomagnification, and toxicity to higher animals, these insecticides have been banned in technologically advanced countries. Besides, many insects have become resistant to these insecticides, making them ineffective for pest control. Now, the replacement chemicals for many pest control operations are organophosphorus insecticides, which are esters of alcohols with phosphoric acid or anhydrides of phosphoric acid with another acid.2 These insecticides having phosphorus as the active nucleus (Table I) were earlier known as organophosphates. However, now they 'Present address: Department of Zoology, Sri Venkateswara College, University of Delhi, Dhaula Kuan, New Delhi 110021, India. 2Chenrical names of organophosphorns insrcticides are listed in the Appendix.

149 hl>VANCES IN APPLIED MICROBIOLOGY, VOLUME 28 Cnpyright 0 1882 by Acdemic Presa, Inc. All rights 01 reproduction t n m y form reserved. ISRN 0-12-(N12fi28-7

150

RUP LAL

TABLE I THE CHEMICALSTRUCTURE OF VAHIOUSGROUPSOF ORGANOPHOSPHORUS INSECTICIDES +Group

Chemical structure

0 Phosphates

-0,h / p\

-0 Phosphorothioates

S

-o,p/

0-

‘0Phosphorodithioates

-0

S

‘P

-0’

//

‘SS

Phosphonothioates

-0,f /

\

0-

Phosphonodithioates

Phosphonates

O\P

//”

are called organophosphorus insecticides because one or more of the oxygens in the phosphoric acid moiety can be replaced. The organophosphorus insecticides possess several unique properties that make them more suitable for control of a variety of pests. They are readily degraded in soil and water by microorganisms. These microorganismslargely bacteria, algae, fungi, and protozoans-are important for their role in the decay of plants and other organic matter, acting as denitrifiers, nitrifiers, ammonium oxidizers, etc. Autotrophic microorganisms are engaged in oxygen evolution and primary production of food. Anything that disrupts their structure and function could be expected to affect the ecological balance in soil and water ecosystems. Thus, because of their importance in sustaining

EFFECTS O F INSECTICIDES ON MICROORGANISMS

151

the ecosystem, microorganisms have been studied extensively in relation to insecticides. The interaction of organochlorine insecticides with microorganisms has been the subject of several reviews (Bollag, 1961, 1974; Pfister, 1972; Ware and Roan, 1970; Cox, 1972; Tu and Miles, 1976; Johnsen, 1976; Butler, 1977; Williams, 1977; Wainwright, 1978; Rup La1 and Saxena, 1980, 1982), but no attempt has been made to bring together the available information on microbial interaction with organophosphorus insecticides.

11. Entry of Organophosphorus Insecticides into Microbial Environments Generally, organophosphorus insecticides are applied to soil and plants and to aquatic environments to kill the target organisms. Considerable quantities of the organophosphorus insecticides reach the soil either by direct application or by wash-off from crops to which they are applied. Smaller quantities of insecticides may enter soil through the feces of animals already containing the insecticides. Eventually, the insecticide from the animals and plants also gets incorporated into the soil on their death and subsequent decomposition. Insecticides can enter the surface water by direct or indirect routes. The two primary sources for the direct entry of insecticides into surface water are the huge amounts added to water for effective pest control (as in mosquito eradication programs) and the enormous quantities discharged into water along with industrial and domestic sewage waters. Organophosphorus insecticides indirectly enter into aquatic environments by drift from aerial or ground application and through water and soil erosion, which includes run-off, wash-off, and leaching from treated lands. The amount of organophosphorus insecticides entering the aquatic environment indirectly is greater than that of organochlorine insecticides, because organophosphorus insecticides are comparatively soluble in water. Small quantities of insecticides may be carried by dust particles and dispersed in the atmosphere to be deposited by rainfall. It seems unlikely that microorganisms in the air will come in contact with air-borne insecticides, since these chemicals are in very low quantities in the vapor form. Hence, insecticide-microorganism interactions are generally confined to soil and aquatic environments. Apart from these two environments, a variety of microbial habitats are offered by animals and plants whose external surfaces and internal tracts are readily accessible to microorganisms. These microbial environments include intestinal microorganisms in both target and nontarget animals, normal leaf

152

HUP LAL

microflora of plants, skin microflora of domestic animals, skin and gut microflora of fish, etc. Organophosphorus insecticides may enter these environments directly or indirectly.

Ill. Accumulation Organophosphorus insecticides are taken up by the microorganisms directly or indirectly via food chains. The uptake of these compounds into inicroorganisms is favored by their high surface area to volume ratio. Once the insecticide is accumulated by the microorganisms it may undergo many changes. Apart from the immediate metabolism and/or toxic effects that may ensue, some further amounts of organophosphorus insecticides may be accumulated and then transferred through several trophic levels in the food chain. Different groups of microorganisms differ markedly in their ability to accumulate organophosphorus insecticides. Ahined and Casida (1958) found that certain bacteria, yeasts, and algae rapidly absorbed thimet and then released it slowly from living and dead cells. Gregory et al. (1969) studied the accuinulation of parathion in the protozoans Paruinecium bursaria, Pnrnineciuin inultiiitirronucleatuin, and Euglena gracilis and the algae Anacysti.s nidulnns and Scencdesmus obliquus. All these organisms were exposed to 1 ppm parathion for 7 days under similar experimental conditions. The concentration of parathion accumulated varied from 50 to 116 ppm (Table 11). The bacteria Pseudomonas cepacia, Xanthomonas sp., Coiiiinoinonas tcrrigera, and Flavobacterium meningosepticurn accumulated malathion more readily than did the fungus Aspergillus mn-yzae (Lewis et nl., 1975); the rate of malathion accumulation in bacteria was approximately 5000 TABLE I1

(:ONCENTRATION OF

PAUTHION RY

ALGAE A N D

O F 1 PPM ( W h ) AND AN

PROTOZOA AFTER A SINGLE

EXPOSURE PERIOD

OF

TREATMENT

7 DAYS" Concentration (ppm)

Number of organisms exposrd pcr niilliliter

Supernatant

Organisms

l . l X 1 0 7 ? 0.09X10' 1 . 3 ~ 1t 0~ 0.44~10' 9.2~10 5 ~0 . 1 5 ~ 1 0 ~ 6.3X10' It 0.11X10' 1.7X102 t 0.02XlO'

0.05 ? 0.010 0.07 t 0.006 0.16 -+ 0.033 0.05? 0.012 0.15 ? 0.023

50? 3 72% 7 62? 2 942 2

1162 2

From Gregory ct ( I / . (1969). Rcprinted with permission from the Journal of Protazoology. Copyright hy thc Soricty of Protozoologists.

EFFECTS OF INSECTICIDES ON MICROORGANISMS

153

times faster than in A . rn-yzae under similar experimental conditions. However, malathion concentration in the organisms declined subsequently since most of the malathion was metabolized by the microorganisms. The bioaccumulation of fenitrothion in algae was directly related to its concentration in the medium (Miyamoto et al., 1979). This was assumed to result from the low excretion rate of algae. Lichtenstein and Schulz (1970) noticed that the volatilization of Dyfonate from distilled water was reduced significantly after the addition of algal cells to the medium. Similarly, loss of Dyfonate due to volatilization was reduced &om medium containing fungi (Flashinski and Lichtenstein, 1974a), perhaps because of the adsorption and/or absorption of the insecticide by the microorganisms.

IV. Metabolism Several physical, chemical, and biological forces act independently or in combination upon insecticides applied to the environment. It is now well established that microorganisms are the major or frequently the only means of degradation for several insecticides in the environment. The microbial degradation of several insecticides, particularly organochlorines, has been studied extensively and reviewed by several workers (Johnsen, 1976; Williams, 1977; Rup La1 and Saxena, 1982), whereas the studies on microbial metabolism of organophosphorus insecticides are more recent and have not been reviewed so far. Organophosphorus insecticides are metabolized by microorganisms because of the microorganisms’ capacity to hydrolyze, reduce, and oxidize these compounds. The reactions involved in the metabolism of organophosphorus insecticides in microorganisms are discussed below.

A. REACTIONS ASSOCIATED WITH MICROBIAL METABOLISM OF ORGANOPHOSPHORUS INSECTICIDES IN MICROORGANISMS

1 . Hydrolytic Reactions Hydrolysis involves the incorporation of water into the substrate. Organophosphorus insecticides containing either the P = 0 (phosphate) or the P = S (phosphorothioate) groups are generally degraded by hydrolytic processes in microorganisms. Mounter and Tuck (1956) showed that Escherichia coli and Propionibacterium pentasaceum hydrolyzed paraoxon and TEPP. Matsumura and Boush (1966) demonstrated that malathion can be hydrolyzed by enzyme preparations from Trichoderma uiride. Pseudonzonas

154

RUP LAL

melophthora converted dichlorovos, diazinon, and parathion to watersoluble products by hydrolysis (Boush and Matsumura, 1967). Phorate is also metabolized through hydrolytic processes in Pseudomonas flumescens and Thiobacillus thioxidans (Ahmed and Casida, 1958). Aspergillus nigw, Penicillium notntum, and Fusurium sp. hydrolyzed trichlorfon (Zayed et al., 1965a,b). One of the hydrolytic products was identified as O-methyl-2,2,2trichloro-1-hydroxyethylphosphoricacid, and a second metabolite was tentatively identified as 2,2,2-tricholoro-l-hydroxyethylphosphonic acid. 0 OH C1 I I1 I H,CO-P-CH-C-Cl

I

OCH,

0 OH

II

I

C1

HO-P-CH-C-C1

I

I

C1

OCH,

Tr ichlorfon

I

I

-

0 OH

I1

I

C1

OH

o-Methyl-2,2,2-trlchloro- l-hydroxyethylphosphoric acid

I

C1

HO-P-CH-C-Cl

I

I

C1

2,2,2-Trichloro-1hydroxyethylphosphonic acid

Microorganisms hydrolyzed parathion to p-nitrophenol (Hasegawa, 1959; Sethunathan and Yoshida, 1972, 1973). This hydrolysis occurred at the nitrophenyl (C-0-P) bond. Many organophosphorus insecticides have been reported to he degraded by hydrolytic reactions in ruminal microorganisms. The insecticides that were rapidly hydrolyzed by ruminal microorganisms included EPN (Cook, 1957; Ahmed et al., 1958) and mevinphos (Ahmed et al., 1958). The compounds that were slightly hydrolyzed included disulfoton, phorate, schradan (Ahmed et al., 1958), and diazinon (Cook, 1957).

2 . Reductive Reactions The reduction of the nitro group to amine has been reported during the metabolism of organophosphorus insecticides in microorganisms. For instance, the major conversion product of parathion in microorganisms is aminoparathion, which is a reduction product (Cook, 1957). Bacillus subtilis H,C,-Oo /\ # - O G N 0 2

H,C,-O

Parathion

-

H,C,-0

/\

Lo

H,C,-0

Am inoparathion

inactivated both parathion and parathion-methyl through reduction of the nitro group to an amino group (Yasuno et al., 1965). Fenitrothion and EPN were converted to their amino derivatives in B . subtilis (Miyamoto et al., 1966). Degradation by reduction was more pronounced than degradation by hydrolysis in this organism. Reductive reactions rather than hydrolysis or

EFFECTS OF INSECTICIDES O N MICROORGANISMS

155

oxidation were also of greater significance in metabolizing organophosphorus insecticides in rumen microorganisms (Cook, 1957; Ahmed and Casida, 1958; Ahmed et al., 1958). The major reactions involved in degradation of parathion in microorganisms are generally hydrolysis and nitro group reduction. However, repeated applications of either parathion or its hydrolysis product, p-nitrophenol, to flooded soil shifted the degradation pathway of parathion from reduction to hydrolysis (Suddakar-Barik et al., 1979). Aminoparathion was recovered as the major metabolite after the first addition of the parathion, but both aminoparathion and p-nitrophenol were identified after the second addition. However, only p-nitrophenol was observed after the third addition. This shift was attributed to the proliferation of parathion hydroIyzing microorganisms that utilize p-nitrophenol as the energy source.

3. Oxidative Reactions The oxidative reactions that are important in degrading organophosphorus insecticides in higher organisms are less common in microorganisms probably because of the lack of a defined mixed-function oxidase system in microorganisms (Matsumura, 1975).In B. subtilis, fenitrothion was metabolized by both hydrolytic and reductive reactions, but no fenitroxon was observed, indicating that oxidative disulfuration did not occur (Miyamoto et al., 1966). However, Ahmed and Casida (1958) found that the yeast Torulopsis utilis and the green alga Chlorella pyrenoidosa could oxidize phorate to corresponding sulfoxide. Stenersen (1969) found that the insecticide bromophos was oxidized by a double dealkylation to bisdemethylated bromophos by the fungi Ahernaria tenius and Trichoderma lignorum. Oxidation reactions involving the conversion of parathion to paraoxon and the opening of the aromatic ring of p-nitrophenol have also been reported in microorganisms (Munnecke and Hsieh, 1974; Suddakar-Barik et al., 1976; Sethunathan et al., 1977).

Br Brom ophos

Bisdemethylated brornophos

B. GENERALMETABOLISMAND THE PATHWAYS OF METABOLISM Organophosphorus insecticides are degraded fairly readily in soil (see Table

HI). The rates of transformation of these insecticides in sterile and nonsterile

156

RUP LAL

soils have been compared in order to predict the role microorganisms play in such transformations (Lichtenstein and Schulz, 1960; Menn et al., 1965).The microbial metabolism of organophosphorus insecticides in soil is affected markedly by biotic and abiotic factors. Some of the biotic factors are the type of microorganisms, their size and development stage, their physiological adaptation to the concentration of insecticide, inherent species difference, etc. Whether the microorganism is studied in isolation for its ability to metabolize a particular insecticide or in combination with other organisms also seems to play an important role. Abiotic factors in soil are temperature, salinity, soil moisture content, etc. (Harris and LichtenTtein, 1961; Lichtenstein and Schulz, 1964; Corey, 1965; Menn et ul., 1965; Whitney, 1967). Recognition of the importance of these modifiers and their accompanying interaction eflect is essential to an understanding of the kinetics of metabolism of organophosphorus insecticides in natural environments. In aquatic environments, the microbial interaction of the organophosphorus insecticides, though less studied, indicates that microorganisms in water certainly play an important role in the degradation of these insecticides (see Table 111).

1 . Parathion (Fig. 1 ) u . Metabolism. Parathion is resistant to chemical degradation and hence might pose a major residue problem. Fortunately, however, it does not, since it disappears in the soil very readily and does not persist in significant amounts. Since chemical and physical factors in soil do not contribute significantly toward parathion degradation, studies were carried out to discover the factors responsible for its disappearance. It has been now established convincingly that microbial metabolism is the major means of parathion degradation. That microorganisms in soil are responsible for the degradation of parathion was supported by the prolonged persistence of parathion in sterilized and dry soils and its lesser persistence in soils with high water content, which favors microbial growth (Lichtenstein and Schulz, 1964; Lichtenstein et al., 1968; Getzin and Rosefield, 1968). Stewart et al. (1971) found that about 0.1% of parathion applied to nonsterilized soil plot5 remained 16 years after the last application. The reason for such a long persistence of parathion was probably that a part of it may be dissolved in lipids of soil organic matter and thus be protected from microbial attack (Tu and Miles, 1976). Microbial dcgradation of parathion has been demonstrated in soils; aminoparathion (Ahmed and Casida, 1958; Lichtenstein and Schulz, 1964; Naumann, 1967; Wolfe et al., 1973) and p-nitrophenol (Hasegawa, 1959)have been identified as the major degradation products. The breakdown of parathion i n flooded cranberry bog soil was attributed mainly to the microflora of such soil (Miller et al., 1967).

EFFECTS OF INSECTICIDES ON MICROORGANISMS

p a r a ffhion hion

I I

OH

157

porooron

0

acid

NH2

OJ

diefhylphosphoric acid

dlothylphosphorlc ocld

P - nitrophenol

1

\OH

0

'NO*

@H

OH

-

hydroquinone

D nitrocotechol

I

1 C02

I

4

NO2

co2

FIG I. Proposed pathways of metabolism of parathion in microorganisms

In addition to aminoparathion, two unidentified metabolites were also detected. In the course of attempts to control Culex pipiens s.l., it was found that B. subtilis was very effective in inactivation of parathion and parathion-methyl (Yasuno et al., 1965). Later studies by Miyamoto et al. (1966) also showed that B. subtilis metabolized parathion and parathion-methyl to their respective amino analogs. In addition, parathion-methyl was also metabolized to desmethyl-parathion and desmethylaminoparathion-methyl in B . subtilis. An extensive study by Hirakoso (1968) showed that several bacteria-B.

subtilis, Bacillus megaterium, Bacillus cereus, Bacillus macerans, Pseudonzonas amuginosu, Pseudomonas ovalis, Pseudovnonas aureofaciens, Alcaligenes uiscolactis, Escherichia freundii, E . coli, Serratia plymuthica, and Achromohacter eurydice--converted parathion to aminoparathion. Boush and Matsumara (1967) observed the degradation of parathion by

Pseudoinonas melophthura, a symbiont of the apple maggot, but the degradation products were not identified. Cultures of the symbiotic nitrogenfixing bacteria Rhizobiurn japonicum and Rhizobium meliloti produced aminoparathion as the major metabolite and p-nitrophenol as the minor metabolite (Mick and Dahrn, 1970).

158

RUP LAL

Parathion degradation was studied in Flavohacterium sp. isolated from diazinon-amended flooded rice soil (Sethunathan and Yoshida, 1972, 1973). Pure cultures of Flavobacterium sp. hydrolyzed parathion to p-nitrophenol. This hydrolysis ceased at the p-nitrophenol stage. However, Raymond and Alexander (1971) reported the production of nitrite froin p-nitrophenol in p-nitrophenol- and chloroform-treated cells of Flavobacterium. Bacillus sp. also liberated nitrite from the hydrolysis product, p-nitrophenol, but not from parathion (Siddaramappa et al., 1973). Penicillium waksmanii isolated from a flooded acid sulfate saline soil converted parathion to aminoparathion and to water-soluble metabolites (Rao and Sethunathan, 1974). The culture containing parathion later showed signs of vigorous growth, which was related to the amounts of polar metabolites produced. Axenic cultures of microorganisms under laboratory conditions are able to metabolize parathion. These observations, however, cannot be extrapolated to natural environments, where the ultimate fate of the insecticide is decided by both biotic and abiotic factors, The nature of microbial populations at the time of application of the insecticide and possibly the balance between the major groups of organisms, which have varying requirements, may influence the degradation of the insecticide as indicated by extensive metabolism of insecticides by mixed microbial cultures under laboratory conditions. For example, a mixed microbial culture, that adapted to growth on parathion, degraded parathion to p-nitrophenol and released nitrite and hydroquinone from p-nitrophenol (Munnecke and Hsieh, 1974). Further studies by Munnecke arid Hsieh (1975)revealed that mixed microbial cultures under aerobic conditions metabolized high concentrations of parathion emulsion very rapidly and they ascertained that degradation was accomplished by metabolic pathways involving hydrolysis, reduction, and oxidation reactions. The metabolic products of parathion under these conditions were p-nitrophenol, diethylthiophosphoric acid, paraoxon, and aminoparathion. In mixed cultures, paraoxon was hydrolyzed faster than parathion. Under low oxygen conditions, parathion was reduced to aminoparathion, which was then hydrolyzed to arninophenol and diethylthiophosphoric acid. Daughton and Hsieh (1977a) also reported the utilization of parathion by a mixed microbial culture and by symbiotic microbial organisms. In this case, parathion was dissimilated by the highly acclimated symbiotic activity of Psetidomonas stuzer grown in a chemostat, which nonoxidatively and cometabolically hydrolyzed the parathion to ionic diethyl thiophosphate and p-nitrophenol. Adaptation to the presence of insecticides may result in a change in species diversity. Populations of the microorganisms may also adapt to the presence of insecticides by adaptation of their enzyme systems, so that an insecticide may be more rapidly metabolized by an already adapted micro-

EFFECTS OF INSECTICIDES ON MICROORGANISMS

159

bial population where the same or related insecticide has been used previously. Daughton and Hsieh (197713) observed that technical parathionacclimated microorganisms were extremely effective in degrading parathion rapidly. The accliinated cells could maintain their technical parathiondegrading ability in nonsterilized soil for at least 8 to 14 days under laboratory conditions; their effectiveness was greatly reduced after 3 weeks. Bacteria capable of hydrolyzing parathion to p-nitrophenol and then metabolizing p-nitrophenol to nitrite and CO, as the end products were isolated from parathion-amended flooded soil (Suddakar-Barik et al., 1976). Even m-nitrophenol, known for its resistance to biodegradation, was degraded by the bacteria isolated from parathion-amended soil. The effect of organic sources on the degradation of parathion by microorganisms in flooded soil was to modify the pathway of metabolism. The addition of organic sources enhanced the nitro group reduction of parathion, but it inhibited the hydrolysis of parathion in soils inoculated with parathion-hydrolyzing enrichment culture (Rajaram and Sethunathan, 1975). Enhanced reduction of parathion in amended soil was due to accelerated soil reduction generally associated with the decomposition of organic matter in flooded soil (Ponnamperuma, 1972). The inhibition of hydrolysis by added organic matter was reported to result from anaerobic decomposition of organic matter that liberated a substance (heat-stable factor) toxic to parathion-hydrolyzing microorganisms (Rajaram and Sethunathan, 1975). The highly acclimated culture of parathion-degrading bacteria was used to study the feasibility of bacteria for degrading the parathion from contaminated soil after parathion spillage or at disposal sites (Barles et al., 1979). The acclimated culture of bacteria capable of utilizing parathion as its sole carbon energy source was inoculated into Yolo silt loam soil to which parathion was applied at rates up to 5000 kghectare. The acclimated culture of bacteria was shown to be capable of completely degrading parathion in soil containing up to 1250 kghectare parathion within 35 days. The ability of acclimated bacteria to degrade parathion deteriorated at application rates higher than 1250 kghectare, probably because of the adverse effects of parathion and its hydrolytic products on bacteria. When considering the role of biotic and abiotic factors in alteration of microenvironments during insecticide-microbe interaction, the case of root environment needs special mention. Among the sites of intense microbial activity in the soil are the root surface and the soil adjacent to it, termed as rhizosphere. In an interesting experiment, Hsu and Bartha (1979) pointed out the role of rhizosphere microorganisms in degradation of parathion. Parathion was added individually (5 wg/gm) to sealed flasks containing soil permeated with either soil system of Phaseolus vulgaris (bush bean) or identical soil without roots. Bush bean plant roots without associated rhizo-

160

RUP LAL

sphere microorganisms failed to produce significant amounts of CO from parathion. The metabolism of organophosphorus insecticides in aquatic environments has been little studied in comparison with that of soil environments. Maleszewska (1974) investigated the metabolism of parathion by bacteria occurring in surface water and sewage and the effects of nutrients on its metabolism. A total of 20 bacterial strains were isolated from water and municipal sewage. Four strains metabolized p-nitrophenol, of which only Bacillus cereus was able to metabolize parathion. The rate of biodegradation was accelerated by the presence of additional organic compounds, and the most rapid degradation was observed in the presence of serine, threonine, asparagine, ant1 alanine. A thin-layer chromatography analysis of cranberry bog flooded waters in laboratory aquaria indicated the presence of aminoparathion and two unidentified metabolites of parathion (Miller et al., 1966). Miller et al. (1966) suggested that microflora present in the aquaria was responsible for the breakdown of parathion. Among the several factors studied, microflora of seawater was found to play a major role in the metabolism of parathion (Weber, 1976). Graetz et al. (1970) showed that parathion was readily metabolized to arninoparathion by niicroorganisrns from lake sediments under both aerobic and anaerobic conditions. Under anaerobic conditions, aminoparathion was not metabolized further, but aerobic conditions favored the metabolism of aminoparathion to a metabolite possessing both phosphoric acid and benzoid moieties of aminoparathion. Not much work has been done on understanding the metabolism of parathion in algae. Chlorella pyrenoidosa was suggested to degrade parathion because only 37%of the original parathion was recovered after 8 days of incubation (Ahmed and Casida, 1958). The presence of algae in rice paddy fields accelerated the degradation of parathion (Sato and Kubo, 1964). Later studies revealed that C. plyrenoidosa wdS responsible for metabolizing parathion around the roots of bean plants, which in turn translocated the sulfur-containing metabolite to the shoots (Mackiewicz et al., 1969). The algal metabolites consisted of 66% aminoparathion and a second product bearing the P-S group and the benzene ring. A trace amount of p-nitrophenol was also observed. However, Zuckerman et al. (1970) reported aminoparathion and three other unidentified metabolites from parathion in C. pyrenoictosa. One of the unidentified metabolites lacked a phen yl ring; the remaining two metabolites contained phenyl rings, but only one contained sulfur. Cultures of a blue-green alga, Anacystis nididans; a green alga, Scenedesinus obliquus; a flagellate, Euglena gracilis; and two ciliate protozoans, Paraniecium bursaria and Paramecium multimimonuc-

EFFECTS O F INSECTICIDES ON MICROORGANISMS

161

leatum, did not metabolize parathion (Gregory et al., 1969). However, the possibility of insecticide metabolism having occurred in these organisms should not be excluded, considering the one-step hexane-extraction method employed.

b. Pathways of Metaholism. The pathways of parathion metabolism in microorganisms essentially involve reduction, hydrolysis, and oxidation. Reduction of the nitro group of parathion forms aminoparathion, which, on further hydrolysis, forms p-arninophenol and diethylphosphoric acid (Munnecke and Hsieh, 1975). Hydrolysis of parathion occurs at the nitrophenyl (C-O-P) bond, which forms p-nitrophenol and diethylthiophosphoric acid (Sethunathan et al., 1977). Further hydrolysis of p-nitrophenol liberates nitrite and hydroquinone or p-nitrocatechol (Munnecke and Hsieh, 1974). Parathion may also enter the oxidation pathway of degradation, thus forming paraoxon, which, on further hydrolysis, yields p-nitrophenol and diethylphosphoric acid (Munnecke and Hsieh, 1975).The process of ring opening of p-nitrophenol has also been reported as evident by CO, evolution (Munnecke and Hsieh, 1974). Whether this is a single-step reaction or some intermediates are involved has yet to be investigated. 2. Diazinon

a. Metabolism. Diazinon is degraded in soil and by microorganisms isolated from soil (Boush and Matsumura, 1967; Matsumura and Boush, 1968; Bro-Rasmussen et al., 1968). Hydrolysis appears to be the major mechanism of degradation of diazinon in microorganisms and yields 0,O-diethyl phosphorothioate and 2-isopropyl-4-methyl-6-hydroxypyrimidine (Konard et al., 1967). Diazinon sprayed into silt loam yielded 2isopropyl-4-rnethyl-6-hydroxypyrimidine (Getzin, 1967). One of the hydrolysis products (2-isopropyl-4-methyl-6-hydroxypyrimidine) was converted to COz, but fumigation of soil with propylene oxide to kill the microorganisms resulted in accumulation of hydrolysis products and less C 0 2 was formed. This observation implies that ring cleavage of diazinon to C 0 2 occurs only in the presence of microorganisms. Also, Trela et al. (1968) found that hydrolysis of diazinon was greatly stimulated by the presence of microorganisms isolated from diazinon-treated soil. One breakdown product was tentatively identified as tetraethyl pyrophosphate. Boush and Matsumura (1967) reported 27% water-soluble and 2% solvent-soluble metabolites of diazinon from Pseudomonas melophthma. In submerged paddy soil in the Philippines, microorganisms played a significant role in the conversion of diazinon to 2-isopropyl-4-methyl6-hydroxypyrimidine (Sethunathan and Yoshida, 1969), and the rate of

162

RUP LAL

conversion to the hydrolysis product was increased on repeated application of diazinon to soil (Sethunathan and Pathak, 1972). However, acidity of the soil seems to have a detrimental effect,since under such conditions diazinon was not susceptible to microbial degradation and the hydrolysis product was not formed (Sethunathan and Pathak, 1972). Axenic cultures of the bacteria Rhixobium sp., Arthrohicter sp., and Flavobactwium sp. also metabolize diazinon (Sethunathan and Pathak, 1971; Sethunathan, 1973; Sethunathan and Yoshida, 1973). The recovery of diazinon in the algae Chlorella, Scenetlesinus, Nitxuchia, Kolida, Actinastruin sp., Monoraphidium, and Golinkiniopsis varied from 62 to 96%, indicating the metabolism of diazinon by the algae (Butler et al., 1975). However, diazinon was not metabolized by cultures of ruminal bacteria or ciliated protozoa (Williams, 1977). Among the soil microorganisms, soil fungi efficiently degrade diazinon. Streptomyces isolated from rice paddy soil under asceptic conditions and in the presence of the energy source, glucose, converted diazinon to COz (Sethunathan and MacRae, 1969a). Soil fungus Zrichodmma uiride also degraded diazinon to water-soluble metabolites tliat were not identified (Matsumura and Boush, 1968). A synergistic relation between two microorganisms to metabolize diazinon has been reported by Gunner and Zuckerman (1968). When in company with Arthrobacter, Streptomyces attacked the pyrimidinyl ring of diazinon and converted it to C02, while Arthrobacter alone could convert only diethylthiophosphoric acid, a metabolite of diazinon, to CO,. The metabolism of diazinon by rhizosphere microorganisms was studied by Hsu and Bartha (1979). Diazinon was added at 5 pg/gm to sealed flasks containing either soil permeated by soil system of bush bean (Phaseolus vulgaris) or identical soil without roots. Bush bean plant roots without associated rhizosphere microorganisms failed to produce significant amounts of CO, from diazinon. From the available data, it is obvious that diazinon is susceptible to degradation in the soil and that the chief causative factor is the microorganisms. However, the degradation is also influenced by many parameters associated with the soil, viz., soil moisture, soil pH, and finally the soil diazinon concentration.

b . Pathways of Metabolism. A complete degradation of diazinon leads to the liberation of CO, (Getzin, 1967; Gunner and Zuckerman, 1968; Sethunathan and MacRae, 1969a; Sethunathan and Pathak, 1972; Hsu and Bartha, 1979). During this process, intermediate metabolites are formed because of hydrolysis of the parent compound. The major metabolites are 0,O-diethyl phosphorothioate and 2-isopropyl-4-methyl-6-hydroxypyrimi-

EFFECTS OF INSECTICIDES O N MICROORGANISMS

163

dine (Konard et al., 1967). As yet, however, nothing is known about how these metabolites are finally converted to CO z .

Diazinon

0,O-Diethyl phosphorothioate

2’-Isopropyl-4 methyl-6-hydroxy pyrimidine

3. SumithionlFenitrothion (Fig. 2 )

a. Metabolism. Bacillus subtilis inactivated Sumithion by reducing its nitro group and forming the amino compound, aminosumithion (Yasuno et al., 1965; Miyamoto et al., 1966). Sumithion degradation in several species of bacteria and fungi, including Bacillus and Fusarium sp., showed that Sumithion was converted to aminosumithion, 3-methyl-4-nitropheno1, and CO, (Miyamoto, 1977). Sumithion was also degraded rapidly by microorganisms in forest soils (Spillner et al., l979a). After 3 days of incubation with Sumithion, two metabolites were detected, 3-methyl-4-nitrophenol and COB.Recently, Roy et al. (1980) have suggested that the disappearance of Sumithion from the aquatic system may be due to its degradation through hydrolysis and oxidation primarily by photolytic or microbial processes.

b. Pathways of Metabolism. Studies with B . subtilis isolated from polluted water samples showed that the bacterium was capable of inactivating Sumithion by reduction of its nitro group (Yasuno et al., 1965; Miyamoto et al., 1966). The metabolite was identified as aminosumithion under culture conditions. Miyamoto et al. (1966) also reported that B . subtilis metabolized aminosumithion at a lower rate than the parent compound to desmethylaminosumithion, desmethylsumithion, and dimethylphosphorothioic acid. The authors proposed that desmethylaminosumithion arose from aminosumithion and dimethylphosphorothioic acid, from Sumithion directly. No sumioxon was detected, indicating that oxidative desulfuration does not take place under the culture conditions in this species or that

164

RUP LAL

S

CH30

>Lo,

CH30

-

S

1I cH30-P-ocH3 I 0

-

S

1I

dimethylphosphorothloic acid

+

surnithion

S

I1

HO-P-OCH3

I

dornet hylsurnit hion

FIG

orninosumithion

4

5

II

HO- P-OCH3

I

desmet hylarninosurni thion

Proposed pathways of metabolism of S u m ...ion in microorganisms.

sumioxon is quickly degraded by esterases. Sumithion is also converted to CO, by microorganisms hut the intermediates formed during the formation of CO, from Sumithion are not known. More exhaustive and detailed studies would shed more light on the pathways of degradation of Sumithion. 4 . Malathion (Fig. 3) a. Metubolism. The metabolism of malathion in soil and microorganisms isolated from soil was studied initially by Matsuinura and Boush (1966), who showed that the bacterium Pseudornonas sp. actively dealkylated the carboxylic acid side-chain of the malathion and carboxylic acid derivatives constituted the major metabolites of malathion. Later studies also showed that Pseudomonus isolated from soil converted malathion to malathion monoacid (Tiedje and Alexander, 1967). In Rhisobiuin leguminosurum incubated with [14C]malathion for 1week, about 25% of the malathion appeared in the form of malathion monoacid, 20% malathion diacid, 5% dimethyl phosphorodithioate, and 10% dimethyl phosphorothioate (Mostafa et a l . , 1972a). About 30% of the malathion was completely mineralized to inorganic phosphate. Degradation of malathion was more rapid in nonsterile Mississippi soil than in sterilized soil, suggesting the involvement of microorganisms (Walker and Stojanovic,

165

EFFECTS OF INSECTICIDES ON MICROORGANISMS

O CH =

i

/

5 cH3-0)1-s-;H-C-O-C

CH3-0

)3- "yi

0

II

-t -0 -Cz H5

CHZ,C -O-%Hs 1 I

0

desmothylmolothton

0

I1

C H rC

H 2 5

-0-C2H5

-

-COOH

P--5-CH CH3-0

I CHZ-CO-C

II

I1

0

0

ma to thi on

dimethylphoiphorodithioic acid

dimcthylphosphOrQthio,C acid

2

H 5

maIothron monoocid

dsmethytphosphoric acid

malathion diacid

FIG3. Proposed pathways of metabolism of malathion in microorganisms.

1973). The different degradation products obtained were malathion monoacid, malathion diacid, dimethyl phosphorodithioate, dimethyl phosphorothioate, and dimethyl phosphate. The authors also isolated Arthrobacter sp. from the soil to prove the involvement of this organism in the metabolism of malathion. It was noticed that Arthrobacter metabolized malathion to malathion monoacid, malathion diacid, dimethyl phosphorodithioate, and dimethyl phosphorothioate. The formation of dimethyl phosphate was a chemical degradation. Trichoderma viride metabolized malathion into a number of carboxylic acid derivatives (Matsumura and Boush, 1966). These metabolites were identified in Penicillium, Rhizoctonia, and Aspergillus as malathion monoacid, malathion diacid, desmethyl phosphorodithioate, dimethyl phosphorothioate, and dimethyl phosphate (Mostafa et al., 1972b). In aquatic systems, Konard et al. (1969) also suggested the involvement of microorganisms in the metabolism of malathion. Recently, it has been shown that bacteria from marine marsh environments are capable of metabolizing malathion to its carboxylic acid derivatives and to phosphothionates (Bourquin, 1977a). Paris et al. (1975a-c) studied the metabolism and kinetics of metabolism of malathion degradation in microorganisms. The bacteria Pseudomonas

166

RUP LAL

cepaca, Xanthomonas sp., Cornmomonas temigera, and Flavobacterium ineningosepticum and the fungus Aspergillus oryzae were found to metabolize malathion. The major metabolite of malathion degradation in both the bacterial and fungal systems was P-monoacid of malathion. Malathion dicarboxylic acid and dimethylphosphorodithioic acid were detected but in minor quantities in both the fungal and bacterial systems. In addition, bacteria metabolized malathion to diethyl malate, which was not detected in fungal system. Malathion was accumulated rapidly in A . oryzae and its concentration subsequently declined, mainly because malathion was metabolized by fungus (Lewis et al., 1975). Malathion degradation at low concentration (I mgil) in batch culture of bacteria followed second-order kinetics as predicted from the Michaelis-Menton theory (Paris et al., 1975~).

11. Pathways of Metabolism. The pathways of metabolism of malathion are not exactly known. Two soil microorganisms, Trichoderina viride and Pseudomonas sp., metabolized malathion via two pathways that did not involve the oxidation of malathion to malaoxon (Matsumura and Boush, 1966). One pathway involved the hydrolysis of malathion to malathion monocarboxylic acid, which was then converted to malathion dicarboxylic acid. The second pathway, as observed only in some strains of Trichoderina viride, involved demethylation of malathion. Bourquin (1977a) proposed similar pathways of metabolism of malathion in bacteria isolated from a salt marsh environment. Metabolism of malathion to dimethyl phosphorodithioate seeins to follow an independent route. Dimethyl phosphorodithioate is then converted to dimethyl phosphorothioate and dimethylphosphoric acid. 5. DichlorvoslDDVP Pseudoinonas irielophthora and Triclzodemzu viricle metabolized DDVP, hut no metabolite was identified (Boush and Matsumura, 1967; Matsumura and Boush, 1968). Dichlorvos disappearance was rapid when Bacillus cereus was added to previously sterilized soil (Lamoreaux and Newland, 1978). The rapid initial disappearance of dichlorvos in sterilized soil was attributed to nonhiological (70%)and bacterial degradation (30%).The microbial population in the soil was suggested to be an important aspect of dichlorvos metabolism (Oshida et al., 1978). 6'. Phorate (Fig. 4 )

Phorate is degraded in soil (Getzin and Chapman, 1960; Deway and Parker, 1965; Bache and Lisk, 1966; Lichtenstein, 1966) and by microorganisms isolated from soil. Tmulopsis utilis and Chlorella p yrenoidosa hydrolyzed phorate, its metabolites phorate sulfoxide and phorate sulfone, and

EFFECTS OF INSECTICIDES ON MICROORGANISMS

+

p h o r o t i culloxide

hydrolytic

H-S-CH2-S-C2H5

products

0

C2H5-0

0

167

P-S-CH2-S-C C2H5-0

II

H 2 5

phorotc sulfone phorphothioot.

rulforidc

FIG4. Proposed pathways of inetabolisin of' phorate in microorganisms.

also oxidized phorate to its sulfoxide (Ahmed and Casida, 1958). Phorate sulfoxide was then slowly converted to phosphorothioate sulfoxide by the microorganisms. The bacteria Pseudomonas fluorescens and Thiobacillus thiooxiclans hydrolyzed phorate but did not oxidize it (Ahmed and Casida, 1958). The bacterium Bacillus inegateriunt and the fungus Aspergillus niger degraded phorate to its sulfoxide and phoratoxon sufoxide (LePatourel and Wright, 1976).

7. DllfonatelFonofos Metabolism. Lichtenstein and Schulz (1970) showed that after 4 days of incubation of Dyfoiiate (12.5 ppm) in distilled water at 30°C, about 42% of the applied Dyfonate was lost. However, the addition of algal cells reduced the volatilization of Dyfonate by GO%, probably because of the accumulation and metabolism of Dyfonate by the algal cells. Among the nine fungi (Aspergillus flavus, Aspergillus fuinigatus, Aspergillus niger, Fusarium oxysporuin,

Mucor alternans, Mucor plurnbeus, Penicilliuin notaturn, Rhizopus arrhizus, and Trichoderma viride) tested for Dyfonate metabolism, M . pluinbeus and R. arrhizus were the most active in metabolizing the insecticide and P. notaturn was the least active (Flashinski and Lichtenstein, 1974a). The activity was measured from the amounts of soluble metabolites produced. The metabolites identified were dyfoxon and methyl phenyl sulfone. Aspergillus produced most of the unidentified metabolites. Rhizopus arrhizus, which was capable of actively metabolizing Dyfonate, was selected to study the metabolism of the insecticide in the natural soil

168

RUP LAL

environment (Flashinski and Lichtenstein, l974b). Rhizopus arrhizus was added to the soil containing [ 14C]Dyfonate. Since it did not support the luxuriant growth of microorganisms because of deficiency in certain nutrients, the soil was enriched with glucose so as to boost the growth of fungus. It was under these conditions that [14C]Dyfonatewas degraded almost completely, as indicated by a recovery of only 12% of the applied insecticide by the production of its oxygen analog, dyfoxon, arid by the appearance of significant amounts of water-soluble metabolites. However, the metabolic pathways of Dyfonate degradation still remain to be elucidated.

8. Triclalorfon IDip terex Relatively little is known about the metabolism of trichlorfon in soil and its associated flora and fauna. The fungi Aspergillus niger, Penicilliuni notaturn, and Fusariuni sp. were quite active in hydrolyzing trichlorfon (Zayed et al., 1965a,b). One of the hydrolytic products identified was O-methyl-2,2,2-trichloro-1-hydroxyethylphosphoric acid. The other proacid. duct is believed to be 2,2,2-trichloro-l-hydroxyethylphosphonic The metabolism of trichlorfon was studied as affected by nodule-forming bacteria (Salama et al., 1975). Monomethyl phosphate and dirnethyl phosphate were identified as metabolites of [ 14C]trichlorfonin the culture media of Rhizobium Ieguininosurunz and Rhixohiunt trqolii. Apart from these two metabolites, I4CO2 was liberated from [ 14C]trichlorfonin rhizobial cultures, which prompted Salama et al. (1975) to suggest that oxidation of trichlorfon occurred during the formation of CO?, but the intermediate compounds were not reported.

9. Miscellaneous Organophosphorus Insecticides Aspergillus and Periicillium were able to grow well on malt medium containing different concentrations of methamidophos, chlorpyrifos, and tetrachlorvinphos (Zidan and Ramdan, 1976). The degradation oftetrachlorvinphos increased with increasing insecticide concentrations, while degradation of other insecticides decreased gradually with increasing insecticide concentration. The fungi Trichoclerina and Alternaria dealkylated bromophos into its desmethyl and bidesinethyl derivatives and hydrolyzed it to dimethyl- and monoinethylphosphorothioic acid (Stenersen, 1969).Bacteria able to utilize Dasanit (fensulfothion), diazinon, malathion, Orthene (acephate), parathion, trithion (carbophethion), dimethoate, Dylox (trichlorfon), parathion- methyl, and Vapona (dichlorvos) as sole phosphorus sources were isolated from soil and sewage (Rosenberg and Alexander, 1979). Extracts of two bacteria derived froin organophosphorus-insecticide-grown cultures enhanced the disappearance of Aspon, Azodrin, Dasanit, Orthene, malathion, parathion, and trithion but not of dimethoate, Dylox, parathion-methyl, and Vapona. The

EFFECTS OF INSECTICIDES O N MICROORGANlSMS

169

extracts formed dimethyl phosphate from Azodrin, dimethyl phosphorodithioate from malathion, diethyl phosphorodithioate from trithion, and diethyl phosphorothioate from Dasanit, diazinon, and parathion. Several organophosphorus insecticides (ethion, trichloronat; fonofos, chlorpyrifos, parathion, chlorfenvinphos, diazinon, and fensulfcjthion)were more persistent in sterile soil than in nonsterile soil (Miles et al., 1979). Microbial degradation played a major role in the degradation of parathion, fensulfothion, chlorfenvinphos, fonofos, and ethion, whereas chemical degradation played a major role in the degradation of chlorpyrifos and trichloronat. This indicates that microorganisms have a selective approach toward the degradation of insecticides, A hydrolytic product of fenthion and fenamifos, 3-methyl-4-(mettiylthio)phenol, was oxidized by a soil isolate of Nocardia sp. in the presence ofa carbon source supporting growth (Rast et al., 1979): When the bacteria were grown on acetate, the compound was mainly oxidized via the 2,3(“meta”)fission pathway without accumulation of the intermediate metabolites. However, in the presence of sucrose as growth substrate, 3-methyl-4-(methylthio) catechol and 4-methyl-5-(methylthio) catechol were accumulated as transient intermediates. The product of ring fission of these metabolites was 2,5dihydro-3-(niethylthio)-4-methyl-5-oxofuran-2-acetic acid which resulted from a 1,2-(“ortho”)-fission of the aromatic nucleus. From the literature it is evident that microorganisms generally employ reductive, hydrolytic, and sometimes oxidation reactions during the inetabolism of organophosphorus insecticides. These reactions are not really energy-yielding reactions and are incidental to the energy production of the corresponding microorganisms. Matsumura and Benezet (1978)have coined the term “incidental metabolism” to describe such reactions because it is merely incidental that the organisins may use these organophosphorus insecticides as a phosphorus energy source in soil. During this process, inicroorganisms may metabolize an organophosphorus insecticide completely to liberate free phosphorus. Rosenberg and Alexander (1979) showed, for instance, that enrichment cultures of Pseurloinolzas were able to use 12 organophosphorus insecticides as the sole phosphorus energy source and organisms isolated from diazinon and malathion enrichments were the most versatile, able to utilize ten and nine compounds, respectively (Table IV).

c. ENZYMESASSOCIATEDWITH MICROBIAL METABOLISM OF

ORGANOPHOSPHORUS INSECTICIDES

Microorganisms have an inherent capacity to metabolize many naturally occurring chemicals by virtue of their comprehensive enzyme systems. However, they may lack the necessary enzymes required for the metabolism

T.4BLE 111 METABOLISMOF ORGANOPHOSPHORUS INSECTICIDES Compound Parathion

Organism Soil microorganisms

IN

~IICROORGASISMS

M e t a M i c products ..\inin oparathion,

p-nitrophenol, ,md dic.th~lthinphosphnricacid

Re fere nce Hasegawa (1959), Lichtenstein rt al. (1966)

Microorg;mi sms from lake sediments

Amirioparatliion

Graetz ct (11. (1970)

PSC14dOmOWls SP.

p-Kitrophenol, nitiitc, anti COY ~~-iiitrophenol p-Yitrophenol. amintrparattiion, paraoxon, nitrite hydroquinone. and COi Aminoparathion and unidentified metabolites Aminoparathion and p-nitmphenol Aminopal-athion and pniirophenol Ainirinparrithion iiiid water-srhhle metul)olitc:s Am inoparat hi on

Siddaramappa et 01. (1973), SethunatliaIi et a!. (1977) Sr:thilnathari arid Yoshirla (1973) Munnwkc and Hsieh (1975:l

F1ar:ohnc t w i u In sp . Mixed IHicrohinl cult III’C with at least ninc bacterial isolates

Chlorella p y-moidosn

Ahmed and Casida (195Y), Mackiewicz et al. (196Ui. Ziickerman et rrl. (1970, Mick and Dahm (1970) Mick and Dahm (1970)

Ran iind Scthunatlia~~ (1974,: Yasuno et (11. (1965), Miyamoto el al. (1966), Hirakoso (1968)

p-Nitrophenol

Anacyrtis nidulans Scenedesmus obliquus E icglena gracilia Paramectrim bwsaria Paramecium multimicronude at u In. Flac.obarteriti~nsp. Ps6!ldrlmtlnlls

Malathion

sp.

Ru.cillrrs sp. Pseudomonas sp.

Arthrobacter sp,

Fungi

Pseudoolnonas repacia, Xanhmorras sp.,

Gregory

Nitrite

Nitrite and 4-nitrocatcchol Nitrite Ualathion monoacid, malathion diacid, and unidentified hydrolytir products Malathion monoacid, malathion diacid, dimethyl phosphorothioate, and dimethyl phosphorodithioate 51ahthion monoacid,

malathion diacid, dimethyl phosphorodithioate, and dimethyl phosphorothioate Malathion monoacid, malathion diacid, desiriethyl pliosphorodithioate. and dimethyl phosphate ~4\lal.lthion-p-monoarid, malathiun diacid,

et al. (1969)

Raymond and Alexander (1971) Siddaramappa r ~ 01. t (1973), Scthllnathali et d.(1977) Siddarainappa et nl. (1973 Matsumura and Boush (1966:11 Ticdjc and Alexander (1967)

Mostafa ~t a!. (1972a)

Walker and Stojanovic (19'73j

Most& et al. (1972b)

Paris ct al. (1975a,b)

TABLE I11 (Contiiiuecl) Compound

Organism

Metabolic products

Refere nce

0.0-Dimethyl phosphorodithioic acid. and diethyl malate

4Ial~tliii~n-~-rrionoat~id.

Bourqiiin ( I 977a)

malathion diacid, desniethylrnalathion, phosphi)rothiunattrs, and c;dmIi dicarboxylic acid #,O-T)isthyl phosphorothioate and 2-ist~pi-op~l-CIiic:tIi~I-

6-hydroxypyrimidine

co, Tetraethyl pyrophosphate ~-1sopropyl-Cmethyl-fi-h~droxy pyrimidine and

Getzin (1967) Trela ct nl. (1968) Sethunathan and Yoshida (1969;. Sethunathan and Pathak (1972)

co, Boush and Matsumura (1967)

Pseudoinonus rnclopli thot-ci

Sumithion

hiinusurnithion, desmethylaminosurnithion,

Matsumura atid Boush jl9@0 SethuTidthdil alld h h C h ? (l%%a) Hsu arid Bertha (1979), Yasuno et Ul. (1965) Miyamoto et al. (1966), Miyamoto (1977)

Brurnophos

Forest soil microorganisms ?In'chodtwui sp and Altrmiofiu

desmethylsumithion, dimethylphosphorothioic acid, 3-methyl-4-nitrophenol. and CO, 3-Methyl-4-nitrophenol and CO, L)esme~bylbroinopl~os I~idesmt.thylt~riiiriophos,

dim~thylphosphorothioi~ atrid; aiid ~ri~~~iomt.tI~ylpl~osphorothkiic acid

Fvnofos

Fungi

uyrcixolI,

ethylethvxyphosghonothioicacid,

Rhitopus arrhizus

3

Phorate

Trichbrfon

Torulopsis utilis and Chlorella pyrenoidosa Bacillus inegaterium and Aspergillus n i g w Rhizobium legurninasarum and Rhimbium trifoii Fungi

ethylethoxyphnnic acid, irietliyl phenyl suliiixide, aIid inethyl phenyl sulfone Dyfoxon and water-soluble metabolites Phorate sulfoxide and phosphorothioate sulfoxide Phorate sulfoxide and phoratoxon sulfoxide Monomethyl phosphate, dimethyl phosphate, and CO, O-Mc:thy1-2,2,1trichloro-J -hydroxytrthylphosphoric arid and 2.2,2-trichl~)ro-l-hydrox~et hyl phosphonic acid

Elashinski and Lichtenstein j1974a)

Flashinski and Lichtenstein (1974b) Ahmed and Casida (1958) LePatourel and Wright (1976) Salama et

QI.

(1975)

174

RUP LAL

TABLE IV ORGANOPHOSPHORUS COMPOUNDS USED AS PHOSPHORUS SOURCES~ Source of bacterial isolate A s p o n (As) Azodrin (Az) Dasanit (Da) Diazinon (Di) Dimethoate (Din) Dylox (Dy) Matathion (Ma) Methylparathion (Me) Orthene (0) Parathion (P) Trithinn (T) Vapona (V)

Compounds used as sole phosphorus source As, Az, Da, Di, Dm, Ma, 0, P As, Az, Da, Di, Dnr, Ma, Mc, 0

Az, Da, Dm, Dy, Ma, Me, 0,V As, Az, Da, Di, Dm, Dy, Ma, Me, 0, P Az, Da, Dm, Ma Az, Da, Di, Dm, Dy, Ma, 0 As, Az, Da, Dm, Ma, Mc, 0, P, V Az, Da, Di, Ma, Me, P As, Az, Da, Di, Ma, 0 Da, Di, Dm, Dy, Ma, 0, P Da, Di, Dm, Ma, T Da, Dm, V ~~

~~~~

From Rosenherg and Alexander (1979).Reproduced with permission from Applied and Enoironmentul M i ~ ~ b i o l o g yCopyright . by thc American Society for Microbiologists.

of synthetic compounds, such as organophosphorus insecticides, that occur in the environment in very minute quantities. This naturally raises the very obvious and logical question as to why microorganisms should bother at all to metabolize these compounds. Organophosphorus insecticides have many structural similarities with naturally occurring compounds. Hence, it is possible that organophosphorus insecticides may be acted upon by the existing enzymes in microorganisms. The enzymes involved in pesticide metabolism may be constitutive (Kaufman, 1965) or may require induction by either the pesticide or an alternate chemical inducer (Alexander, 1965a,b). Many microorganisms are able to “adapt” to one of a variety of substances added to the culture medium by forming an enzyme system that is not already present when the organism is grown in the absence of the added substance. This phenomenon is termed “enzyme induction” and the substance that initiates the response is the enzyme “inducing agent.” Audus (1960) suggested that microorganisms can develop the ability to degrade pesticides either by enzyme induction or by chance mutations. Preliminary observation on enzymatic metabolism of a compound comes from the indirect observations following enzyme inhibition by heat denaturation. Autoclaving the soil to kill the soil microorganisins (Kishk et al., 1976) and mycelia of fungi (Flashinski and Lichtenstein, 1974a)reduces the ability

EFFECTS OF INSECTICIDES ON MICROORGANISMS

175

of microorganisms to metabolize insecticides, thus suggesting the involvement of microbial enzymes. Enzymatic hydrolysis of parathion has been demonstrated in cell-free extracts of adapted mixed or pure cultures of microorganisms (Sethunathan and Yoshida, 1972, 1973). Cell-free extracts of Flavobncteriuin sp. showed exceptional ability to hydrolyze parathion and diazinon, as did the whole cells of the same bacterium. With respect to parathion, the reaction ceased at the p-nitrophenol stage. The enzyme involved in the hydrolysis was suggested to be constitutive (Sethunathan et nl., 1977). However, Munnecke arid Hsieh (1974) isolated an inducible enzyme, parathion hydrolase, from a mixed microbial culture adapted for growth on parathion. A cell-free enzyme preparation from this culture hydrolyzed parathion at the rate of 416 nmol/minute/ng of protein. The enzymatic hydrolysis of parathion was 2450 times faster than that of chemical hydrolysis in 1 N sodium hydroxide solution at 40°C. Other organophosphorus insecticides-Triazophos, paraoxon, EPN, diazinon, methyl-parathion, Dursban, fenitrothion, and cyanophoswere also enzymatically hydrolyzed with the same enzyme preparation at rates significantly higher than those of the chemical hydrolysis with sodium hydroxide (Munnecke, 1976). However, three organophosphorus insecticides-azinophos-M, ethion, and fenthion-were resistant to enzymatic degradation. The organophosphorus insecticide detoxification procedure by the consumer involves the use of strong alkali (1 N or more) for chemical hydrolysis of the phospho-alkylester bond, with subsequent burial of the treated waste solution (Munnecke, 1979). This method is slow and large amounts of salts are required. Therefore, Munnecke (1979) suggested that microbial enzyme preparations can be used as an alternative in waste disposal technology. The crude enzyme preparation that was able to detoxify many organophosphorus insecticides was thus examined for its ability to hydrolyze residual parathion in industrial waste waters (Munnecke, 1979). The immobilized enzyme system was able to hydrolyze 95% or more of the parathion added to industrial waste waters. The enzyme was sufficiently stable and active for use in industrial waste treatment. However, it was susceptible to deactivation by temperatures above 50°C or by a pH above 10 or below 6. These studies were further extended to understand the ability of crude enzyme extract to hydrolyze commercial insecticide formulations of emulsifiable concentrates of parathion (48%), Dursban (50%), diazinon (25%), cyanophos (50%), and parathion-methyl wettable powder (Munnecke, 1980). The microbial enzyme preparation could hydrolyze these insecticides faster than the chemical hydrolysis, and the hydrolysis was not inhibited strongly by the detergent and solvent ingredients in commercial formulations, Spain (1979) isolated an enzyme from particulate cell fraction of extracts of

176

RUP LAL

Moraxella sp. This enzyme catalyzed the conversion of p-nitrophenol to hydroquinone with release of the nitro group as nitrite. Oxygen and reduced nucleotides were required for p-nitrophenul oxidation, and flavin adenine dinucleotide stimulated the activity. The enzyme was specific for 2-pnitrophenol and did not release nitrite from any other member of related nitroaromatic compounds. Enzymatic hydrolysis of malathion in Trichodmna virirle was demonstrated by Matsuinura and Boush (1966). The enzyme was prepared by an acetone powder extraction procedure. They suggested the presence of powerful carboxyesterases because carboxylic acid derivatives of malathion constituted the major portion of the metabolites. Paris et al. (1975a) a150 suggested the involvement of carboxyesterase in the metabolism of malathion in bacteria and fungi. The presence of this enzyme was confirmed from the assumption that the products of' degradation of inalathion should be malathion-p-monoacid and ethanol. Bourquin (19774 observed desmethylmalathion, phosphorothionates, and four carbon dicarboxylic acids as the degradation products of malathion as a result of phosphatase activity in microorganisms. Trichlorfon was metabolized to monomethyl phosphate and dimethyl

MAXIMUM

ACTIVITY OF

TABLE V EXTRACTSOF P S l ~ U t / U n b O n ( i S 7 AND VARIOUSINSECTICIDES~

CELI.-PREE ON

PSeudll?lloflas28

Maximum activityb Pseirclurnonr~s28

Substratca

Diazinon

Malathion

Diazinon

Malathion

Aspon Azodrin Desanit Diazinon Malathinn Ortliene Pal-athion Tritliion

62" 92' 238" 283r 222" 52" 189' 142'

110' 108" 212" 250 275' 41r 200" 125"

44c 121' 205" 301' 257" 37' 259' 138"

88' 133' 210" 263' 288' 32' 212" 117d

"From Rosenberg and Alexander (1979). Reproduced with permission from Applied and Encironrnentd M i c r o l h l o g y . Copyright by the American Society for Microbiologists. * Nanomoles of substratc destroyed per i n i n peI nig of protein. "At 10.0 hours. " A t 7.0 hours. 'At 3.5 hours.

EFFECTS OF INSECTICIDES O N MICROORGANISMS

177

phosphate by the enzyme preparations of Rhizobium trifolii and Rhizobium leguminosarum (Salama et al., 1975). Crude extracts of cells of Nocardia sp. grown on acetate as cosubstrate contained considerable catechol 2,3dioxygenase and low catechol 1,2-dioxygenase activity (Rast et al., 1979). These enzymes were probably responsible for the metabolism of 3methyl-4-(1nethylthio)phenol, a hydrolytic product of fenthion and fenamiphos. A detailed study on the enzymatic degradation of organophosphorus insecticides in Pseudomonas sp. was carriedout by Rosenberg and Alexander (1979). Resting cells of Pseudomonas derived from the culture grown on diazinon or malathion metabolized diazinon and malathion to a significant extent. Cells derived from the culture grown on one of the insecticides also metabolized the other. Extracts from both diazinon and malathion grown cells of Pseudomonas catalyzed the initial phases of degradation of 8 of the 12 organophosphorus insecticides (Table V). The metabolic products formed by the crude enzyme preparations were ionic dialkylphosphorus and thiophosphorus esters. On the basis of these results, phosphatase or phosphotriesterase enzymes were suggested to be involved in the hydrolysis of the aryl (P-0) bond.

V. Effects of Organophosphorus Insecticides on Microorganisms A. GENERALEFFECTSON SPECIESPOPULATIONAND MICROBIALACTIVITIES Insecticides influence the density and composition of microbial populations in natural environments as well as in pure cultures. Soil fertility depends upon the delicate balance between the various types of microorganisms involved in nitrification, denitrification, ammonium oxidation, and carbon and other mineral cycles. The addition of insecticide may disturb this equilibrium and thus, the fertility of the soil. In an aquatic ecosystem, these insecticides can pose a serious threat by affecting microorganisms, particularly those acting as food for filter feeding organisms and those involved in the primary production of food. This section describes the effects of organophosphorus insecticides on microbial population and on the activities of microorganism in relation to their environment.

1. Parathion Parathion is one of the most commonly used organophosphorus insecticides. Because parathion is highly toxic to higher organisms, systematic screening studies have been carried out in order to ascertain its effects on

178

RUP LAL

microorganisms, It is evident from such studies that the effects of parathion on the growth of bacteria, fungi, and algae is dose dependent, with very low concentrations having no effect at all and higher concentrations drastically arresting the multiplication of the microorganisms. Parathion applied at 10 lblacre for 3 successive years had no effect on the number of bacteria or fungi (Bollen et al.. 1954). Similarly, application of parathion up to 100 Ib/acre had no evident effect on nitrifying and denitrifying bacteria in soil, except a small temporary reduction observed at the highest concentration (Lichtenstein and Schulz, 1960). In a clay-loam soil under greenhouse conditions, 50 ppm parathion had no effect on the numbers of nitrifiers, cellulose decomposers, or other groups of microbes (Kasting and Woodard, 1951). Certain bacteria and yeast were not affected by as much as 200 ppm parathion (Naumann, 1967). In nonsterile sandy loam and organic soils, 10 ppm parathion had no effect on bacteria or fungi (Miles et ul., 1979). Gregory et al. (1969) reported that 1 ppm parathion had no detrimental effect on Paramecium bursaria, P . inultimic7-onucleatuni, Euglena gracilis, Anucystis nidulans, and Scenedesinus ohliquus. It was later shown by Moore (1970) that a concentration of 1.2 ppin parathion also did not have any effect on E . gracilis, but Poorman (1973) reported that even concentrations above 1.2 ppm and as much as 100 ppm of parathion did not alter the growth of E . gracilis. Parathion is known to inhibit the population growth of different microorganisms such as bacteria at high concentrations such as 200 ppm (Naumann, 1960) and 50,000 ppm (Wolfe et al., 1973). Mick and Dahm (1970) reported that micromolar quantities of parathion were sufficient to suppress the population growth of Rhizobium japonicum and Rhixobiurn nwliloti. Inhibition of Chlorella by parathion has also been reported (Cole and Plapp, 1970). However, reports on the effect of parathion on E . gracilis are conflicting: Moore (1970) observed that as little as 10 ppm killed the cells within 1 to 2 hours, but Poorman (1973) reported that concentrations up to 100 ppm did not have any detrimental effect on the population growth even after 24 hours of treatment. While such discrepancies may be explained in experiments conducted under natural conditions, it is disturbing that they should also occur in pure culture conditions, illustrating the inherent variability of microbial response to the insecticide. The sensitivity of different fungi to the toxic effects of parathion varied, and at 40 ppm all the fungi except Aspergillus fumigatus were af€ected (Cowley and Lichtenstein, 1970). The same workers also showed that the addition of yeast extract, asparagine, and ammonium salts to parathioncontaining medium reduced the susceptibility of the fungus to parathion. This clearly implies that the eficacy of parathion is also dependent on the nutrient status of the environment.

EFFECTS O F INSECTICIDES ON MICROORGANISMS

179

It is interesting to note that parathion, apart from having no effect at all or inhibiting the growth of microbial populations, may also stimulate, i.e., accelerate, the growth of the population. Naumann (1960) observed that parathion increased the number of various physiological groups of bacteria, such as nitrifiers and nitrogen fixers, and the increase in population was more prominent in the presence of moisture. Even a concentration as high as 10,000ppm was stimulatory to population growth provided parathion was applied in combination with chernozen (Naumann, 1960). The cell number of E . grucilis was also slightly stimulated 7 days after treatment with 100 ppm parathion (Poorman, 1973).

2 . Parathion-methyl Parathion-methyl [O. 15 kg active ingredient (AI)hectare] increased the population of nitrogen-fixing azotobacteria in a loam soil and higherconcentrations (15, 150, and 300 kg Allhectare) decreased the population initially but increased it afterwards (Naumann, 1970a).The population of actinomycetes in a loam soil was significantly reduced on treatment with parathion-methyl at 0.15, 1.5, and 15 kg AIkectare, and higher concentrations, such as 150 and 300 kg AI/hectare, decreased the number of microorganisms as well as changed the species composition (Naumann, 1970a, 1971).The effect was not permanent because during the first 15 days of treatment with parathionmethyl at 300 kg AIhectare, the number of soil actinomycetes was depressed but subsequently it increased to nearly twice as much as in the untreated soil. The number of soil bacteria was also increased at 0.3 kglhectare parathionmethyl (Yurovskaya and Zhulinskaya, 1974). Both temperature and soil type seem to influence the interaction of parathion-methyl with microorganisms (Naumann, 1972). An increase in temperature from 10" to 20°C increased the duration of incubation from 15 to 35 days. In loam soil in the field, parathion-methyl at 1.5 kg AIhectare suppressed the actinomycetes, but in loam soil with vegetation cover, the same concentration increased the population of actinomycetes significantly.

3. Diazinon Diazinon is less a potent organophosphorus insecticide than is parathion. Its inhibitory effect on cell populations has been documented for bacteria (Tu, 1970) and Penicillium (Draughon and Ayres, 1978). Diazinon at 1000 ppm caused a significant decrease in the population of E . coli in pure culture conditions, and higher concentration of 10,000 ppm resulted in total mortality of E . coli (Ballington et al., 1978). However, diazinon at 3-4 kghectare did not affect the fungal population in soil (Gunner et al., 1966; Gunner, 1970). Preplant incorporation treatment of 2 and 4 kghectare of diazinon had no eft'ect on root colonization by mycorrhizal fungus (Glomus

180

RUP LAL

mosseae) in soybean, planted in an Andovery clay-loam (Burpee and Cole, 1978). In nonsterile sandy loam and organic soils, diazinon at 10 ppm did not have any effect on bacterial and fungal populations (Miles et d., 1979). The effects of diazinon on algal and bacterial populations revealed that concentrations lower than 1pg/liter did not alter the cell number of bacteria and algae (Murray and Guthrie, 1980). Earlier, Singh (1972)had reported that diazinon in nitrogen-free and nitrate-nitrogen media had no effect on the blue-green algae Cylinclrosperinuin sp. and Aulosira fmtilissima. Similarly, the growth of another blue-green alga, Phanothece pallida, was not reduced significantly at concentrations of 50 to 400 ppm (Kannaiyan, 1980). The stiinulatory effect of diazinon on the growth of algae (Sethunathan and MacRae, 1969b) and actinomycetes populations (Gunner et al., 1966; Gunner, 1970) has also been documented. In addition, diazinon has been reported to affect microbial activities in soil. Microorganisms in diazinon-treated sandy loam soil (10 and 100 pg/gm) showed an increase in ammonium production, a slight depression in nitrification, and an increase in oxygen consumption (Tu, 1970). Diazinon has also bcen reported to increase glucose utilization in effective strains of Hhizobiuin trifolii (Salem et al., 1971). However, in Azotobacter chroococcum and Azotobacter agile, diazinon at 50 ppin inhibited glucose utilization and nitrogen fixation initially and stimulated it afterwards (Salem and Gulyas, 1971).

4 . Fenitrothion Addition of fenitrothion to forest soil changed the population of aerobic and anaerobic bacteria, actinomycetes, and fungi after 1 year (Salonius, 1972). However, fenitrothion at 0.75, 10, and 50 kghectare in silt-loam soil had no effect on microbial activities, but higher concentrations showed an initial decrease and a subsequent increase in nitrification (Ross, 1974). Spillner et (11. (l979b) also showed that microflora of forest soil was not affected by fenitrothion (7.4 ppm in wet soil), and the insecticide was readily degraded in soil, suggesting that this insecticide is comparatively safer than many other insecticides in forest soil.

5 . Malathion Malathion at 4.2 and 5 kglhectare decreased the number of many soil bacteria (Swaminathan and Sullia, 1969). Malathion above 100 ppm reduced the growth of Azotobacter and Chroococcum, whereas about 300 ppm malathion was required to produce an inhibitory effect on Rhizobiurn trifolii (Mendoza, 1973). A population of malathion sole-carbon-degrading bacteria in laboratory salt marsh environment increased significantly with increasing treatment levels and in sediments with repeated treatment (Bourqnin, 1977b).

EFFECTS O F INSECTICIDES ON MICROORGANISMS

181

A commercial malathion solution prepared on distillation at less than 155°C yielded malathion and a solvent fraction (Stanlake and Clark, 1975). These fractions were then used to study their effect on soil microorganisms in a natural community and in axenic cultures under laboratory conditions. Each fraction was applied at 0.44pg/gm to soil and 400 pg/liter to log-phase bacterial cultures maintained axenically. Neither fraction significantly affected microbial growth in natural community. The malathion fraction had no bacteriocidal effect on the axenic bacterial cultures either. The solvent &action, on the other hand, had a significant bacteriocidal effect. The growth of the alga Chlorogloeae fritschii was reduced at 200 ppm malathion (DaSilva et al., 1975). Salk (1976) reported the inhibition of growth at SO ppm malathion in 10 algal isolates. 6. Chlorpyrgos

The literature dealing with the side effects of chlorpyrifos on microflora indicates that this compound is particularly damaging to microbial populations. Chlorpyrifos at 1.5pglgm reduced the population of ammonium oxidizers, denitrifiers, and nitrate oxidizers in soil (Sivasithamparam, 1969), and at field concentrations in a clay-loam, it inhibited the aerobic nitrogen-fixing population of bacteria and increased the actinomycetes and algal population (Sivasithamparam, 1970a,b). This effect was characterized by an increase in microbial activity. Tu (1977) also showed that chlorpyrifos alone or in combination with lindane caused significant inhibition of the growth of Rhizobium japonicum. The microflora of the soil was affected by chlorpyrifos as observed by reduction in population growth (Tu, 1978a). However, chlorpyrifos in nonsterile sandy loam and organic soils at 10 ppm did not have any effect on bacterial or fungal populations (Miles et al., 1979). The diatom population in water in the fields was reduced by chlorpyrifos treatment (Nelson et al., 1976). At concentration of 10 pg/gm (Tu, 1970) or 50 pg/gm (Lin et al., 1972) of soil, commercial formulation of chlorpyrifos (about 48% active ingredient) was found to inhibit nitrification slightly. A concentration of 500 pg/gm of commercial formulation of chlorpyrifos in soil did not inhibit acetylene reduction in pure cultures of Azotobacter vinelandii (Wood and MacRae, 1974). However, that chlorpyrifos alone or in combination with lindane caused a significant reduction in the nitrogen-fixing ability of microflora of soil (Tu, 1977, 197%). Inhibition of nitrogenase has also been observed in a number of asymbiotic nitrogen-fixers by chlorpyrifos during the first week of incubation, followed by stepwise recovery to normal levels (Hegazi et al., 1979). A study of the effects of chlorpyrifos in freshwater ponds provided clear proof of the role of insecticide in increasing phytoplankton population (Hurlbert et al., 1972). Three successive treatments spaced at %week intervals

182

RUP LAL

increased the population of phytoplankton both in ponds treated with 0.5 pprn and in ponds treated at five times the rate at which this insecticide is normally used in mosquito-control operations. In three of the four ponds treated at higher rates, Anubaena was a principal contributor to phytoplankton blooms. Chlorpyrifos also suppressed the zooplankton counts. Hurlbert (1975) concluded that the suppression of the zooplankton population by chlorpyrifos is a major factor contributing to the algal blooms. This conclusion was also supported by Hughes et uZ. (1980), who observed a similar relationship between the zooplankton population and algal bloom formation in chlorpyrifos-treated ponds.

7. Dimethoote Dimethoate inhibited the growth of rhizobia cultured on wet or dry agar plates (Daitloff, 1970). Above 100 ppm, dimethoate reduced the growth of Azotobucter, Chroacoccurn, and Rhizobium trijolii (Mendoza, 1973). Bacteriocitasis was obtained at a concentration of 1000 ppm in Azotobucter, whereas the higher concentration of 3000 ppm was required to produce a similar effect in Rhizobiunz. Dimethoate at 1.89 ppm allowed normal growth of bacteria, whereas 3.78 ppm completely inhibited two strains of Rhizobium meliloti (Staphorst and Strijodom, 1974). However, Congregado et al. (1979) reported an increase in bacterial propagules in soil containing 10 and 100 &gm of dimethoate.

8. Disulfoton Disulfoton applied at the rates of 0.5, 1, and 2 gin AIL00 gm seed inhibited the microbial population of actinomycetes, nitrifying bacteria, and anaerobic nitrogen fixers (Mahnioud et ul., 1972). The effect was also directly related to the concentration of the insecticide used. However, disulfoton at 1.7, 8.4, and 16.8 kghectare in oven-dried soil had no effect on Rhizobium japonicum (Kapusta and Rouwenhorst, 1973).

9. Fensulfothion Fensulfothion (20 kghectare) decreased the number of nodule-forming microorganisms in a loam soil (Kulkarni et al., 1974), whereas in nonsterile sandy loam and organic soils, fensulfothion at 10 ppm did not have any effect on bacterial growth (Miles et al., 1979). 10. Gurdona and DDVP

Gardona (tetrachlorvinphos) and DDVP were applied to Sabouraud's medium in 10, 30, 60, and 120 ppm amounts and the effects on size and morphology of colonies of a number of soil fungi were observed (Jakubowska and Nowak, 1973). Significant inhibition of growth was caused by DDVP

E F F E C T S O F INSECTICIDES O N MICROORGANISMS

183

followed by Gardona, except in Sepedonium chr!ysospermuni, which was equally affected by both the insecticides and in Geotrichuiri cancliduni,which was more sensitive to Gardona than to DDVP. These insecticides also had a growth-stimulating effect on Ftisarium javanicum, Mucor fragilis, Mucor hiemelis, and Penicillium aurantiocandidum but showed no effect on Geotrichum candidum, Sepedonium, and Fusarium sumbucinum. Some species-Mycelia sterilia, Geotrichum candidum, Sepedonium chrysospermum, Fusuriuin javanicum, and Fusariuin sumbucinum-showed some alterations in their morphologies, since their colonies were white with more fluffy surface and low, dense growth. Gardona at 50 and 250 ppm also inhibited the nitrogenase activity of Azotobacter vinelandii (Rodell et al., 1977). In the fungus Penicillium citrinum, DDVP inhibited growth and citrinin production (Draughon and Ayres, 1978), and in Aspergillus niger, DDVP aEected growth and fermentation activity (Rahmatullah et al., 1978, 1979). 11. Fenthion

The effects of fenthion at I00 ppb was studied in the algae Skeletonerna, Cyclotella, Dunaliella, and Phaeodactyluin (Derby and Ruber, 1970). This concentration inhibited growth, and maximum inhibition was observed in Skeletonemu, followed by Cyclotella, Phaeodactylum, and Dunaliella. Higher concentrations of fenthion (10 to 40 ppm) also inhibited growth and fermentation in Aspergillus niger (Rahamatullah et a l . , 1978).

12. Fonofos In sandy loam soil, fonofos (11 kg AIihectare) decreased fungal and actinomycetes populations during the first 2 weeks, and these microorganisms returned to pretreatment levels afterwards (Gawaad et al., 1972, 1973). In nonsterile sandy loam soils and organic soils, bacterial and fungal populations were not affected by 10 ppm fonofos (Miles et al., 1979). Fonofos at 50 ppm inhibited glucose utilization in an effective strain of Rhizobium trifolii and increased glucose utilization in an ineffective strain (Salem, 1971). However, fonofos at 50 pprn initially inhibited glucose utilization and nitrogen fixation in Azotobacter chroococcum and Azotobacter agile, but later these activities were stimulated (Salem and Gulyas, 1971). 13. Phorate Phorate at 40 ppm was toxic to most fungi isolated from prairie soil, except in Aspergillusfumigatus, where phorate treatment stimulated growth (Cowley and Lichtenstein, 1970). The addition of yeast extract, asparagine, and ammonium salts to phorate-containing media reduced the susceptibility of the fungi to phorate. The fungal population was stimulated by phorate treatment (Visalakshi arid Nair, 1980). However, a population of actinomy-

184

RUP LAL

cetes was suppressed by higher residual levels of phorate, whereas a population of bacteria was stimulated at higher residual levels and suppressed at lower residual levels. The growth of the blue-green alga Aphanothece pallida was not adversely affected by the phoraie even at 50 to 400 ppm (Kannaiyan, 1980). Phorate (8 kgihectare) decreased the number of nodules on plants in a loam soil, presumably because of the reduction in number of bacteria (Kulkami et al., 1974). However, phorate treatment increased the number of nodules per plant on beans in clover soil under laboratory conditions, and under field conditions, phorate did not affect nodulation in clover soils (Gawaad et al., 1972). Swaniiappan and Chandy (1975) also reported an increase in nodulation upon treatment with phorate at 1.1A1 kghectare.

14. Trichlmfon The growth of Rhizobiurvi leguininosnrunz was inhibited at 1285 ppin trichlorfon (Salama et a l . , 1973). However, trichlorfon did not have any effect on the behavior or cell population growth of hygienic indicator microorganisms at 5" to 6"C, whereas at 20" to 22"C, trichlorfon at 1000 ppm inhibited the growth of E . coli (Yurovskaya, 197%). Trichlorfon at 100 ppm inhibited the growth of Dunaliella, Phueodactyluna, Monochrysis, Protococcus, and Chlm-ella by 51, 75, 62, 83, and 73% respectively (Ukeles, 1962).

15. hliscellaneous Organophosphorus Insecticides Among the many fungal strains isolated from soil to study the effect of' organophosphorus insecticides, Aspergillus arid Penicilliuin proved to be quite resistant to the effect of insecticides (Zidan and Rarndan, 1976). These organisms were able to grow well on malt extract medium containing different concentrations of insecticides, especially methamidophos, chlorpyrifos, and tetrachlorvinphos. Phosphamidan at 10 to 40 pg/ml decreased the total dry weight of mycelia of Aspergillus niger (Rahmatullah et a l . , 1978). Nitrogen fixation by soybean nodules and Azotohacter virielandii was studied using the acetyl reduction technique with regard to the effects of phosmet, methidathion, and trichlorfon (Rodell et ul., 1977). These insecticides did not inhibit the activity of Azotobacter significantly. However, nodulation was affected b y inonocrotophos and trichlorfon, siiice these insecticides inhibited the microorganisms engaged in nodule formation in soybeans (Salem et n l . , 1977). Thionazin and trichloronat at 1 to 10 kdhectare in sandy loam soils decreased the number of bacteria initially; these bacteria recovered afterward (Tu, 1969). Mevinphos inhibited growth and citrinin production in Penicilliuin citrinuin (Draughon and Ayres, 1978). Metaphos, carbophos, and their mixtures did not cause any change in the behavior and population kinetics of E . coli, Entwococci, Streptococci, Shigella, and Salmonella at levels found in

EFFECTS O F INSECTICIDES ON MICROORGANISMS

185

the natural environment (Yurovskaya, 1975a). However, metaphos and carbophos affect E . coli, and Streptococcus faecalis at levels exceeding those found under natural conditions (Yurovskaya, 1975b). In sandy loam soil, leptophos, phorate, and terbufos decreased the bacterial number in early stages of treatment; these bacteria recovered afterward (Tu, 1978b). Recently Miles et al. (1979) have studied the effects of eight organophosphorus insecticides (ethion, fonofos, trichloronat, chlorfenvinphos, fensulfothion, parathion, chlorpyrifos, and diazinon) on microorganisms in sterile and nonsterile mineral and organic soils. Compared with untreated soil, none of the insecticides had significant long-term effects on populations of bacteria or fungi in nonsterile mineral and organic soils. Microbial populations in nonsterile soil declined only after a 24-week test period. In sterile soil, no significant bacterial ( > 300 gm/soil) or h n g a l ( > 50 gm/soil) activity was noted until the sixteenth week. By the twenty-fourth week, most sterile samples were contaminated with bacteria and fungi, thus making it difficult to assess the effects further. Various organophosphorus insecticides inhibited the growth of phytoplanktons to varying degrees (Sweeney, 1968). Guthion and Meta-Systox did not have any effect, but methyl-trithion inhibited the natural population of phytoplankton significantly, as shown in Table VI. Walsh and Alexander (1980)have described a simple marine algal bioassay for short- and long-term studies of organophosphorus insecticides (EPN, carbophenthion, ethoprop, DEF, parathion-methyl, and phorate). Skeletonema costutum was grown in optically matched culture tubes that fit directly into a spectrophotometer, allowing population density to be estimated by absorbance without removal of samples. The 96-hour ECS0values reported were EPN, 340 pgiliter; carbophenthion, 109 pgiliter; DEF, 366 TABLE VI EFFECTOF ORGANOPHOSPHORUS INSECTICIDES ON PHYTOPLANKTON POPULATION

Organophosphorus insecticide Bayer 29493 (Baytex) Diazinon Dibrom Di-Syston Ethion Grithion Meta-Systox Methyl-trithion Systox

Percent decrease in phytoplankton population at 1 pprn 7.2 6.8 55.6 55.2 69.0

0.0 0.0

85.9 7.11

186

RUP LAL

pdliter; ethoprop, 8.4 mg/liter ; parathion-methyl, 5.3mg/liter ; and phorate, 1.3 mg/liter. From the available data on the effect of organophosphorus insecticides on growth and activities, it is extremely difficult to make any meaningful conclusion as to which particular insecticide is the most potent. The reason for this is that effect is dose dependent, is variable from microbe to microbe, and is influenced by extraneous factors such as temperature, moisture, and nutritional status of the environment in which the organism is growing.

B.

CYTOLOGICAL AND BIOCHEMICAL EFFECTS

The primary target of action of organophosphorus insecticides in insects is the nervous system, more specifically, the inhibition of the enzyme acetylcholinesterase at the synaptic junction. As a consequence, acetylcholine, the neurotransmitter responsible for the propagation of a nerve signal from one neuron to another across the synaptic junction, gets stranded, resulting in total disruption of nervous control. By contrast, microorganisms display a multitude of potential targets for organophosphorus insecticides, making it extremely difficult to understand the mode of action of the insecticides. However, it can be generalized to a certain extent that the target of action depends both on the nature of the microorganism (i.e., whether it is autotrophic or heterotrophic) and on the chemical nature of the insecticide (i.e., whether or not it can function as an alkylating agent). Targets of action so far identified are the photosynthetic system, nucleic acids, and enzymes involved in various essential metabolic pathways.

1 . Effects on Photosynthesis The process by which the carbohydrates are synthesized from CO, and water is unique to green plants and autotrophic microorganisms. In these microorganisms, it appears that the detrimental effect of organophosphorus insecticides is probably mediated through photosynthesis. Insecticides interfere with photosynthetic activity by means of (1)the development and structural integrity of chloroplast, (2) photochemical pathways involved in the conversion of radiant energy to chemical energy, and (3)many biosynthetic pathways that are involved in the production of output products. Malathion at 1mg/liter has been reported to inhibit photosynthesis by reducing chlorophyll production in Vuucheria geminata, Tribonerna sp., and Oscilluturiu Zuteu (Torres and O’Flaherty, 1976).However, malathion did not inhibit chlorophyll production in Chlorococcum hypnosporum. Lower concentrations of malathion (0.1 and 0.5 pg/liter) even stimulated chlorophyll production in Clzlwella, Tribonerna, and Oscillatmia. Other cytological changes produced by malathion treatment in V. geminata were the formation of

EFFECTS OF INSECTICIDES ON MICROORGANISMS

187

brown crystals inside the cytoplasm, formation of a second cell wall inside the first, and distortion of gametangia. Torres and O’Flaherty (1976) also observed a synergistic interaction among the two triazine herbicides and malathion in their influence on algae. Chlorophyll production was reduced to a greater extent when treated with a combination of pesticides than could be expected independently. Inhibition of photosynthesis and growth of Chlorella pyrenoiclosa caused by parathion increased with decreasing cell concentration (Cole and Plapp, 1970). In estuarine phytoplanktons, photosynthesis was inhibited by malathion and diazinon at I ppin, as indicated by a decrease in carbon fixation (Ware and Roan, 1970). On the contrary, Stadnyk et al. (1971)reported that diazinon at 1ppm had no significant effect on photosynthesis and biomass of Scenedesmus quadricaudu. Derby and Kuber (1970) used oxygen output as the variable to study the effect of 100 ppb fenthion and temephos on the photosynthetic activity in Skeletonema, Cyclotella, Dunaliella, and Phaeoductylum. Inhibition of oxygen output after treatment with fenthion was maximum in Skeletonemu, followed by C yclotella, Phaeoclactylum, and Dunahella, whereas temphos brought the maximum reduction in oxygen output in Phaeodactylunt, followed by Dunalella, Skeletonemu, and Cyclotella. The effect of diazinon on ATP production in three species of photosynthesizing fresh water algae-Chlorella ellipsoidea, Chlamydomonas sp., and Euglena gracilis-revealed that diazinon inhibited ATP production, presumably by interfering with the photosynthetic activity of these organisms (Clegg and Koevening, 1974). It was further suggested that the inhibition of photosynthetic activity results from the interference of diazinon with photophosphorylation in the light reaction of photosynthesis.

2 . Mutagenic Eflects

Of all available organophosphorus insecticides, only DDVP and malathion have been studied with regard to mutagenic effects. The mutagenic action of DDVP was reported for the first time in E . coli (Lofroth et al., 1969), and it was later confirmed using the same organism (Lofroth, 1970; Bridges et al., 1973; Lawley et nl., 1974; Wennerberg and Lofroth, 1974; Green et al., 1974). DDVP has also been reported as mutagenic in Salmonella and Streptomyces (Carere et al., 1978). As compared with DDVP, malathion is not a very potent mutagenic agent (McCann et al., 1975). Shiau et al. (1980) tested the DNA-damaging and mutagenic activity of malathion in Bacillus subtilis and Salinonelln typhirnurium. Malathion was found to be mutagenic and did not require metabolic activation. Bacillus subtilis was superior to S. typhimurium for the detection of mutagenic activity (Fig. 5). In an attempt to understand the mutagenic action of DDVP and mala-

188

500

RUP LAL

1

I

0

50

100

150

200

250

300

CONCENTRATION OF MALATHION ( re /plats )

FIG5 . Comparison of Bacillus subtilis strain TKJ6321 (0)and Salnionella typhi7nurium strain TA1535 (0)mutations induced by malathion without nietabolic activation by rat liver 59 mix. From Shiau et al., (1980).Reproduced by permission from Mutation Research, Vol. 71, p. 169. Copyright by Elsevier/North-Holland Biomedical Press.

thion, many investigations were carried out to ascertain whether the nucleic acids (i.e., DNA and RNA) are the targets of action of these compounds. Both DDVP and malathion efficiently damaged DNA in microorganisms by inducing DNA strand breaks (Green et al., 1974; G r s i n , 1978). Apart from this, DDVP, which is a potential methylating agent, methylated isolated DNA and RNA from E . coli (Lofroth et al., 1969; Lofroth, 1970). Bridges et al. (1973) compared DDVP action with that of methyl methanesulfonate, a known methylating agent, and showed that DDVP activity was identical to that of methyl methanesulfonate and the results were consistent with their known ability to alkylate DNA. A further attempt to study the detailed process of alkylation was made by Lawley et al. (1974), who failed to detect the methylation product, 3-methyl guanine, in DNA from DDVP-treated E . coZi cells, although it was present in DNA treated in an isolated state. These findings were further confirmed in the same system by Wennerberg and Lofroth (1974). However, Green et al. (1974) observed that DDVP and methyl methanesulfonate do not differ in the type of strand breakage they cause, and they suggested that the major DNA damage in microorganisms resulting from DDVP treatment arose indirectly through alkylation and con-

EFFECTS O F INSECTICIDES ON MICROORGANISMS

189

sequent uncontrolled nuclease attack on DNA. Carere et al. (1978) attributed the mutagenicity to the presence of a vinyliden chloride group in DDVP, contrary to the suggestion of Bridges et al. (1973) that mutagenicity results from the phosphoric moiety of the molecule.

3. Eflects on Enzymes, Catabolic Pathways, and ATP Synthesis Plate cultures on the slime mold Polysphonclylium violaceum on exposure to vapors of DDVP or eserine, a specific inhibitor of enzyme acetylcholinesterase, showed an increase in the number of aggregation centers and sorocarps as shown in Fig. 6 (Clark, 1977). On the basis of these findings, Clark (1977) proposed that DDVP inhibits some enzymes, such as acetylcholinestrase or serine estrase, that are instrumental in determining the number of aggregation centers. However, Clark (personal communication) now does not believe that the DDVP effect on aggregation centers is mediated through acetylcholinesterase, because Polysphondylium violaceum produces a number of enzymes with esterase activity during development, and while some of these enzymes are DDVP-inhibited, none of these is cholinesterase. Indirect evidence for the involvement of malathion in the enzymes of catabolism comes from observations in Bacillus subtilis where malathion accelerated starch and saccharose breakdown but delayed the breakdown of sorbitol (Maleszewska, 1974). It has been shown that the phosphatase and dehydrogenase of the bacterial respiratory cycle are also susceptible to the inhibitory action of chlorofenvinphos (Rahmatullah et at., 1978). Some of the organophosphorus insecticides, such as diazinon, phorate, and chlorpyrifos, inhibited the ability of microorganisms to metabolize herbicides such as propham (isopropyl phenyl carbamate), chloropropham (isopropyl3chlorophenyl carbamate), diallate [ S-(2,3-dichloro-Z-propenyl) bis(l-methylethyl) carbamothioate], and triallate [S-(2,3,3-trichloro-2-propenyl) bis(1methylethyl) carbamothioate], as shown by several workers (Kaufman et al., 1970; Kaufman, 1977; Anderson and Domsch, 1980), thereby lengthening herbicidal activity. This effect can be attributed to the competitive inhibition of enzymes (involved in metabolism of herbicides) by organophosphorus insecticides. The inhibitory effect malathion exerted on citric acid production in Aspergillus niger was postulated to result from the direct action of insecticide on ATP synthesis (Rahmatullah et al., 1978). To test this hypothesis, ATP was introduced into growing cultures of fungi to which malathion had been added at 10, 50, or 90 pg/ml, and the citric acid content were measured (Rahmatullah et al., 1979). Malathion inhibited citric acid production in a dose-dependent manner. It was therefore concluded that malathion, by in-

FIG.6. Enhancement of center formation in Polysphondylium t;iolaceutn by DDVP and acetylcholine. (a) PvD7206 on Bonner’s Salt Solution (BSS); M acetylcholine chloride. All cultures were grown from spore suspensions spread on Cerophyl (b)PvD7206 on 3 X M DDVP; (c) PvD7206 on 3 X agar plates with Escherichiu coli B/r. Postvegetative cells were harvested in cold BSS. 50 pl of this suspension (2 x cells) were deposited on Millipore filters. Cultures were allowed to develop on filter pads saturated with BSS, with BSS containing 3 x A i acetylcholine chloride, or with BSS containing 3 X l W 3 A f DDVP. From Clark(1977). Reprinted by permission from Nature (London), Vol. 266, p. 171. Copyright 1977Macmillan Journals Limited.

EFFECTS OF INSECTICIDES ON MICROORGANISMS

191

hibiting the oxidative metabolism of A. niger, reduced the production of cellular ATP, resulting in the decrease in citric acid production.

VI. Summary and Future Prospects The accumulation, metabolism, and effects of organophosphorus insecticides have been reviewed. Organophosphorus insecticides have received little attention with regard to their ability to accumulate in microorganisms. Since some of the microorganisms are known to accumulate organophosphorus insecticides at levels many times higher than those in the surrounding medium, there is reason to believe that such activities may be far more common than has been assumed by the investigators. Microorganisms undoubtedly have the ability to metabolize organophosphorus insecticides. While an impressive beginning has been made in learning about the metabolism of some of the organophosphorus insecticides, the subject is still at a rudimentary state. Large gaps in our knowledge persist and the metabolic story for most of the organophosphorus insecticides now in common use is at best fragmentary and in need of being augmented. For instance, many of the metabolites have been characterized chemically, but certain water-soluble and solvent-soluble metabolites have been reported and remained unidentified. The difficult job of identifying these metabolites must be undertaken. Further, our ability to predict either the routes or rates of metabolism of organophosphorus insecticides in the microorganisms is also far from satisfactory. There are reports on microbial fission of benzene rings of insecticides (especially in case of parathion and diazinon). This ring fission is evident by the evolution of 14C02from 14C-radiolabeled ring carbon atom. However, there have been relatively few studies in which the routes and intermediates have been elucidated. It is also evident from some of the reports that have recently appeared in the literature that in microbial environments, the metabolism of the insecticide cannot be viewed with respect to microorganisms in isolation, but chemical, photochemical, physical, and biological factors may provide some different routes for the metabolism of insecticides in microorganisms. Hence, the subject of microbe-insecticide interaction has to be viewed in the light of biotic and abiotic factors. A synergistic relationship between microorganisms to metabolize organophosphorus insecticides has been reported. If such synergistic interactions are common, then there is every possibility that this may be responsible for the inability of many microorganisms to metabolize organophosphorus insecticides in pure cultures that are readily degraded in mixed microbial culture. By comparison, aquatic microorganisms (especially algae and protozoa)

192

RUP LAL

have received little attention with regard to their ability to metabolize organophosphorus insecticides. With the exception of some unicellular algae and some diatoms, data are lacking on their interaction with organophosphorus insecticides. Generally, the soil and the aquatic environments are considered more significant with respect to microorganisms, since inicroorganisms are more abundant here and play a significant role in inany key processes. Microorganisms 'are also present on the surfaces of animals and plants and are also common in the gastrointestinal tracts of higher animals. This group of microorganisms also needs some attention with respect to their role in the metabolism of organophosphorus insecticides. Organophosphorus insecticides are metabolized by enzymes in microorganisms. These insecticides also seem to be potential inducers of enzymes in inicroorganisms. Where some progress has been made to characterize hydrolase enzymes responsible for the metabolism of several. organophosphorus insecticides, data are lacking on the nature of the enzymes involved during oxidative and reductive reactions. An immobilized enzyme system was examined for its ability to hydrolyze residual insecticides in process wastewaters before their discharge into waste. An enzyme preparation was obtained from a bacterial mixed culture previously enriched for growth in parathion. The crude enzyme extract had the biochemical characteristics required for its industrial use with regard to temperature and pH optimum, stability, activity, and salt tolerance, and it was able to hydrolyze many other organophosphorus insecticides in addition to parathion. Thus, an enzymatic hydrolysis procedure for the organophosphorus insecticides would prove a significant improvement ovcr chemical hydrolysis (which is a slower process and requires large amounts of salt) in waste technology. It is also envisioned that the enzyme preparation could be commercially distributed directly with insecticide and could be used at a later date for container detoxification. However, the economics of this system cannot be evaluated until the industrial pilot-scale results indicate the operational stability of the immobilized enzyme system and what auxilliary pretreatment steps are required to maintain the same chemical and physical operating conditions required for enzyme stability. It is not possible to reach a general conclusion regarding the effects of organophosphorus insecticides on microorganisms, since the available information will not permit it. In general, few organophosphorus insecticides have great or prolonged adverse effects on the total microbial population in soil or in water. An adverse influence of insecticide occurs only at applications of higher concentration. The usual pattern is one of an initial decrease in total number followed by a return to normal or even increase in number. Organophosphorus insecticides have also been shown to reduce or increase

193

EFFECTS OF INSECTICIDES ON MICROORGANISMS

the number of dinitrifiers, ammonium oxidizers, nitrate oxidizers, etc., which will influence microbial activity in relation to the environment. Most of the work on the effects of insecticides on microorganisms has been interpreted in response to microorganisms in isolation, but physiochemical and biological factors that are of utmost significance have not been considered. Further specific data can be obtained only by using axenic cultures under controlled experimental conditions, but care must be taken when relating such findings to what may occur in nature under natural conditions. Little is known about the cytological and biochemical action of organophosphorus insecticides in microorganisms. In autotrophic microorganisms, organophosphorus insecticides interfere with photosynthetic activity by reducing chlorophyll production or by inhibiting photosynthetic phosphorylation, thereby reducing ATP production. In fungi, organophosphorus insecticides reduce energy production by their interference with the respiration cycle. Organophosphorus insecticides such as DDVP and malathion are mutagenic and capable of alkylating DNA, which may alter the synthesis of many enzymes and their activity, producing many morphological and physiological changes, as reported by Clark (1977) in Polysphondylium violaceum on DDVP treatment. Low concentrations of organophosphorus insecticides have been found to partially inhibit the metabolism of herbicide in microorganisms. A common explanation indicating the competitive inhibition of enzymes has emerged recently. This may be correct, as it seems, but there is no experimental data to support it, To evaluate the general occurrence and possible ecological significance of this pesticidal effect, studies are also needed to determine the agriculturally feasible combinations of chemicals applied to soil at recommended concentrations and in a chronological sequence similar to that needed in the field. Appendix CHEMICAL DESIGNATION OF THE ORGANOPHOSPHORUS INSECTICIDES MENTIONED IN TEXTBY COMMON on TRADENAMES Common or trade name

THE

Chemical name ~

Acephate Aspon Azinop hos-M Azodrin

BaytedBayer 29493

0,s-Dimethyl acetyl phosphoramidothioate O,O,O,O-Tetra-n-propyl dithiopyrophosphate 0,O-Dimethyl S-(4-oxobenzotriazine-3-methyl) dithiophosphate 0,O-Dimethyl 0-(2-methylcarbarnoyl-l-methylvinyl) phosphate 0,O-Dimethyl 0-(3-methyI-4-methylthiophenyl) phosphorothioate

194

RUP LAL

Corninoir

01

trade namc Hron1ophos Carbophenothion Carbofos Chlorfcanvinphos Chlorpyrifos C:yallophos

Dasanit DEF Diazinon 1Iil)ruin Dichlorvos Diinc+hoatr Uipteres Disullhton Ui-Syston Diirshan Dylilnatl. Dylox t.: PN Kthion E tho["'"p Fenumiphos Feniti-othion Fensulfothion Fcnthion Fonofos Gardonn Grithion L(ytopll0s Malathion

Metaphos M<4a-Sy\tox

Methyl p m i t h i o n Mcthyl trithion Mwinphos

Monocrotophos ()rthcw~

Parathion P~ir2ithion-inetliyl Pliorate

Phosmc~t

Chemical name

0-(4-Bromo-2,5-dichlorophenyl) 0,O-diethyl phosphorothioate S-(4-Chlorophenylthiomethyl) O,O-diethyl phosphorodithioate S -1 1,2-Di(ethoxycarhonyl)ethyl]dimethyl phosphorothiolothionate 2-Chloro-1-(2,4-dicl~lorophe11yl)vinyl diethyl phosphate O,O-Diethyl 0-(3,5,6-trichloro-2-pyridinyl)phosphorothioate 0 ,O-llimethyl 0-(~1-cyanophenyl)thionophosphate Diethyl 4-(methylsulfinyl)pheny1 phosphorothioate hutyl phosphorothioate 0.0-Diethyl O-(2-isopropyl-6-methyl-4-pyrimidinyl) phosphorothioate O,C)-Dinrethyl 0-(1,2-dibrorno-2,2-clichloroethyl)phosphate 2.2-Dichlorovinyl dimethyl phosphate Dimethyl S-(M-tnethylcarbamoylmethyl) phosphorothiolothionate phosphonate Ihnethyl 2,2,2-trichloro-l-hydroxyethyl O,O-Diethyl S-[Z-(ethylthio)ethyl] phosphorodithioatte Sec Ui-Syston See Chlorpyrifos O-Ethyl S-phenyl ethyl Phosphoilodithioate See Dipterex 0 - E t h y l 0-4-nitrophenyl phenyl phosphonothioate 0,0,0',0'-Tetraethyl S,S '-methylene bisphosphorodithioate 0-Ethyl S,S-dipropyl phosphorothioate 0-Methyl 0-(3-methyl-4-methylthiophenyl)isopropyl phosphoramitlate O,O-Dimethyl 0-(3-methyI-4-nitrophenyl) phosphorothioate See Dasanit See BaytedBayer 29493 see Dylunate (Z)-2-Chloro-1-(2,4,5-tetr~chlorophenyl)et~1enyl dimethyl phosphate See Azinophos-M O-(4-Rromo-2,5-dichlorophenyl) 0-methyl phetryl phosphonothioate See Carbofos 0,O-l)imethyl 0-p-nitrophenyl phosphorothioate Mixture of O,O-tlimethyl 0-(2-ethylthio)ethyI phosphorothioate and 0,O-dimethyl S-(e-ethylthio)ethyl phosphorothioate 0.S-Dimethyl phosphorainidothioatc S-[(5-Methoxy-~-oxo-1,3,4-thiadiazol-3(II)-yl)~nethyl] 0,O-dimethyl phosphorodithioate See Metaphos O,O-Dimethyl S-(p-ch1orophenylthio)methyl phosphorodithioate 2-Mothoxycarhonyl-1-methylvinyldimethyl phosphate See Azodrin See Acophato O,O-l>iethyl 0-p-nitrophenyl phosphorothioate

O,O-Diethyl S-(ethylthio)methyl Pliosph0'0dithioat.c~horo~ithioat~ 0,O-Dimethyl phthalimidoinetllyl pliosphoroditl~ioate

EFFECTS OF INSECTICIDES ON MICROORGANISMS

Common or trade name Phosphamidon Schradan Systox TEPP Temephos Terhufos Tetrachlorvinphos Thimet Thionazin Triazophos Trichlorfon Triclrloronat Trithion Vapona

195

Chemical name 2-Chloro-3-(diethylamino)-l-methyl-3-oxo-l-propenyl dimethyl phosphate Octanrethyldiphosphoramide 0,O-Diethyl 0-ethylmercaptoethyl thiophosphate Tetraethyl pyrophosphate 0,0’-(Thiodi-4.l-phenylene)bis(0,O-dimethyl phosphorothioate) S-(t-Buty1thio)irietliyl 0,O-diethyl phosphorodithioate See Gardona See Phorate Diethyl 2-pyrazinyl phosphorothionate 0,O-Diethyl O-(l-phenyl-1,2,4-triazol-3-y1) phosphorothioate See Dipterex Ethyl-2,4,5-trichlorophenyl ethyl phosphonothionate See Carbopheriothion See Dichlorvos

The author wishes to express his sincere thanks to Dr. S. Shivaji for his valuable comments which have done much to improve thc manuscript. Thanks are also due to Dr. S . Gupta for reading the manuscript; to Dr. H . S . Attri for making the recent literature available; and to Drs. Ravi Kant, J. P. Khuraria, B. V. Prasad Reddy, and S . K. Khurana for their dedicated assistance during the preparation of this manuscript.

REFEHES(:C:S

Ahmed, M. K., and Casida, J . E. (1958). J . Econ. Entomol. 51, 59-63. Ahmed, M . K . , Casida, J. E., and Nichols, R. E. (1958). J. Agric. Food Chein. 6, 740-746. 7 , 35-30, Alexander, M. (1965a). A&. A p p l . Mi~~obiol. Alexander, M .(1965b). Proc. Soil. SOC. Atti. 29, 1-7. Ali, M. S., Rahlnatullah, M., Jahan, R., Yusuf, H. K. M., and Chowdhury, A. A. (1979). Enzyin. Microh. Techno!. 1, 127-128. Anderson, J. P. E., and Domsch, K. €1. (1980). Arch. Enuiron. Contam. Toricol. 9, 115-123. Audus, L. J. (1960). I n “Herbicide and the Soil” (E. K. Woodford and G. R. Sager, eds.), pp. 1-19. Blackwell, Oxford. Bache, C. A . , and Lisk, D. J . (1966). 1. Assoc. Off A n d . Chem. 49, 647-650. Ballington, P. E., Skipper, H. D., and Hegg, R. 0. (1978). J . Enairon. Qual. 7, 262-264. Barles, R. W., Daughton, C. G . , and Hsieh, D. P. H. (1979). Arch. Enuiron. Contam. Toxicol. 8, 647-660. Bollag, J . M. (1974). Adz;. A p p l . Microbial. 18, 75-130. Bollag, W. B. (1961). Annu. Rev. Microhiol. 15, 59-92. Bollen, W. B., Morrison, H. E., and Crowell, H. H. (1954). J. Econ. Entomol. 47, 302306. Bourquin, A. W. (I977a). A p p l . Enuiron. Microhiol. 33, 356362. Bonrquin, A. W. (1977h). J . Enairon. Quai. 6, 373378.

196

RUP LAL

Boush, G. M., and Matsumura, F. (1967). J . Econ. Entorno!. 60, 918-920. Bridges, B. A,, Mottershead, R. P., Green, M. H. L., and Gray, W. J. H. (1973). Mutat. Res. 19, 2955303. Bro-Rasmussen, F . , Noddegaard, E., and Voldum-Clansen, K. (1968). J. Sci. Food Agric. 19, 278-282. Burpee, L. L., and Cole, H. (1978). Bull. Enoiron. Contain. Toxicol. 19, 191-197. Bushway, R. J. (1978). Diss. Abstr. Znt. B38, 5329. Butler, G. L. (1977). fiesidue Reu. 66, 1962. Butlcr, C . L., Deason, T. R . , and O'Kellry, J. C. (1975). Bull. Enoiron. Contain. Toricol. 13, 149-152. Carere, A , , Ortali, V. A , , Cardamone, G . , and Morpurgo, G . (1978). C h e m Biol. Interact. 22, 297308. Clark, M . A. (1977). Nature (Lontlon) 266, 170-172. Clegg, T. J . , and Koevening, J. L. (1974). Bot. Gaz. (Chicago) 135, 368372. Cole, D. R., and Plapp, F. W. (1970). Enoiron. Entomol. 3, 217-220. l. Congregado, F., Simon-Pujol, D., and Jaurez, A. (1979). Appl. Enoiron. M i ~ ~ o b i o37, 169-171. Cook, J. W. (1957). 1. A y i c . Food Cheni. 5, 859-863. v K112-114. J~. Corey, R . A. (1965). J . Eco11. E ~ L ~ ~ 58, Cowley, G . J.. and Lichtenstein, E. P. (1970). J . Gen. hficrohiol. 62, 2 7 3 4 . Cox, J. L. (1972). Residue Reo. 44, 23-28. Daitloff, A. (1970). Aust. J . E x p . Agric. Anim. Hush. 10, 562-567. Daughton, C. G., and Hsieh, D. P. €1. (1977a). Bull. Rnoiron. Contain. Tmicol. 18, 48156. Daughton, C. G . , and Hsieh, D. P. H. (1977h). Appl. Enoiron. Mimohiol. 34, 175-184. DaSilva, E. J., Henrikisson, L. E., and Henriksson, E. (1975). Arch. Enoiron. Contam. Toxicol. 3, 193-204. Derby, S . B., and Ruher. E. (1970). Bull. Enoiron. Contum. Toxicol. 5, 553-558. Deway, J. E., and Parker, B. L. (1965). J . Ecoti. E n t o d . 58, 491497. Draughon, F. A., and Ayres, J. C. (1978). 1. Food Sci. 43, 576-578. Flashinski, S. J . , and Lichtenstein, E. P. (1974a). Can. J. Microbiol. 20, 399-411. Flashinski, S. J., and Lichtenstein, E. P. (1974b). Can. J. Miwobio1. 20, 871472. Gawaad, A. A. A . , El-Minshawy, A. M., and Zeid, M. (1972). Zentralbl. Bakteriol. 127, 290-295. Gawaad, A. A. A., Hammad, M . H . , and El-Gayar, F. H. (1973). Agroketn-Talojtan 22, 161168. Getzin, L. W. (1967). J. Econ. Entoinol. 60, 505-508. Getzin, L. W., and Chapman, R. K. (1960). J . Econ. E n t o n d 53, 47-51. Getzin, L. W., and Hosefield, I. (1968). J. Econ. Entomol. 50, 512-516. Graetz, D. A., Chesters, G., Daniel, T. C . , Newland, L. W., and Lee, C. B. (1970). /. Wutw Pollut. Control Fed. 42, 76-94. Grecn, N. H. L., Medcalf, A. S. C . , Arlett, C. F . , Harcourt, S. A., and Lehmann, A. R. (1974). M u t o t . Res. 24, 365377. Gregory, W. W., Reed, 1. K., and Priester, L. E. (1969). J . Protozool. 16, 69-71. Griffin. D. E. (1978). iMutat. Res. 52, 161-169. Gundersen, K., and Jensen, H. L. (1956). Acta Agric. Scond. 6, 100-114. Gunner, H. B. (1970). Medetl. Fac. Zdantlbouww. Rzj'ksunio. Gent 35, 581-597. Gunner, H. H . , and Zuckerman, B. M. (1968). Nature (London) 217, 1183-1184. Gunner, H. B . , Miller, C. B., Deubert, K. H . , and Longley, K. E. (1966). Plant Soil. 25, 249-264. Harris, C. R . , and Lichtenstein, E. P. (1961). J. Econ. Entoniol. 54, 1038-1045.

EFFECTS OF INSECTICIDES ON MICROORGANISMS

197

Hasegawa, T. (1959). Fukuoka Acta Med. 50, 1900-1906. Hegazi, N . , Momb, M., Beld, M., Amer, H., and Farag, R.S. (1979). Arch. Enoiron. Contam. Toxicol. 8, 629-635. Hirakoso, S. (1968). World Health Organ. WHOiVBC 68, 1 4 . Hsu, T. S., and Bartha, R. (1979). Appl. Entiiron. Microbiol. 37, 36-41. Hughes, D. N., Boyer, M. G . , Papst, M. H., and C. D. Fowle (1980). Arch. Enuiron. Contam. Toxicol. 9, 269-279. Hurlbert, S . H. (1975). Residue Reo. 57, 81-198. Hurlbert, S. H., MulIa, M. S., and Willson, H. R. (1972). Ecol. Monogr. 42, 269-299. Jakubowska, B., and Nowak, A. (1973). Zesz. Nauk. Akad. R o h Szczecinie. 10, 141-150. Johnsen, R. E. (1976). Residue Reu. 61, 1-28. Kannaiyan, S. (1980). Proc. S y m p . Pesticide Residues Enoiron. India pp. 492-494. Kapusta, G., and Rouwenhorst, D. L. (1973). Agron. J. 65, 112-115. Kasting, R., and Woodard, J. C. (1951). Sci. Agric. 31, 133-138. Kaufman, D. D. (1965). J. Agric. Food Chem. 15, 582-591. Kaufman, D. D. (1977). Soil Biol. Biochem. 9, 49-57. Kaufman, D. D., Kearney, P. C., VonEndt, D. W., and Miller, D. E. (1970). J . Agric. Food Chetn. 18, 513-519. Kishk, F. M . , El-Essawi, T., Abde-Ghafar, S . , and Abou-Donia, M. B. (1976). J . Agric. Food Chenl. 24, 305307. Konard, J. D., Armstrong, D. E., and Chester, G. (1967). Agron. J . 59, 591-594. Konard, J . D. Chester, G., and Armstrong, G. (1969). Soil Sci. Soc. Am. Proc. 33, 259-262. Kulkarni, J. H., Sardeshpande, J. S., and Bagyaraj, D. J. (1974). Plant Soil 40, 169-172. Lal, Rup and Saxena, D. M. (1980). Residue Reu. 73, 4 9 4 6 . La], Rup and Saxena, D. M. (1982). Microbial. Reu. 46 (in press). Lamoreaux, R. J., and Newland, L. W. (1978). Chemosphere 7, 807-814. Lawley, P. D., Shah, S. A , , and Orr, D. J. (1974). Chem. Biol. Interact. 8, 171-182. LePatourel, G. N. J . , and Wright, D. J. (1976). Comp. Biochem. Physiol. 53, 73-74. Lewis, D. L., Paris, D. F., and Baughton (1975). Bull. Enuiron. Contam. Toxicol. 13, 596-601. Lichtcnstein, E. P. (1966). Natl. Acad. Sci. Natl. Res. Counc. Publ. 1402, 221-229. Lichtenstein, E. P., and Schulz, K. R. (1960). /. Econ. Entomol. 54, 517-522. Lichtenstein, E. P., and Schulz, K. R. (1964).J . Econ. Entomol. 57, 618-627. Lichtenstein, E. P., and Schulz, K. R. (1970). J . Agric. Food Chem. 18, 814-818. Lichtenstein, E. P., Fuhremann, T. W . , and Schulz, K. R. (1968).J . Agric, Food Chem. 16, 870. Lin, S. C., Funke, B. R., and Schulz, J. T. (1972). Plant Soil 37, 489-496. Lofroth, G. (1970). Naturwissenschaften 57, 393394. Lofroth, G . , Kim, C., and Hussain, S . (1969). Enuiron. Mutagen. Soc. News Lett. 2, 21-27. McCann, J., Choi, E., and Ames, B. N. (1975). Proc. Natl. Acad. Sci. U.S.A. 73,51354139. Mackicwicz, M., Deubert, K. H., Gunner, H. B., andzuckerman, B. M. (1969). J. Agric. Food Chem. 17, 129-130. Mahmoud, S . A. Z . , Taha, S . M., Abdel Hafez, A. M., and Hamed, A. S. (1972). Egypt. J . Microhiol. 7, 39-52. Maleszewska, T. (1974). Pol. Arch. Hydrobiol. 21, 163-171. Matsumura, F. (1973). In “Survival in Toxic Environments” (M. A. Q. Khan and J. P. Bederka, Jr., eds.), pp. 129-152. Academic Press, New York. Matsumura, F. (1975). I n “Toxicology of Pesticides,” pp. 325354. Plenum, New York. Matsumura, F., and Benezet, H. S. (1978). In “Pesticide Microbiology” ( I . R. Hill and S. J. L. Wright, eds.), pp. 623-667. Academic Press, New York. Matsumura, F . , and Boush, G . M. (1966). Science 153, 1278-1280.

198

RUP LAL

Matsumura, F., and Boush, G. M. (1968). J. Econ. Entotnol. 16, 610-612. Mentfoza, C. (1973). An. lnst. Nac. Inuest. Agrar. Ser. Cen. 2, 2 1 3 5 . Menn, J. J . , McBain, J. B., Anderson, A. J., and Patchett, G. G . (1965). J. Econ. E n t o d . 58, 875-878. Mick, K. L., and Dahm, P. A. (1970). I . Econ. Entomol. 63, 1115-1159. Miles, J. R. W., Tu, C. M., and Harris, C . R. (1979). Bull. Enoiron. Contam. Toxicol. 22, 312318. Miller, C. W., Zuckerman, B. M., and Charig, A. J . (1966). Trans. A m . Fish Soc. 95,345349. Miller, C. W., Tomlinson, W. E., and Norgren, R. L. (1967). Pestic. Monit. /. 1, 4 7 4 8 . Miyamoto, J. (1977). Natl. Res. Counc. Can. Ottawa pp. 459-496. Miyamoto, J., Kctagawa, K., and Sato, Y. (1966). J p n . J. E x p . Med. 36, 211-225. Miyamoto, J., Takimoto, Y., and Mihara, K. (1979). ACS S y m p . Sex 99, 3-20. Moore, R. B. (1970). Bull. Enukun. Contam. Toxicol. 8, 129-137. Mostafa, I. Y., Fakhr, I. M. I., Bahig, M . R. E., and El-Zawahry, Y. A. (197%). Arch. Mikrobiol. 86, 221-224. Mostafa, I. Y., Bahig, M. R. E., Fakhr, I. M . I., and Adam, Y. E. (197213). Z. Naturfmsch. 27, 1115-1116. Mounter, L. A., and Tuck, K. U . (1956). J. B i d . Chem. 221, 537-542. Munriecke, D. M. (1976). Appl. Enoiron. Microhiol. 32, 7-13. Munnecke, D. M. (1979). Biotech. Rioeng. 21, 2247-2261. Munnecke, D. M. (1980). J. Agric. Fuod Chein. 28, 105-111. Munnecke, D. M., and Hsieh, P. II. (1974). Appl. MinobioE. 28, 212-217. Munnecke, D. M., and Hsieh, P. H. (1975). Appl. Mi~rohiol.30, 575-580. Murray, H. E . , and R. K. Guthrie (1980). Bull. Enuiroii. Contain. Toxicol. 24, 535-542. Naumann, K. (1960). Soils Fert. 23, 1356. Naumann, K. (1967). Photopathol. Z. 60, 343357. Naumann, K. (1970a). Zentralbl. Bakteriol. Abt. 124, 743-754. Naumacin, K . (1970b). Zentralbl. Bokteriol. Abt. 124, 755-765. Naumann, K. (1971). Pedobiologia 11, 286-295. Naumann, K. (1972). Zeiitralbl. Bakteriol. Abt. 127, ,379396. Nelson, J. H., Stoneburner, D. L., Evans, E. S., Jr., Pennington, N. E., and Meisch, M. V. (1976). Bull. Enoiron. Contum. Toxicol. 15, 630434. Oshida, H., Tochinloto, H., Sasano, H., Nakamura, H., Tomorrari, H . , Kikuchi, Y., and Ikejima, N . (1978). Tokyo Toritsu Eisei Kenkyusho Neinpo 29, 362366. Paris, D. F., Lewis, D. L., Barnett, J. T., Jr., and Baughinan, G. L. (1975a). Report No. EPA 660/3-75.007. Environmental Protection Agency, Cincinnati, Ohio. Paris, D. F . , Lewis, D. L., and Barnett, J. T. Jr. (1975b). Notl. Tech. Inform S m . 293, p. 54. Paris, D. F., Lewis, D. L., Barnett, J. T., and Bangham, G . L. (1975~).Notl. Tech. Inform. Sero. 293, p. 45. Pfister, R. M. (1972). Crit. Rev. Microhiol. 2, 133. Ponnanipctruma, F. N. (1972). Ado. Agron. 24,29-96. Poorman, A. E . (1973). Bull. Enoiron. Contam. Toxicol. 10, 25-28. Rahmatvrllah, M., Jahan, R . , Chowdhury, A. A , , and Faruk, A. A. M. (1978). J. Fermeut. Technol. 56, 169-172. Rahmatullah, M . , Ah, M. S . , Yusuf, 11. K. M . , Jahan, R., Asuduzzaman, J., and Chowdhury, A. A. (1979). J. Ferment. Technol. 57,379381. Rajaram, K. P . , and Sethunathan, N. (1975). Soil Sci. 119, 296300. Rajaram, K. P., and Sethunathan, N. (1976). Plant Soil 44, 683-690. Rao, A. V., and Sethunathan, N. (1974). Arch. Mierobiol. 97, 203-208. RdSt, H. G., Engelhardt, G., Wallnoefer, P. R . , Ochlmann, L., and Wagner, K. (1979). J.

E F F E C T S OF INSECTICIDES ON MICROORGANISMS

199

Agric. Food. Chein. 27, 669-702. Raymond, D. G. M . , and Alexander, M . (1971). Pest. Biochein. Physiol. 1, 123-130. Rodell, S., Funke, B. R., and Schulz, J. T. (1977). Plant Soil 47, 375381. Rosenberg, A , , and Alexander, M . (1979). A p p l . Environ. ht‘iwubiol. 37, 886-891. Ross, D. J . (1974). N.Z. J. Agric. Res. 17, 9-17. Roy, G., Kasturi, L. D., and Pearl, W. (1980). I . Agric. Food Chern. 28, 102-105. Salama, A. M., Mostafa, I. Y., and El-Zawhry, Y. A. (1973). Acta B i d . Acnd. Sci. Hung. 24, 2530. Salaina, A. M., Mostafa, I. Y . , and El-Zawahry, Y. A. (1975). Acta B i d . Acad. Sci. Hung. 26, 1-7. Salem, S. H. (1971). Agrokenz. Talajit 20, 368-376. Salem, S. H., and Gulyas, F. (1971). Agrokmiz. Tnlajit 20, 377388. Salem, S. H., Szegi, I., and Gulyas, F. (1971). Agrokem. Talnjit 20, 581-582. Salem, S. H . , Harned, A. S.,Zohdy, L., and Sahab, A. F. (1977). Acta Phytopccthol. Acnd. Sci. Hung. 12, 2 3 3 0 . Salk, M . S. (1976). Diss. Abstr. Znt. B36, 3749. Salonins, P. 0. (1972). J. Econ. Lntoinol. 65, 1089-1090. Sato, R., and Kubo, H. (1964). Ado. Watw Pollut. Res. 1, 95-99. Sethunathan, N . (1973). Residue Rev. 47, 143-165. Sethunathan, N . , and MacRae, I. C. (l969a). J. Agric. Food Chenr. 17, 221-225. Sethunathan, N . , and MacRae, I. C. (1969b). Plant Soil 30, 109-112. Sethunathan, N., and Pathak, M . D. (1971). Can. J. Microhiol. 17, 699-702. Sethunathan, N . , and Pathak, M . 13. (1972). J . Agric. Food. Chern. 20, 586-589. Sethunathan, N . , and Yoshida, T. (1969). J . Agric. Food CAem 17, 1192-1195. Sethimathan, N . , and Yoshida, T. (1972). Proc. Znst. Enoiron. Sci. N.Y. M e t . 18, 255-257. Sethunathan, N . , and Yoshida, T. (1973). Can. J . Microbid. 19, 873-875. Sethunathan, N . , Siddaramappa, R., Rajarain, K. P., Barik, S . , and Wahid, P. A . (1977). Residue Rev. 68, 91-122. Shiau, S . Y . , Huff, R. A , , Wells, B. C . , and Felkner, I. C. (1980). Mutat. Res. 71, 169-179. Siddaramappa, R . , Rajaram, K. P., and Sethunathan, N . (1973). A p p l . Mimohiol. 26,846-849. Singh, P. K . (1972). Proc. 13th Annu. Conf. Assoc. Microbial. Zndia 56. Sivasithamparam, K. (1969). Proc. Ce!ylonAssoc. Ado. Sci. 25, 1. Sivasithamparain, K. (1970a). J . Gen. Micwhiol. 42, 293300. Sivasithamparam, K. (1970b). Riso 19, 339346. Spain, J. C. (1879). Diss. Abstr. Znt. B40, 1167-1168. Spillner, C. J . , DeBaun, J. R . , and Menn, J. J. (1979a). /. Agric. Food Chenr. 27, 1054-1060. Spillner, C . J., Thamas, V. M . , and DeBaun, J. R. (1979b). Bull. Enoiron. Contain. Toxicol. 23, 60 1-606. Stadnyk, L., Camphell, R. S., and Johnson, €3. T. (1971). Bull. Environ. Contain. 7uxicol. 6, 1.3, Stanlake, G. J., and Clark, J. B. (1975). A p p l . Miwobiol. 30, 3 3 5 3 3 6 . Staphorst, J. L., and Strijodom, B. W. (1974). Ph!ytophyZactica 6, 205-208. Stetierseti, J. (1969). Bull. Rnuiron. Contarn. Toxico!. 4, 104-112. Stewart, D. K. R . , Chisholm, C., and Rogab, M . T. H. (1971). Nature (Imirlon) 229, 47. Suddakar-Barik, and Sethunathan, N. (1978). J. Enoiroit. Qutrl. 7, 346348. Suddakar-Barik, Siddaramappa, R., and Sethunathan, N . (1976). Aritonie Van Leeuwenhoeck 42, 461470. Suddakar-Barik., Wahid, P. A , , Rarnakrishna, C., and Sethunathan. N. (1979). J. Agrit.. Food C,’he~n.27, 1391-3392. Swamiappari, N . , and Chandy, K. C. (1975). Cum. Sci. 44, 558.

200

RUP LAL

Swaminathan, R., and Sullia, S . B. (1969))). Curr. Sci. 38, 282-284. Sweeney, R. A. (1968). Algae as indicators of pesticides. AlBS Meet. Columbus, Ohio, Sept. 4 pp. 1-13. Tiedje, J. M., and Alexander, M. (1967). Agron. Abstr. 94. Torres, A. M. R., and O'Flaherty, L. M. (1976). Phyculogin 15, 2536. Trela, J. M., Ralston, W. J., and Gunner, H. B. (1968). Bacteriol. Proc. p. 6. Tu, C. M. (1969). Bucterid. Proc. p-3. Tu, C. M . (1970). AppE. Microbid. 19, 479484. Tu, C. M. (1977). Bull. Enoiron. Contam. Toxicol. 18, 190-199. Tu, C. M. (1978a)).Bull. Enoiron. Contam. Toxicol. 20, 212-218. Tu, C. M. (1978b). Conimun. Soil Sci. Plant A n d 9, 629-6313, Tu, C;. M., and Miles, J . R. W. (1976). Residue Reo. 64, 1745. Ukeles, R. (1962).Appl. Microbid. 10, 532537. Visalakshi, A , , and Nair, M. R. G. K. (1980). Pruc. Sy7n. Pesticide Residue Enoiron. India pp. 481-486. Wainwright, M. (1978). J . Soil Sci. 29, 287-298. Walker, W. W.. and Stojanovic, B. J. (1973). J . Enoiron. Quai. 2, 229-232. Walsh, G. E., and Alexander, S. V. (1980). Water Air Soil Pollut. 13, 45-55. Ware, G. W., and Roan, C. C. (1970). Residue Reu. 33, 1545. Weber, K. (1976). Water Res. 10, 237-241. Wennerberg, H.,and Lofroth, G. (1974). Cheni. B i d . Interact. 8, 339348. Westacott, D., and Wright, S . J. L. (1975). Pest. Sci. 6, 6 1 4 8 . Whitney, W. K. (1967). J . Econ. Entoniol. 60, 68-74. Williams, P. P. (1977). Residue Reo. 66, 63-135. Wolfe, H. R., StaiE, D. C., Armstrong, J. F., and Comer, S . W. (1973). Bull. Enuiron. ContaTtt. Toxicol. 10, 1-9. Wood, P. A,, and MacRde, I. C. (1974). Bull. Enuiron. Contam. Toxicol. 12, 26. Yasuno, M . S. H . , Sasa, M., and Uchida, M. (1965). J. Exp. Med. 35, 545-563. Yurovskaya, Y. M., and Zhulinskaya, V. A. (1974). Soils Fert. 38, 5026. Yurovskaya, E. M . (1975a). Gig. Snnit. 9, 102-103. Yurovskaya, E. M. (1975b). J . Hyg. Epidemiol. Mi~7obiol.Iinmunol. 19, 10-16. Zayed, S . M. A. D., Hassan, A., and Hussein, T. M. (19652). Z. Naturfmsch. 20, 587-591. Zayed, S . M. A. I)., Mostafa, I. Y., and Hassan, A. (196%). Arch. Mikrobiol. 51, 118-121. Zidan, Z. H., and Ramdan, E. N. (1976). Egypt. J . Microbid. 11, 93-99. Zuckerman, B. M., Deubert, K., Mackiewicz, M . , and Gunner, H. (1970). Plant Soil 33, 273-281.

Solid Substrate Fermentations K. E. AIDOO,~R. HEN DRY,^ AND B. J. B. WOOD^ BiotechnoEogy Unit, University of Strathchyde, Glasgow, Scotland

......... ...................... A. Pre-Pasteur Perio ........................ tit I~evelopments. . . . . . . . 111. Design Considerations and Types ~f Solid-State Fermen tors . . . . . . . . . . ........................ I . Introduction . . . . .

..........

11.

IS

IV. Characteristics of Solid-State Fcrincntations Physiological Aspects of Microbial Growth on Solid Substrates . . . . . . . . . . . . . . V . Advantages and Disadvantages of So VI . Future Developments of Solid-state Fermentation Systems , . VII. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . ...

201 202 202 208 224 226 227 229 231 232 233

I. Introduction From the beginning of his history, man has made use of microorganisms for his benefit, and solid-state fermentation processes have played an important role. The term “solid-state fermentation” is defined as any fermentation process in which the substrate is not a free liquid. “Solid-state” and “solidsubstrate” fermentations are terms used by different workers but they are essentially the same. For the purpose of this review, solid-state fermentation is used to mean any fermentation that takes place on solid or semisolid substrate or that occurs in a nutritionally inert solid support, which provides some advantage to the microorganism with respect to access to nutrients. Such processes have been employed in the production and preservation of a variety of foods, and the production of enzymes, organic acids, antibiotics, and other microbial products. The type of solid substrate fermentation in which organisms grow on or in solid particles floating in a liquid medium is not considered in this review. Fermentors used in solid-state fermentation systems vary from a simple open pan or tray to mechanized rotating drums, used at one time for the production of gluconic acid. Over the last 20 years, deep-tank or submerged-culture methods have been used for the production of hngal ‘Present address: Department of Biological Sciences, University of Science and Technology, Kumasi, Ghana. 2Department of Chemical and Process Engineering. 3Department of Applied Microbiology. Author to whom inquiries regarding this contribution should be addressed. 20 1 ADVANCES IN APPLIED MICROBIOLOGY, VOLUME 28 Copyright 0 1982 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-002628-7

202

K. E. A I D 0 0 ET AL.

enzymes instead of surface-culture methods or solid-state fermentations, and, according to Arima (1964), this is because deep-tank methods allow better control of environmental factors such as temperature and pH. Solidstate fermentations may be processes with or without continuous rotation of the fermenting mass. In our studies (Aidoo, 1979) on some solid-state fermentations with mechanized solid-state fermentors, we found that the coniparative ease of running solid-state fermentors and particularly their low energy requirements in comparison with stirred tank reactors suggest that these ferinentors have potentially great economic importance, for both the industrialized and the developing nations. At present, solid-state fermentation processes are used on a commercial scale for the production of different types of fermented foods, particularly in the Orient and in some developing countries (Platt, 1962; Hesseltine, 1965; Yong and Wood, 1974). In addition to their use with foods, such processes have been used successfully in recent years for large scale production of hngal metabolites (Hesseltine, 1972, 1977) and for bioconversion of plant, animal, and domestic wastes into useful products (Anthony, 1971; Smith, 1973; Han et al., 1974, 1975, 1976; Rhodes et al., 1975; Rolz, 1978; Stanton, 1978; Detroy and Hesseltine, 1978; Moo-Young et al., 1979). This review is an attempt to trace the history of the growth of microorganisms-fungi, yeast, and bacteria-on solid substrates, a process that has led in recent years to the development of‘ the term “solid-state fermentation,” and to assess its importance to man. Some attention has been given to the contribution of fungi to man’s food supply through solid-state fermentations. The historical development is presented in two parts: (1)from prehistoric times to about 1860, i.e., the pre-Pasteur period, and (2) from about 1860 to the present day, although some of the fermentation processes classified under this group may well have existed before 1860-for example, the fermented foods found in Africa, Asia, and South America.

II. History of Solid-state Fermentations A. PRE-PASTEUR PERIOD The use of microorganisms in the preparation of foods (bread, cheese, and many others) and directly as food (edible fungi) dates back many centuries and these are some examples of solid state fermentation systems. Breadmaking is one ofthe oldest arts known to man. Archaeological discoveries indicate that the earliest “milling” implements, dating back some 75,000 years, consisted merely of cup-shaped stones to hold the grain and round stones to pound it (Horder et al., 1954).It is still not known when the Egyptians started making bread, but the discovery of the fermentation is

203

SOLID SUBSTRATE FERMENTATIONS

TABLE 1 EXAMPLESOF ~

~~

Examples Bread dough formation Cultivation of mushroom Common mnshroom Padi-straw mushroom Japanesc mushroom Cheese (Roquefort) Production of Takadiastase Koji process Soy sauce Miso

SAC Tempeh Red rice Ontjom Soy cheese Ragi Production of citric acid Production of aflatoxin Rotting and decay of wood Vinegar production Leaching of sulfide minerals Secondary treatment of sewage in trickling filter beds Oil degradation in soil

~

FERMENTATION'

SOLID-STATE ~

~

Microorganisms involved

Saccharom yces cerevisiue , Lactohucillus sanfrunciscob Bacteria and fungi involved in compost formation Agaricu.r hispnrus

Volvariellu vnlvuceae Lentinus edodm Penicillium roqueforti Aspergillus oryzue

A . oryzue A . oryzae, Sacchuromyces rouxii A . oryzue Rhizopus strains Monascus purpureus Neurospora sitophilrr Actinomucor eleguns, etc. Mucor, Rhizopus, and yeast A . nigw

Certain strains of A . jlcwus and A . parasiticus Higher fringi

Principal energy source Carbohydrates of dough mix

Hemicellulose of strawmanure component Rice straw Lignin and polysaccharide of wood Milk curd Wheat bran

Wheat and soybeans Rice and other cereals Rice Soybeans Rice Peanut press cake Soy curd Rice Cane sugar, beet sugar, molasses Peanut, wheat, rice

Mixed bacterial and protozoan population

Polysaccharides and lignin of wood Ethanol Sulfur and iron content of mineral Organic components of sewage

Many bacteria, yeast, and fungi

Hydrocarbons and derivatives

Acetoliacter species Thiobucitlus species

"Modified, after Ralph (1976), with the permission of Professor B. J. Ralph and Food Technology in Austrulia. "In sour-dough bread levens.

204

K. E. A I D 0 0 ET AL.

attributed to them, and by the year 2600 BC, they were making bread by methods essentially similar to those of the present time. The history of breadmaking has been well presented by Sheppard and Newton (1957) in their book “The Story of Bread.” The use of mushroom-type fungi as food and the superstition and folklore connected with it also date back many hundreds of years. An early history and a description of present-day developments in the cultivation of Agaricus bispurus, a commonly cultivated edible mushroom, have been presented recently by Hayes and Nair (1975). Food fermentations such as cheesemaking and Oriental fungus-processed foods, preservation of fish and animal products, and production of vinegar and gallic acid are also types of solid-state fermentation systems that have existed for many hundreds or thousands of years.

I . Cheesemaking Cheese has been used as a food for many centuries and records of its use date back to biblical times. In Asia and Europe, cheese had been prepared for several hundred years, at least, before the birth of Christ. Roquefort cheese has been known for almost 1000 years. It originated in southern France and is made only from the milk of ewes. The sheep graze in the Cevennes region and their milk is made into cheese and transported to the vicinity of Roquefort for ripening. Roquefort cheese is made by inoculating the curd with a dried bread product rich in spores of Penicilliuin r o y u e f ~ r i ,a blue-green mold. Thom and Currie (1913) reported that pure cultures of the same organism, P . roquefmrti, occurred in all Roquefort and related types of cheese. Molded bread is prepared by growing P . r o q u e f i i in the interior of the bread in a moist, cool place until the bread is covered with a moldy mass containing a vast number ofspores. The molded bread is dried, ground to a powder, and used as the inoculum. Ripening takes place in caves, mineshafts, and rooms with controlled relative humidity and temperature; the temperature for ripening must not be much higher than 10°C and the relative humidity should be high (about 90 to 96%). Holes are punched in the curd to facilitate the development of the mold throughout the cheese. Although aerobic, P . roquefurti grows well under oxygen limitation in the narrow air spaces of the curd. After the mold has developed to the desired extent, the cheese is wrapped in foil and stored at approximately 4”C, at which temperature the mold enzymes are active but the mold growth is inhibited. Many types of cheese are now made by processes similar to that for Roquefort, including a cheese commonly known as “blue cheese,” made from cows’ milk. Babel (1953) described the production of “blue cheese” on the commercial scale. Although the mold-containing cheeses of the type discussed above have

SOLID SUBSTRATE FERMENTATIONS

205

the most obvious similarities with activities of the type normally subsumed under the title “solid-state fermentations,” a case can be argued for including all of the 400 or so known varieties of cheese under this heading. Immature cheese provides on environment high in solids and rather low in available water, in which bacteria appropriate to the variety of cheese being produced grow and carry out their metabolic activities. Cheddar cheese is a hard cheese prepared from whole milk, originating in Cheddar, England; the name now refers to a family of cheeses rather than a single variety. The biochemistry and microbiology involved in the manufacture of cheddar cheese and the current trends and future prospects of cheddar cheese production have been reviewed by Scott (1967). 2. Koji Process Of great historical importance to modern technology is the “koji process,” which has been used in the Orient for many centuries. Koji is an enzyme preparzition produced by growing a mold such as Aspergillus oryzae on steamed rice or other cereals. It is used as a starter in the soy sauce (shoyu) industry and in fermentation of miso (a semisolid cheeselike food) and many other Oriental foods. Koji is also used in the brewing of the Japanese rice wine (sakk). Hesseltine (1965) noted that, according to information supplied by a major shoyu manufacturer, shoyu has been produced in Japan for over 1000 years. Although the making of soy sauce originated in China, Japan has probably the largest soy sauce plant in the world, the Noda Mati Plant of the Kikkoman S h o p Company Limited, founded in 1764 (Yong and Wood, 1974). Shurtleff and Aoyagi (1976) state in “The Book of Miso” that the Chinese originated “Chiang” (a forerunner of miso) about 2500 years ago and that the manner of preparation was brought to Japan by Buddhist priests in the seventh century. Bush (1959) noted that Buddhism, which was well established in China and Korea by the fourth century, was introduced to Japan between 500 and 600 AD. In Japan, miso has been prepared for over 1000 years (Shibasaki and Hesseltine, 1962), and the traditional drink, sak6, is believed to have been made at least since the eighth century (Miall, 1975). Sufu, the Chinese soybean cheese, is also of great antiquity. The process of producing tofu, the bland, unfermented soybean curd, is reported as the invention of Liu Ap, a Chinese emperor, who ruled from 179 to 122 BC. The fermentation of sufu from tofu is reported in annals of the Ching Dynasty (1644 to 1912) (Peppler, 1967). Other Oriental fermented foods prepared by the koji process are tempeh, a popular Indonesian food, prepared by inoculating Rhizopus oligosporus on either soybean (tempeh kedelee) or coconut meal (tempeh bongkrek) (Van Veen, 1957; Djien and Hesseltine 1961; Martinelli and Hesseltine, 1964); and hamanatto, a Japanese fermented soybean product using A . myzae, which is also called “tu su” by

206

K . E. A I D 0 0 ET AL.

the Chinese and “tao-si” by the Filipinos, (Hesseltine, 1965). Hesseltine and Wang (1967) outlined a process for making hamanatto based on information from Dr. A. Kaneko of Nagoya University. Ontjom, a food unique to the East Indies, is prepared by fermentation of peanut press cake with Neurospura sitophila. Ang-kak, a product made by fermenting rice with certain strains of Monascus purpureus, is used for coloring various foods, including fish and Chinese cheese, and for manufacturing red wine in the Orient. Natto (apparently the only fermented food in Japan in which a bacterium is the sole organism responsible for the fermentation) is made with Bacillus subtilis ( B . natto). Fermented and other fungus-processed foods from the Orient have been reviewed by some workers from the Occident (Church, 1920; Lockwood, 1947; Shibasaki and Hesseltine, 1961a; Yong and Wood, 1974); however, most essays and reviews on the subject from the Orient are in Japanese and other Oriental languages.

3. Preservation of Fish and Animal Products Preservation of fish, meat, and other animal products with the help of fermentation and added salt, starch, and sugar also has a very long history. Records go back some 2500 years, but sausages as a fermented product probably predate agriculture (Stanton, 1978). In Southeast Asia, rice has been used as an alternative to salt in the preservation of fish. Fish fermentation, one of the oldest fermentation methods, is still practiced in the Far East and some parts of Africa. “Garum,” or fermented fish guts, was used by the Romans (Stanton, 1978). In Japan, Aspergillus species are used to ferment bonito fish to a dried product called “katsuobushi” (Hesseltine and Wang, 1967). The fermented fish product is dark in color, very dry and very hard, resembling wood. The dried fish is shaved into ribbons over a box containing a blade much like a plane. The shavings are then added to food for flavoring. Other fermented fish products from the Orient are fish sauce and fish paste. Fish sauce is prepared by mixing small, uneviscerated fish with salt at the ratio of 3:l.The mixture is placed in concrete tanks built into the ground to control temperature. The tank is filled with fish-salt mixture, sealed, and left for 6 months, at which time the fish has liquefied, and the liquid is drained off and filtered. The filtrate is placed in earthenware containers and allowed to ripen in the sun for 1 to 3 months. The finished product is a clear dark-brown liquid with a distinctive aroma and flavor. The “faseich” or pungent fermented fish of Sudan is prepared by degutting herring and adding capsicum (hot chili or pepper) to the fish. It is believed that the addition of capsicum inhibits the growth of pathogenic bacteria. The fish with the chili are left in containers containing 4-6% (wlv) salt and are allowed to ferment for 2 to 4 months at 28-36°C. The principal species of

SOLID SUBSTRATE FERMENTATIONS

207

microorganism isolated from “faseich” were halophilic bacteria (Lewis 1977). In Ghana, the fish Tilapia is degutted, soaked in sea water for a day or two and allowed to ferment on the beaches at about 2532°C for a week. After fermentation, the fish is sun-dried for several weeks to a dried product called “kobi” with a very strong ammoniacal or pungent smell. “Kobi” is usually used as a flavoring agent. We are not aware of any report on the microflora of “kobi” fermentation.

4 . Vinegar Production Although vinegar production has been known for thousands of years, its microbiological nature was not realized until in the nineteenth century. In 1837, Kutzing reported that the conversion of ethanol to acetic acid was brought about by living microorganisms. Fifteen years earlier, Persoon (1822) had given the name Mycoderina to the film that formed on liquids in which acetic acid fermentation was taking place. It was for Louis Pasteur (1868) to confirm Kiitzing’s opinion. Pasteur, however, believed that the fermentation was caused by a single species of bacteria, Mycoclerma aceti. Hansen (1878) showed that more than one species of bacteria was involved in the souring of beer, i.e., the oxidation of ethanol to acetic acid. H e isolated and named Bacterium aceti and Bacterium pasteurianuin and later isolated Bacteriuin kutzingianurn. Since the time that Pasteur described the first culture of an acetic acid bacterium, over 60 species have been established. Frateur (1950) reduced the number of species to 10 and devised a method for their identification (Leisinger and Muller, 1967). Vinegar is used as a food condiment and also as an antiseptic (McCullough, 1945). Vinegar production involves conversion of sugar in fruit juice and other sugar-containing media to ethanol, and acidification of the latter with acetic acid-producing bacteria on supporting medium in the presence of an adequate supply of oxygen. It is reported (Prescott and Dunn, 1959) that Boerhave discovered in the early part of the nineteenth century that when wine was permitted to trickle down through a tall receptacle containing loosely packed pomace, vinegar was rapidly produced. In 1823, a German named Schutzenbach modified the method of Boerhave by introducing other types of porous material in order to obtain maximum contact of the organism with air. The method used by Schutzenbach is the basis for modern methods of manufacture using the vinegar generator. The methods of deep batch and continuous production of vinegar have been discussed and compared economically with the older process (White, 1966).

5 . Gallic Acid Production of gallic acid is another early mold fermentation process; its discovery was made in 1787 or earlier by Scheele (Prescott and Dunn, 1959), who was studying the effect of a mold on a water infusion of gall nuts. Gallic

208

K. E. A I D 0 0 ET AL.

acid is used in the tanning, printing, and other industries. Van Tieghem (1867) carried out classical studies in connection with gallic acid fermentation. He was the first to establish the importance of Aspergillus niger as the predominant mold in the fermentation, and gave it the name gallic acid. In one of the oldest methods used for the production of gallic acid by fermentation, tannin-containing subtances were piled up in heaps and moistened with water. Mold developed throughout the heap, which was stirred occasionally and maintained at a temperature of about 30°C. After a fermentation period of about a month, the gallic acid was leached from the heap. Present-day methods of production of gallic acid make use of liquid-extract fermentations, and Ikeda and co-workers (1972) reported that A. niger is still the most satisfactory organism for production of gallic acid.

B . POST-PASTEUR PERIOD AND CURRENT DEVELOPMENTS Between 1860 and 1900, trickling filters for sewage disposal were invented, but these were an adaptation of the already known vinegar generator (Johnson, 1971). During the next 20 years, from 1900 to 1920, came developments in the production of fungal and other microbial enzymes. The new fermentation introduced from 1920 to about 1940 was the production of gluconic acid, and the importance of this fermentation to solid-state fermentation processes at present lies in the rotary drum fermentor used at that time. The fermentation industry exploded between 1940 and 1950 with the discovery and exploitation of antibiotics. The first clinically useful antibiotic, penicillin, was produced by both surface- (solid-state) and submergedculture methods. Although no longer in use, a surface-culture method was used for the first commercial production of penicillin (Sylvester and Coghill, 1954). During the decade from 1950 to 1960, steroid transformation by fungal spores was developed. The spores were produced by solid state fermentations. Since 1960, important developments in the new solid-state fermentation processes include the production of mycotoxins and the treatment and re-use of animal, plant, and domestic wastes.

1. Production of Mold Enzymes The koji mold, A. uryzae was first isolated in 1878 by a German, Dr. H. Ahlburg, while a teacher at the Tokyo Kaisei School (the forerunner of the University of Tokyo), and Atkinson (1881) was the first to demonstrate its strong diastatic activity. Takamine (1894) succeeded in improving the diastatic activity of the mold and prepared from it a medicament having a strong digestive activity-Takadiastase. He was probably the first to realize the

SOLID SUBSTRATE FERMENTATIONS

209

technical possibilities of producing enzymes from mold and to introduce such enzymes into industry. In 1914 he introduced into the United States a mold-bran process used in enzyme production for saccharification of starchy substances; it is thought that he got his idea from the koji process in soybean fermentation in Japan. Underkofler and co-workers (from 1939 to about 1947) developed three different methods of production of mold-bran using Takamine’s technique; they reported higher yields of ethyl alcohol from grains saccharified by molded bran than from those saccharified by the use of barley malt (Underkofler et al., 1939, 1946, 1947; Hao et al., 1943). Hao et al. (1943) used 27 strains of mold of the genera Aspergillus, Mucor, Penicillium, and Rhizopus, and they found strains of A . oryzae to be the best suited for industrial use because of the ease of handling, consistency of results, and high yields of alcohol from the saccharified mash. The methods used by Underkofler and co-workers for the production of mold-bran utilized drum, pot, and tray equipment (Prescott and Dunn, 1959). a. Drum Method. A dry “seed inoculum” was first prepared by steamsterilizing ground corn (10 gm), wheat bran (100 gm), and 60 ml of 0.2N hydrocholoric acid containing Z n S 0 4 * 7H 2 0(0.62 ppm), F e S 0 4 - 7H 2 0 (0.63 ppm), and C u S 0 4 * 5H,O (0.08 ppm). After cooling, the moist, sterilized substrate was inoculated with a culture of A . oryzae and incubated at 30°C. After sporulation reached a maximum, the culture was rapidly dried. The dried product (110 gm), greenish in color, was used to inoculate a much larger charge (1.0-1.2 kg) of sterile bran contained in a 5-gallon drum. Air was passed slowly into the drum, which was maintained at about 30°C (never above 35°C)to favor growth of the mold. During the germination period, the drum was rotated for not more than 15-20 minutes every 2 hours, and after germination the drum was rotated continuously at not more than 1rpm for 40-45 hours. The moldcovered bran was removed, dried at room temperature, ground in the same manner as malt, and added to corn mash.

b. Pot Method. Hao, Fulmer, and Underkofler developed this method in 1943 and said that it was superior to the rotating drum used earlier in the following respects: The equipment required less space and was less complicated; the mold mycelium was not disturbed during growth; and uniform aeration was obtained, resulting in rapid growth of the mold. The method consisted of packing about 750 gm of moist wheat bran into a 3-quart aluminum pot with holes at the bottom. The bran was sterilized, cooled, and inoculated with 5-10 gm of a well-sporulated culture of the mold (usually a strain of A. oryzae, prepared as described above). The seeded bran mash in the pot was placed on a layer of cotton batting and incubated at 30°C. Air was passed through the mass to aerate and to control fermentation tempera-

210

K. E. A I D 0 0 ET AL.

ture. Aeration continued for 12-24 hours and the product was then removed from the pot and treated as described above. c. Tray Method. As with the pot method (Ha0 et al., 1943), no agitation was used in the tray method and it was very similar to the old conventional tray koji method (Hesseltine, 1977). Equal amounts of wheat bran and water were mixed and spread in layers 1 in. deep in aluminium trays. The moistened bran was steam sterilized, cooled, and inoculated with a culture of A . oryzae. The seeded bran was incubated at 25°C for about 4 days and the mold spores were destroyed by adding 95%ethanol to the mold-bran mixture in the proportion of 1500 ml ethanol to 1.0 kg moist bran. The alcohol extract was removed with a hydraulic press and the resulting dried, ground press cake was used as the enzyme preparation. This appeared to be the best of the methods studied by Underkofler and associates. Mold enzymes may be produced by a variety of methods (Prescott and Dunn, 1959; Beckhorn, 1960): 1. Production in tanks under submerged-culture conditions (Balankura et al., 1946; Le Mense et al., 1947, 1949; Adams et n l . , 1947; Pool and Underkofler, 1953) in a manner similar to that used for the production of antibiotics. In the submerged-culture process, the raw materials are mixed in a tank, cooled, sterilized by a batch or continuous process, cooled to the optimum temperaturc for the growth of the mold, and pumped or allowed to flow by gravity to a fermentor. The sterile material is seeded with the desired mold and given aerobic conditions to produce enzymes. At the time of maximum enzyme activity, the mold mycelium and the enzymes are separated by filtration. Practically all of the microbial enzymes offered commercially today are produced by variations of this procedure. 2. Production by growing the mold on shallow layers of solid substrates in trays (Underkofler, 1939; Roberts et al., 1944; Jeffreys, 1948). In order to eliminate the labor normally required for handling trays in the older processes, Jeffreys (1948) described a method for producing mold enzymes by a continuous-tray method. Spores of A. oryzae are prepared by inoculating a sterile, moist bran medium in %quart jars. After adequate sporulation has takcn place, the spores are used to inoculate a prepared substrate, the inoculum amounting to about 0.5%on a dry-weight basis. The substrate is prepared by first feeding it onto a conveyor by a percentage feeder, where it is mixed with the required amount of water and heated to 88°C or higher with steam for about 15 minutes. The steamed mixture is dropped onto another conveyor, where it is cooled and sprayed with spores suspended in sterile water. The substrate and the spores are thoroughly mixed together and dropped onto a spreader that distributes it uniformly in sterile trays. The trays are conveyed to a loading point and loaded onto a truck that holds 20 trays, 4 in. apart. The trucks are pushed into culturing tunnels (special

SOLID SUBSTRATE FERMENTATIONS

21 1

incubators) on overhead trolleys. Air is circulated in the culturing tunnels by means of fans that are located 6 ft apart between the ceiling and subceiling and that draw in the air, fresh or recirculated. During incubation, the temperature of the inoculated substrate is controlled by the circulation of cool, humidified air through the culturing tunnels. When it becomes necessary to warm the air of the tunnels, it is accomplished by the use of “thermostatically controlled steam jets.” After the growth on the substrate reaches an optimum, usually after 2 4 3 0 hours, the trucks and their contents are transported into the drying tunnels, which consists of primary and secondary stages with a central exhaust. The air is made to flow parallel to the direction of movement of the trucks in the first stage and perpendicular to their direction in the second stage. The temperature of the enzyme-containing product is not allowed to exceed 43°C during the drying process. With the moisture content reduced to about 8%, the dried material is ground and bagged or extracted. 3. Production in rotating drums (Underkofler et al., 1939, 1947; Hao et al., 1943). Underkofler et al., (1939, 1947) described a method for producing amylases by the use of rotating drums with controlled aeration and temperature (detailed above); products made by this method were high in activity. The koji process for making enzymes needed in soybean fermentation in Japan traditionally uses wooden trays in which soybeans and cereals, such as rice and wheat, are inoculated with the koji mold (usually A . oryzae) and incubated for about 3 days. The trays are stacked vertically with about a 4-in. gap separating them, and each tray is filled to a depth of about 2 to 3 in. Terui and co-workers (1958, 1959, 1960) developed koji incubation chambers that involve automated inoculation of solid substrates, controlled environmental conditions, automated turning of the solid fermenting substrate, and the automated harvest of the finished product. These automated incubation chambers are now being used in many commercial plants in Japan. They use three types of incubators. Incubator I or the sectional heap incubator is divided into 10 sections by wooden drawers with nylon-netting false bottoms, and vinyl-coated copper-pipe assemblies are laid between the sections. The incubator, with a total capacity of 600 liters, has a water circulation system with gear pump and thermostat; humidified air is introduced from the bottom. Zncubator IZ is a wooden box (60 X 60 x 60 cm) furnished with vinyl-coated %-in. iron pipes alternately arranged and a vinyl-coated wire gauze rack for supporting the culture medium. Other parts are similar to incubator I. Zncubator ZZZ is a taller form of incubator 11; known as the high-heap process, such incubators are used for citric acid production and are also applied to koji fermentation. Arima (1964) reported that the old, conventional koji method, although

212

K. E. A I D 0 0 ET AL.

considerably improved, is still used in soy sauce manufacture and in the Japanese sakh industry, and that the factories that produce enzymes adopt either tray culture, the rotating drum, or a modified tray culture method. H e reported on disadvantages associated with the tray and the rotating drum methods as follows: (1)Tray method. It is difficult to control temperature in the koji culture, and the layer of koji bran cannot be very deep, about 2-4 cm, although this method had been successfully used for production of amyloglucosidase from Rhizopus strains. (2)Rotary drums. When the drum rotates, it cuts the growing mycelium. Low enzyme production is experienced with A . oryzae and particularly with coenocytic genera such as Rhizopus. Also, the useful space in the drum is only 30%of the total, which is another disadvantage of the drum-type fermentor. Arima stated that, although the rotary drums are used in some enzyme plants, it is only for incubating the inoculated substrate for the first 10-20 hours, after which the koji culture is distributed on trays and incribated as under tray culture. Hesseltine (1977) noted that with the advent of deep-tank fermentation, the bran process became neglected in the West. Since the discovery of antibiotics, practically all new fermentor equipment and new plants in the United States are based on the conventional deep-tank fermentor for producing microbial products. An exception is the new Kikkoman plant in Wisconsin built to produce soy sauce; the koji used is incubated in large rooms with occasional stirring. In addition to koji, Armia (1964) reported that enzymes other than koji that are produced on a large scale of solid-state fermentation processes are Takadiastase and amyloglucosidase of Rhizopus, pectinase of Sclerotinu, and acid-resistant proteases of A . niger. Hesseltine (1977) reported that some commercial enzymes, such as microbial rennet, are made by growing Mucor pusillus on a solid substrate. Apart from the animal digestive tract, some sources of rennet are plants and microorganisms. Sardinas (1969) reported that, although no vegetable rennets were marketed at that time, they seemed to have economic potential. Prins and Nielsen (1970) reported that production of rennet from Mucw rniehei, Endothia parasitica, and M . pusillus was highly successful, and these enzymes now have a large market. Solid-state fermentation processes have also been used for the production of bacterial enzymes, particularly amylases. Beckford et al. (1945) described a method for producing bacterial amylase on wheat bran. High levels of amylase production from B . subtilis were obtained with a medium consisting of sterilized wheat bran moistened with dilute phosphate buffer (1.5 gm .KHsP04 and 3.5 gm. K2HPO4*3H2Oper liter). Maximum amylase production was achieved at 37"C, with a ratio of 1 part wheat bran to 2.5 parts buffer and after about 48 hours incubation. Beckford listed the advantages of the method as (1) availability of large surface area for the growth of

SOLID SUBSTRATE FERMENTATIONS

213

the organism and (2) the ability of the material to be air-dried rapidly and rendered relatively stable. In commercial practice, this method of production has been superceded by stirred tank reactor fermentations. Amylases and proteases were the major hydrolytic fungal enzymes produced by solid-state fermentation, and the organisms that are the most widely used sources of fungal enzymes are A . oryzae and A . niger, although species of Mucnr, Rhizopus, Penicillium, and others have also been used. Fungal and other microbial enzymes are now widely used in the food, pharmaceutical, textile, chemical, and other allied industries, and solid-state fermentations have played an important role in the production of the enzymes, although current production favors the stirred tank reactor. 2 . Production of Organic Acids a. Citric Acid. Wehmer (1893) first described citric acid as a product of mold fermentation. Two Penicillium-like molds, which Wehmer designated as Citromyces pfeferianus and Citromyces glaber, produced the acid from nutrient sucrose solution containing calcium carbonate. In 1891, Wehmer had shown oxalic acid to be produced by A . nigw but he did not discover that this mold also produces citric acid and in larger quantities than the Citromyces species. In 1917, Currie published his first results on production of citric acid by a strain of A . niger. Although large numbers of fungi ( A . niger, Aspergillus clavatus, Penicillium luteum, Penicillium citrinum, Paecilomyces divaricatum, Mucor pirijbrmis, and others) have been used to produce citric acid either on laboratory, pilot-plant, or commercial scale, only strains of A. niger appear to be of industrial importance. Although citric acid had been produced on commercial scale by submerged-culture methods, Cahn, in 1935, advocated a rather unusual method of citric acid production. Solid material, such as cane or beet pulp, was impregnated with sugar solutions-sucrose or molasses. The unsterilized mass was inoculated with a mold culture (A. niger), the age of which was said to be relatively unimportant, and fermentation proceeded rapidly over the relatively large surface at a temperature of 2035°C. The fermentation was usually completed in 4 or fewer days, at which time a yield of 55% citric acid based on sucrose originally present, or 45% citric acid calculated on the basis of total sugar originally present in the molasses, was obtained. He found that, whereas beet pulp could be used only once, it was possible to use cane pulp more than once, and he reported that 0.45 kg of citric acid could be obtained from 2.56 kg of molasses and 1.24 kg of beet pulp. Based on Cahn’s (1935) method, successful production of citric acid on solid inert materials using A . niger were reported by Terui et al. (1957) and Lakshminarayana et al. (1975), using sawdust and sugar-free sugar cane

214

K. E. A I D 0 0 ET AL.

bagasse, respectively. It appears that there was no published information on citric acid fermentation on solid inert materials between 1957 and 1975. Terui and co-workers (1957) reported that during citric acid fermentation by a submerged process, the yield of the acid is generally much more susceptible to the impurities of raw materials, and the power consumption rate is believed to be much higher than in the case of surface- or solid-culture processes. Terni (1944) proposed the high-heap aeration process whereby moistened and steamed raw material was mixed with porous substances such as rice hulls, inoculated with spores, and packed in a large incubator box to form a high heap through which conditioned air was passed. He and his co-workers (1957) used the high-heap process for citric acid production from molasses-sawdust mixture, using incubators equipped with horizontal water-cooling pipes buried in the heap. For citric acid production, blackstrap molasses was first diluted and rice- or wheat-bran was added. The mixture was heated at 90-95°C for 30 minutes, then cooled below 40°C. The sugar concentration was adjusted by a second dilution. Inoculum, prepared by cultivating a strain of A. niger on wheat-bran or molasses-sawdust medium at 30°C for 4-5 days, was added to the diluted molasses to a concentration of 1%, and then mixed with supporting materials in order that the culture medium could form a porous heap. As supporting material, sawdust, sometimes in combination with rice hulls, was used. Sawdust was first washed and sterilized by steaming before being added to the medium. Humidified air (almost 100% in relative humidity) was supplied at 30°C and warm water (about 33°C) was passed through the water pipes occasionally, especially when the culture was still on the lag phase. Lakshminarayana and co-workers (1975) reported higher yields of citric acid by solid-state fermentation techniques than by surface- and submerged-liquid cultures. They used two liquid media (sucrose and molasses) and also studied the effects of methanol on citric acid production. For the surface- and submerged-culture experiments, 75-ml portions of the liquid medium were distributed in 500-ml Erlenmeyer flasks, and then sterilized, cooled, and inoculated with spores of A. niger. For solid-state fermentation, 10 gm of chopped sugar-free sugar cane bagasse was introduced into 500-ml Erlenmeyer flasks containing 75 ml of the liquid medium; then it was sterilized and inoculated. In experiments in which the effect of methanol was tested, 3% methanol was added. Fermentations were carried out at 30°C for 6 days; for the submerged-culture method, the flasks were placed on rotary shaker operating at 210 rpm with a 2-in. amplitude throw. In tray experiments (a larger scale solid-state fermentation technique), 150 gm of bagasse was mixed with 1 liter of sucrose medium, sterilized in a stainless steel container, cooled, and inoculated with spore suspension. Methanol (30 ml) was added and the material was spread in an enamel tray

215

SOLID SUBSTRATE FERMENTATIONS

(12 in. X 15 in. X 2 in.) presterilized with ethanol. The trays were covered with polyethylene sheets and also incubated at 30°C for 6 days. After fermentation, citric acid was extracted with distilled water in the case of solid-state fermentation methods; for surface and submerged methods, the contents of the flasks were filtered to separate the mycelia from the citric acid. The results (Table 11) clearly show the improvement in yield by solidstate cultures under their operating conditions. Various workers have given an account of the development of the citric acid fermentation industry (Prescott and Dunn, 1959; Meyrath, 1967; Miall, 1975).

h. Kojic Acid. Kojic acid, used as an insecticide, antibiotic, and analytical reagent, was first isolated by Saito in 1907 as a by-product of the growth of A . oryzae on steamed rice by solid-state fermentation. A Japanese biochemist, Yabuta (1912) investigatcd Saito’s acid, named it “kojic acid,” and, in 1924, elucidated its chemical structure. In 1914, Traetta-Mosca TABLE I1 CITRIC ACIDPRODUCTION BY A. nigc.1. (STRAIN 3/1) UNDER DIFFERENT CULTURAL CONDITIONS“

Cultural condition

Medium

Methanol 3%

Residual sugar

(”/.I

Citric acid”

(%I

Citric acid‘ (%)

Sucrose Suhmerged Surfiace

Solid-statc

-

+

10.0 13.1 20.7 24.3 12.5 12.5

4.5 3.8 5.2 22.9 14.1 62.8

5.0 4.3 6.6 30.2 16.1 71.8

Molasses Srll~nlerged

Surfice

Solid-state Tray rxprriments (sucrow medium)

27.1

4.9

-d

-d

-I

20.7 10.0 2.7 3.9 2.1

9.4 30.8 18.4 56.4 78.6

11.8 34.3 18.9 58.7 80.2

6.7

‘Adapted from Laksliiniiiarayana et ul. (1975), with the permission of Biotecltuolog!, crnd Bioeugiiieeriiig.

* Based on the initial sugar concentration in the fermentation inediuin. “Based on the actual sugar consumed. “Poor growth.

216

K. E. A I D 0 0 ET AL.

showed that kojic acid was produced from different sugars, and Tamiya (1927) pointed out that different species of Aspergillus had the ability to produce the acid from sucrose. Although kojic acid was first produced by solid-state fermentation, liquid or submerged-culture methods are now used for production. 3 . Fermented Foods

Apart from the Oriental fermented foods, several other fermented foods, some of them also having very long histories, are known in many parts of the world, particularly in Africa, Asia, and South America. Although reviews of such fermented foods prepared by solid-state fermentation processes have been published by various workers (Platt, 1962, 1964; Hesseltine, 1965; Stanton and Wallbridge, 1969; Batra and Millner, 1976; Hesseltine et al., 1976), there is very little information on historical developments of such fermented foods in comparison with the Oriental types. Cereals, beans, and starchy roots (tubers) are some of the main solid substrates that are prepared and used for microbial fermentations. Among the cereals, wheat, rice, maize (corn), millet, and sorghum are the main types commonly used. A Turkish fermented food, tarhana, is prepared fiom parboiled wheatmeal and yogurt in the ratio of 2:1. Vegetables are added and the mixture is allowed to ferment for several days, after which it is sun-dried (Platt, 1964). Dosai (or idli) is a food prepared in South India by fermenting rice and black gram or beans with lactic acid bacteria and an unspecified yeast (Rao, 1961; Batra and Millner, 1976). Agidi (Ghana, Nigeria, Sierra Leone), kenkey (Ghana), and ogi (Nigeria) are some of the fermented foods made from maize (corn), sorghum, or millet in West Africa (Banigo, 1969; Christian, 1971; Muller and Nyarko-Mensah, 1972); similar types are found in Brazil, Peru, the Caribbean islands, and New Zealand. The corn dough is fermented by molds, lactic acid bacteria, and unspecified yeasts and the fermentation process involves at least two stages. During the first stage, Aspergillus and Penicilliurn species predominate and produce enzymes to saccharify starch and other carbohydrates, while in the second stage, alcohol and organic acids (particularly lactic and acetic acids) are produced by yeasts and lactic acid bacteria. Cocoa fermentation, an example of solid-state fermentation, is regarded as the most convenient way of removing adhering pulp and obtaining the cocoa beans in a state suitable for drying; it is also a process necessary for obtaining cocoa of the finest flavor and aroma. Cocoa fermentation is a two-stage process. The first involves conversion of sugars to alcohol by a yeast, Saccharomyces theobrornoe (first isolated and named by Preyer in 1901), and, during the second stage, acetic acid is produced from the alcohol by Acetobacter aceti (Knapp, 1920; Whymper, 1921). In Ghana, the traditional

SOLID SUBSTRATE FERMENTATIONS

217

process of cocoa fermentation is carried out by splitting open the ripe cocoa pod; the beans with the adhering pulp are then distributed on banana or other broad leaves. Small holes are made in the leaves to drain off the cocoa “sweatings” produced during the fermentation. The beans are covered with banana leaves and allowed to ferment for about a week. The fermenting mass is turned or mixed occasionally by hand or by a wooden spade. During the fermentation, temperatures rise from about 30°C on the first day to about 50°C by the sixth day. After fermentation, the beans are sun-dried. Similar techniques are used in Brazil, the Ivory Coast, Trinidad, Ceylon, and other West African, Caribbean, and South American countries. Dawadawa is a fermented food made in West Africa from the African locust bean (Parkiafilicoidea). The bean is boiled, dehulled, covered with leaves, and allowed to ferment for 2 3 days. We are not aware of any published information on the microbiology of dawadawa fermentation. The fermented mass is pounded into paste, made into small balls, sun-dried, and used as flavoring agent. “Gari” (a dietary staple in some West African countries),“kokonte”(Ghana), and “lafun” (Nigeria) are some of the fermented foods made from cassava (Manihot utilissima) (Stanton and Wallbridge, 1969).

4 . Transformation of Organic Compounds by Fungal Spores Various workers (Sehgal et al., 1966; Singh et al., 1965, 1967, 1968; Vkzina et al., 1968; Sansing and Ciegler 1973) have used solid-state fermentation techniques for mass production of spores needed in the transformation of organic compounds such as steroids, antibiotics, fatty acids, and carbohydrates. Sehgal et al. (1966) and Singh et al. (1968) described the use of Fernbach flasks for laboratory-scale and the tray method for semi-industrial production of fungal spores. With Fernbach flasks, sporulation was carried out on “pot” barley. The flask (2.8-liter capacity) containing 200 gm of moistened “pot” barley was sterilized at 121°C for 1 hour, cooled to about 28”C, and inoculated with a spore suspension of Aspergillus ochraceus. The flask was incubated at 28°C for 6 days with 98% relative humidity, and spores were then harvested. They obtained 6 gm of spore paste, 30%dry weight, consisting of 5 x 10 l 1 conidia. For larger scale studies, hard wheat bran (24 kg) and 9 liters of tap water were mixed and sterilized at 121°C for 1hour. The sterile bran was cooled and inoculated with 1.2 liters of spore suspension of A . ochraceus and distributed in aluminium trays, each tray measuring 762 mm x 457.2 mm x 44.5 mm and offering a surface area about 12 times that available in a Fernbach flask. Twenty-four trays were stacked in a truck that occupied 6 square feet of floor space and were incubated at 28°C with about 98% relative

218

K. E. A I D 0 0 ET AL.

humidity for 6-7 days. Spores were harvested by suspending and mixing the moldy bran in 120 liters of water containing 0.01% “Tween 80,” centrifuged at low speed; the spore suspension was then filtered through glass wool and the spores were collected in a “Sharples” supercentrifuge. Each tray yielded 7 x 10” spores of A . ochraceus. Vi.zina and Singh (1975) reported that the solid-state fermentation technique for production of fungal spores had advantages over submergedculture methods because the former was simple and gave higher yields and also homogenous and pure spores.

5 . Production of M ycotoxins Gray (1970) and Butler (1975) have given comprehensive accounts of mycotoxicoses; they reported that the toxic substances, usually of fungal origin, may be produced on a wide range of solid substrates such as leaves, stems, grains, and seeds. Aflatoxiris, probably the most publicized of the mycotoxins, are produced by certain strains of Aspergillus flavus and Aspergillus parasiticus (Sargeant et al., 1961; Asao et al., 1965; Hesseltine et d.,1966; Detroy et al., 1972). Apart from peanut meal, from which aflatoxin was first isolated, Hesseltine and co-workers (Hesseltine ct al., 1966, 1968; Shotwell et u l . , 1966; Stubblefield et aZ., 1967) have shown that aflatoxins may be produced by solid-state fermentations on a wide range of‘foodstuffs such as rice, wheat, and oats and also on forages. Hesseltine (1972, 1977) reported high yields of aflatoxin (more than 1 gm per kilogram substrate) using a technique similar to the koji process. At first, h e and his associates tried to produce aflatoxin by both still- and shaking-liquid culture methods and the yields were very low. They then adopted a continuous shaking of solid substrates in Fernbach flasks and obtained high yields of aflatoxin. An inoculum of A . parasiticus was produced by inoculating agar slants of potato dextrose agar and incubating them for 1 3weeks at 28°C. The conidia of A . parasiticus were removed by adding 3 ml of sterile water containing 0.0005% Triton X-100 to each slant. Initial fermentations were carried out in 300-ml Erlenmeyer flasks containing 50 gm of polished rice or in 2.8-liter Fernbach flasks containing 300 gm of rice. After 25 ml of tap water was added to the rice (50 gm) in the flasks, the mixture was allowed to stand at room temperature for 2 hours with frequent shaking. The flasks were autoclaved, cooled, inoculated with 0.5 ml of inoculum, and placed on a New Brunswick shaker set at 28°C and 188 rpm. Small amounts of sterile tap water were added to the mixture during the first and second days, just enough not to cause the individual rice grains to adhere to one another. At the fifth or sixth day of continuous shaking, the flasks were steamed to destroy the mold, then chloroform was added to the fermented rice, and, with two extractions, over 95% of the total aflatoxin was

SOLID SUBSTRATE FERMENTATIONS

219

removed. Hesseltine and co-workers used the method described above to produce aflatoxin on wheat, oat hulls, groats (dehulled oats), whole oats, and forages by strains of Aspergillus. Recently, Silman et al. (1979) reported on large-scale production of aflatoxin in corn by a solid-state fermentation process. Prior to carrying out the large-scale fermentation, preliminary laboratory studies were run by flask and column fermentations. Yellow dent corn was used and the mold employed was A . flavus (1 x lo3 propagules/gm). Flask experiments were conducted in a 500-ml Erlenmeyer flask with 75 gm of corn (moisture ranging from 18-26% and incubated at 29 or 33°C. The purpose of these experiments was to establish the viability of the indigenous population and to determine effects of moisture, time, and temperature on production of aflatoxin. An aflatoxin level of 5750 ppb (dry basis) was obtained in 7 days at 22% moisture content and at 33°C. Column fermentation was conducted in glass or plastic units, 4 in. in diameter by 5 ft tall, that held up to 8 kg corn. Temperature control was achieved either by operating in a controlled-temperature room or by controlling water temperature in a jacket fitted around the column. Air, humidified to 8045%, was introduced at either the bottom or the top. Corn samples (350 gm) were taken from the columns by emptying the column, mixing the corn in a rolling drum for 5 minutes, sampling directly from the drum, and returning the remaining bulk to the column. Aflatoxin levels as high as 6200 ppb (dry basis) were obtained in 10 days. Large-scale production of aflatoxin was carried out in a standard Butler-type corn storage bin of 18-ft diameter equipped with a slotted floor raised about 1ft above a concrete pad and with a single auger stirator that followed a helical path in and out from the center, thereby mixing the corn once every 24 hours. Fermentation was carried out for about 11 days and a target level of 1000 ppb aflatoxin was attained. Moisture level of the corn, fermentation temperature, and air-flow rate were the critical operational parameters that governed the success of large-scale production of aflatoxin by A . flavus on corn. Ochratoxin, another mycotoxin, was also produced by a continuous shaking method (Hesseltine et al., 1972). Isolates of Penicillium viridicatum were grown on 150gm of sterile pearled wheat in shaken flasks at 20°C for 12 days, and yields of 1578 and 1584 p g ochratoxin Ngm and 110 and 83 p g ochratoxin B/gm were obtained. These amounts were much higher than those obtained in a liquid yeast extract sucrose medium with similar fermentation conditions (Hesseltine, 1977). In the same research laboratory, Hesseltine (1977) studied the production of ochratoxin with A . ochraceus in a four-chambered fermentor and reported high yields of ochratoxin, regularly obtaining 20002500 pg/gm wheat and occasionally, 3000-4000 pg/gm wheat. The highest rate of agitation, 16 rpm, gave the highest ochratoxin yields (2860 pg/gm) but

220

K . E. A I D 0 0 ET AL.

considerable time (12-19 days) was required to reach the peak; at 1 rpm, a yield of 2400 pg ochratoxinlgm was obtained in 8-9 days. The four chambers of the fermentor were made of Plexiglas, 7.6cm wide and 33 cm in diameter and each attached to a common shaft. For the most effective agitation, baffles were fitted of a size and shape that would permit all of the grain to be elevated and, subsequently, dropped at some point during each revolution. These baffles were 7.6 cm long and bent upward to a 30°C angle in the direction of rotation. Each chamber carried a 1-kg load and could be rotated at a speed from 1 to 40 rpm. All chambers contained a 3.7-cm round port for inoculating and removing product. The four-sectional fermentor was equipped to allow continuous passage of sterile air through each section. The shaft of stainless steel pipe had four 0.48 cm holes drilled through the pipe walls to allow passage of air forced through the hollow shaft.

6 . Processing of Plant, Animal, and Domestic Wastes a. Fermented Plant Material as Feed. Conversion of agricultural wastes such as crop residues by fermentation to more useful products has received much attention in recent years. Residues from the processing and harvesting of some major agricultural commodities (plants and grain crops) include bagasse, sawdust, straw, stalks, husks, corn-cobs, and wheat and rice bran, and they have great potential as feeds after upgrading by fermentation. Han and co-workers (1974, 1975) fermented rice straw and sugar cane bagasse with cellulose-digesting organisms such as Cellulomonas and Alculigenes species. Rice straw and sugar cane bagasse were dried, ground, and passed through a 20-mesh screen; treated with alkali, acid, and other chemicals; and tested for digestibility of cellulose with Cellulomonas species. Treatment with 4% sodium hydroxide for 15 minutes at 100°C increased the digestibility of cellulose from 29.4 to 73%; treatment with 5.2% ammonia increased digestibility to 57.0%. Treatment with sulfuric acid only solubilized the cellulose but did not increase the digestibility, and grinding or high-pressure cooking of the rice straw and the bagasse had little effect on increasing the digestibility by the Cellulomonas species. Later (1975), they developed a large-scale, semisolid fermentation process involving the growth of Aureobasidium pullulans, Candida utilis, and Trichoderma viride on straw using a cement mixer, and they obtained a product useful in its entirety as animal feed. Rye grass straw was sun-dried and ground to pass through a 20-mesh screen, and the ground straw was first hydrolyzed; one part of straw was mixed with three parts of0.5 N H2S04 and heated at 121°C for 15 minutes or kept at room temperature (25°C) for 1hour. For every 100 gm of hydrolyzed straw, 30 ml of 4 N ammonia solution was added to adjust

SOLID SUBSTRATE FERMENTATIONS

22 1

the pH to between 4.5 and 5.0. The pretreated straw was transferred to a 30-gallon (about 114 liters) capacity cement mixer, inoculated with a suitable organism such as A . pullulans, C. utilis, or T . viride, and rotated at 1rpm at room temperature for 2 3 days. Fermentation was under nonaseptic conditions. The best result in terms of increasing protein and digestibility of straw was obtained with C. utilis. They found this semisolid fermentation to be less costly than the submerged culture methods. The basic advantage of this process is that the absorptive properties of the straw are used for carrying out the fermentation. In the acid treatment step, the portion of the straw that is not hydrolyzed retains its fibrous nature and acts as a matrix to hold water, sugar, and other soluble solids. When this mass is ammoniated in the next step, the ammonium sulfate is held in the fibrous matrix with the other soluble components. Thus a substrate containing water, mineral salts, and sources of carbon and nitrogen is made available for the growth of the microorganisms (Han and Anderson, 1975). Because the pretreated fermented straw had a potential as an inexpensive animal feed of acceptable nutritive value, Han et al. (1976)developed a continuous process large-scale fermentation of straw to animal feed with A . pu2lulans or C . utilis. The pretreatment of the straw and the fermentation process were essentially the same as described above. Moo-Young et al. (1979) have developed a novel fermentation process for the bioconversion of agricultural and forestry wastes into proteinaceous feed/food products, based on mass microbial cultivation of a new cellulolytic hngus Chaetomium cellulolyticum in a solid-state fermentation system. The process uses a three-stage operation: (1) thermal and/or chemical pretreatment of a cellulolytic material, (2) aerobic fermentation of the pretreated material with nutrient supplements, and (3)separation of the suspended solids (the product) from the fermented medium. The cellulolytic material was the main carbon source for the fermentation, and nutrient supplements (nitrogen, phosphorus, potassium, etc.) were derived from commercially available fertilizer blends or animal manures. Where manure was used, it was pretreated by anaerobic fermentation to produce methane fuel gas as a byproduct that supplied operating energy. Kokke (1977) reported on the use of solid-state fermentation process for upgrading carob (locust tree) pods as a cattle feed additive. Water (50 ml) containing 2.5 gm (NH,), . H P 0 4was added to 50 gm of chopped carob pod in a petri dish (4-cm diameter), which was covered and autoclaved at 110°C for 20 minutes, during which the liquid was completely absorbed. The sterile material was inoculated with either Rlzizopus oligospmus or Monuscus ruher and incubated at 30°C for 3 days. The fermented product, suitable for animal feed, contained 73-83% of the original carob sugars, and the original

222

K. E. A I D 0 0 ET AL.

high tannin content of the pod (about 1.8%)was completely removed. Kokke found the method comparatively inexpensive, easy, and suitable for utilization in villages and farms in developing countries. The fermentation process had been used by Brook et al. (1969) to remove residual toxicity while enriching the protein content of cassava, a staple diet in some developing countries. Their method involved converting cassava to a coarse-grade flour and mixing it with mineral salts and a source of nitrogen. A fungus inoculum was added and the moist paste was fermented as a spaghetti-like extrusion. They noted that success of such fermentation depended on the type of microorganism used, size of inoculum, and control of additives and environment. Raimbault et al. (1977) and Senez (1978) have reported a simple and inexpensive process of solid-state fermentation for protein enrichment of starchy waste materials from cassava, banana, potatoes, and other sources potentially available in tropical and temperate regions for direct animal feed. Fermentation was carried out in a commercial bread-making blender of 10-kg capacity and under nonaseptic conditions. The coarsely ground material with about 3035% moisture was gently steamed for 15-20 minutes so as to break the starch granules. After cooling to about 40"C, the preparation was mixed with water containing spores of A . niger (a selected strain having high amylolytic activity and suitable amino acid composition), nitrogen sources (ammonium sulfate and urea), and mineral salts, to give about 60% final moisture content; the fermentation temperature was 3540°C. Aeration was done by passing humidified air through the perforated bottom of the tank. Water was occasionally sprayed onto the fermenting mass to maintain the moisture level. After 30 hours of incubation, the product obtained had an average of 20% true protein, 25% residual reducing sugar, 63% moisture, and the extent of conversion of carbohydrates to protein was 20-25% (Senez,

1978). Another recent report on the use of a solid-state fermentation process for enrichment of protein in plant materials has come from Bajracharya and Mudgett (1979). They pressed 15 gm of fresh alfalfa leaves and the solid residue was autoclaved in a 250-ml Erlenmeyer flask at 121°C for 20 minutes. The contents of the flask were allowed to cool, inoculated with Aspergillus niger and Rhizupus nigricans, and incubated on a Gyrotary shaker at 400 rpm at 28°C and relative humidity of 75-80% for 4 days. Upon reimbibing and second pressing, total protein recovery from alfalfa was increased to 47.2%for unfermented control samples and 64.5%for fermented samples. Up to 50% more protein could be recovered by such fermentation than by conventional mechanical extraction processes. They reported that such protein is generally suitable for human consumption in fabricated foods and the fermented residual cakes could serve as animal feed.

SOLID SUBSTRATE FERMENTATIONS

223

b. Fermented Animal Waste Material as Feed. In the past, farm animal wastes have not been a problem since they were used as soil conditioner, but intensive livestock rearing has changed this situation. Anthony (1971) and Smith (1973) have reviewed the use of animal waste, primarily as a nitrogen source in the feeding or refeeding of livestock. Solid-state fermentation systems have been used to convert feedlot waste liquid (FLWL) combined with corn as cattle feed (Rhodes et al., 1975; Hrubant, 1975). The liquid obtained (about 7% nitrogen) when feedlot waste (FLW) was screened to remove fibrous material, was mixed with corn to give a mixture with a moisture content of 3542%; FLWL (175 gm) and corn (350 gm) were mixed in a 2-liter Erlenmeyer flask. The flask was held at 9" from horizontal on a board rotating at 0.6 rpm and allowed to ferment at 28°C using the organisms naturally present. Preparation of FLWL and large-scale fermentation procedures were reported by Rhodes and Orton (1975). It was found during this two-stage silage-like fermentation that lactobacilli and yeasts became the predominant flora after coliform bacteria in the FLW died out as a result of production of lactic acid during the fermentation. Possible health and pollutant hazards inherent in the waste were reduced in the early heterolactic phase of the fermentation. Homolactics dominated the later phase and yeasts also increased (Hrubant, 1975). This process conserves most of the nitrogen in the FLWL, while converting the waste to a feed with a more desirable amino acid composition. Hrubant and co-workers (1976) reported on a continuous process of solid-state fermentation of FLWL and corn using a three-chambered horizontal drum. The FLWL contained in a reservoir was added to cracked corn from a miniature bin to give the correct moisture level. The FLWL-corn mixture was added at 30-minute intervals into the first of the three chambers of a horizontal drum rotating at 0 . 5 rpm. The mass was slowly rotated through three compartments, each with baffles arranged so that the moist corn tumbled and gradually moved to the second chamber and to the third. From the third chamber, the fermented mass was collected and was fed to cattle moist or dried for storage. This nonaseptic fermentation was run at 18, 24, and 36 hours through-put time and each chamber contained 2.3 kg of FLWL-corn mixture (3:2 w/w) at 42% moisture. Fermentation was started in the first chamber and during the 36-hour period, lactobacilli and yeasts from the FLWL grew exponentially to 10"' and 10' organisms per gram dry weight, respectively. This population remained stable in all chambers at all retention times throughout the subsequent 62 days of continuous operation. Hesseltine (1977) reported on the use of a standard 70-liter cement mixer to ferment several kilograms of FLWL-corn mixture. The mixture was adjusted so that the bowl rotated at 0.5 rpm and it was charged with 23 kg of grain and 11-13 kg of FLWL. The interior of the bowl was sandblasted and painted

224

K. E. A I D 0 0 ET AL.

with an epoxy paint to eliminate rust formation due to acid formed in the fermentation. After being charged, the bowl was tilted and operated at 3035°C for 36 hours, after which the product was dried by blowing 60°C air into the rotating bowl. The major acids formed during the fermentation were lactic, 65.7%; acetic, 22.5%; n-butyric, 5.4%; propionic, 3.9%; unidentified, 1.8%; n-valeric, 0.96%; n-caproic, 0.83%; and isovaleric, 0.43%. Loehr (1974, 1977) has reviewed methods of biological treatments of farm wastes and Knapp and Howell (1979) have also given an account of the alternative approaches to treatment and reuse of livestock waste and sewage sludge. c. Coinposting Methods that have been used to dispose of municipal solid waste include barging to sea, incineration, land fill, and composting. Composting, a fermentation process, modifies but does not dispose of refuse. Reviews on composting were published by Kershaw (1968) and Gray et al. (1971, 1973). Composting may be considered as a two-phase fermentation. During the first phase, more readily available simple sugars, for example, are fermented by diverse organisms, leading to production of heat, which induces the second phase, whereby thermophilic organisms attack less readily available polysaccharides, for example, with further production of heat. Schulze (1962) published a detailed study of the process of continuous thermophilic composting. Different methods of composting, including mechanical and semimechanical processes, have been developed for production of odorless compost from municipal refuse. Examples of the development of compostmaking plants are the “Dano system” from Denmark, the “Verdier system” from France, and the “Beccari system” from Italy (Kershaw, 1968). The operations of the “Schulze composter” and the “Dano system” have been presented in detail by Schulze (1962) and McCrae (1969), respectively.

I l l . Design Considerations and Types of Solid-state Fermentors Mukhopadhyay and Pathak (1973) noted that in the design and fabrication of solid-state fermentors, some factors have to be considered; these include the techniques of inoculation, sampling, and transfer systems; sterilization of equipment, air, and fermenting medium; and monitoring, measurement, and control of various parameters. There is remarkably little information on monitoring and measurement of fermentation parameters in solid-state fermentations. However, Ramstack et al. (1979) have recently reported on the use of the gas chromatographic technique for analysis of headspace gas in a solid-state fermentation process. For a pilot-plant or large-scale fermenta-

225

SOLID SUBSTRATE FERMENTATIONS

tion of moist solid substrate, overheating becomes a problem; incorporation of suitable heat exchangers into the design of the fermentation vessel to remove excessive heat is of great importance. These considerations, among such others as materials and methods of construction of the fermentation vessel itself; and agitation, rotation, or shaking arrangement, could lead to the development of reliable commercial fermentation equipment offering greater uniformity and understanding of the process. Different workers have used difTerent equipment designs (fermentors) to TABLE I11 TYPESOF SOLID-STATE FERMENTATION VESSELS~ Fermentor (capacity) Wooden trays Rotating drum 55-gallon

5-gallon

Fermentation process Koji fermentation with A. o q z a e

Church (1923)

Composting of typical garbage mixture Koji fcrrnentation with A . oryzae Enzyme production with A . oryzae

Schulze (1962)

Fernbach flask (2.8-liter)

Production of aflatoxin with A . parasiticus Production of fungal spores

Aluminum pot (3.4-liter) Horizontal vessel (4chambered; 26 litcrs) Horizontal drum (3chambered) Cement mixer 70-litcr

Enzyme production with A . oryzae

114-liter

Bread-making blender (10 ka) Corn storage bin; Butler-type (18-ft diameter containing 1266 bushels of corn) A horizontal vessel with paddles (20-liter)

Reference

Production of ochratoxin with A. ochraceus Continuous, using feedlot wastes and corn Large scale of feedlot waste and corn fermentation Fermentation of straw with Aureobnsidium pullulans, Candida iitilis, and Trichoderma uiride Protein enrichment of starchy materials with A . niger Production of aflatoxin with A. flavus

Koji fermentation with A. oryzae

"With permission, from John Wiley and Sons, Inc., and Professor Tanro.

Arima (1964) Underkofler et 01. (1939) Hesseltine at al. (1966, 1968) Sehgal et a2. (1966) Hao at (11. (1943) Lindenfelser and Ciegler (1975) Hrubant et al. (1976) Hesseltine (1977) Han et al. (1975) Raimbault et al. (1977); Senez (1978) Silman et al. (1979)

Aidoo (1979)

226

K . E. A I D 0 0 ET AL.

carry out solid-state fermentations, and Table 111 summarizes different types of solid-state fermentation with different fermentors. All these fermentors and many others have at least one common feature: they offer simple and inexpensive processes. Fulfilling the requirement for simple, less complex equipment on laboratory, pilot-plant, and large-scale operations is an advantage of solid-state fermentation.

IV. Characteristics of Solid-state Fermentations In solid-state fermentation, several factors must be taken into consideration to achieve high efficiency of the overall process; some of the general characteristics follow (Hesseltine, 1972, 1977).

1. Commonly used substrates include cereal grain, legumes, or other plant and animal products with high carbohydrate and/or protein content. 2. The solid substrate must be in a form so as to allow free circulation of air; it should neither become agglomerated with mycelium or gummy so that individual particles adhere together. 3. Usually the only other medium component required is water, although occasionally other nutrients such as nitrogen sources or minerals may be added. For instance, Kokke (1977) sterilized carob pods in ammonium salt solution before fermenting them. 4. Because of relatively dry substrate, there is a much reduced possibility of bacterial contamination. 5. Control of temperature is sometimes critical, and control of the composition of the atmosphere in the culture vessel with respect to oxygen, carbon dioxide, and volatile metabolite concentrations may be needed. Besides these characteristics regarding the nature of the substrate, other problems will have to be solved before a continuously agitated solid-state fermentation process can be used on a commercial scale to produce a variety of products. According to Hesseltine (1972), some of the general problems associated with the use of solid substrates are as follows: 1. Inoculum should be produced in the form of spores in large quantities to germinate rapidly and uniformly at better than 95%. In the production of spores of Aspergillus sojae and A. oryxae in the old Japanese process, wood ashes, especially of certain species of trees, were added to brown rice or wheat bran to obtain spores that had a germination rate of 95% or better. 2. The moisture level of the solid substrate has to be determined for each species used and probably for each strain as well.

SOLID SUBSTRATE FERMENTATIONS

227

Ralph (1976) considered two principal types of microbial behavior under solid-state fermentations: (i) that in which a solid material serves as the main source ofnutrients for an organism which intimately associates itself with the surface and interstices of the material; (ii) that in which a nutritionally inert solid support provides soinc advantage to the nrganisrn in respect to access to nutrients.

According to Ralph, these two categories are not generally clearcut in practice, since the majority of solid materials involved in interactions with microorganisms are complex mixtures of nutritional and inert components and the functions overlap. However, some reports have been published on high yields of organic acids, enzymes, and other fungal metabolites by the use of solid inert materials. An example is the production of citric acid by A. nigw on solid inert materials. Meyrath (1965, 1966) studied production of amylase dextrinogenic activity with A. cwyzac using different culturing methods (surface, submerged, bran, and a synthetic solid inert material, vermiculite). He found that the rate of enzyme production on vermiculite impregnated with 4% starch solution was as high as on bran and the yield almost doubled; both the liquid- and submerged-culture methods gave lower rates of amylase production. H e noted that a technological advantage of vermiculite is the fact that very high concentrations of substrate can be handled in this way; a high proportion of starch granules can be mixed with a slightly humidified absorbing agent and sterilized, and the remaining part of the sterile liquid substrate can be added along with the inoculum. In liquid media, starch solutions above 4% are highly viscous and in submerged cultures, these high concentrations of substrates will also result, of course, in heavy growth, too heavy to be able to give homogenous aerobic conditions throughout the fermentor. PHYSIOLOGICAL

ASPECTS OF MICROBIALGROWTHON SOLIDSUBSTRATES

Moisture content of the solid substrate is an important factor during microbial growth in a solid-state fermentation process. It was found during our study and also from published information as shown in Tables IV and V, that fermentation of moist solid substrate produces large quantities of heat. It appears that there are very few published data on the physiological aspects of microbial growth on moist solid substrate. Wang (1976) pointed out two major characteristics of the solid-state fermentation process as (1)limitation of water required for microbial growth and (2) limitation of mobilization of microbes and nutrients. With regard to limitation of water, there is either a definite loss of moisture from the sub-

~.

-

.

Total inctabolic heat

ol-gaI>isnI

A , oryzue

Bacillus omylosckens

Medium composition (parts by weight) Wheat bran, 1.0; rice hulls, 1.0; water, 2 . 6 Wheat bran, 6.0; soybean oil meal, 1.5; rice hulls. 2.5; water, 10.0

Moisture content (percentage)

(kcdkip)-dry- coinputed on the basis of

Culture prrid jh(>unj

Initial

Final

lieat balllarice

Respiratory activity

Metabolic heat removed by aeration computed on the basis [if heat balance

41

63.1

55.8

610

598

55.1

33

63.0

57.2

538

592

451

"Adapted from Terui c'f a / . (l959), with prrmiscion from Osaka University and Profvssor G. Terui.

SOLID SUBSTRATE FERMENTATIONS

229

TABLE V HEATGENERATION OF KOJI CULTUREa

Kinds of koji culture

A . uryzue, wheat bran koji A. oryzue, wheat bran koji, rod shaped A. niger, wheat bran koji B. subtilis, wheat bran koji (one-third bean cake) A. oryzae, rice koji for miso A. oryzue: rice koji for saki. A. oryzae, koji for soy sauce (wheat bran) A. niger, citric acid fermentation (sawdust with molasses) A. niger, citric acid fermentation (starch cake, rice bran)

Total heat generation (kcah4

475-625 470 570-625 500520 110-180

100 440 540

570

a Adapted from Arima (1964), with permission from Almqvist and Wilsell International and Professor Arima.

strate (for instance, in the koji process and in composting, which are open systems) or little or no moisture loss from the substrate as in mushrooin cultivation and the Kaoliang brandy brewing process in which solid mash is inoculated with carbon dioxide-tolerant saccharifying molds and the mash sealed with a polyethylene sheet (closed systems). Recently, Knapp and Howell (1980) have presented kinetic models of microbial growth on solid substrates. They studied the principles and fundamental similarities of microbial growth on solid substrates and also considered the fermentation of cellulosic substrates to produce single-cell protein.

V. Advantages and Disadvantages of Solid-state Fermentation There are many advantages of solid-state fermentation processes over the conventional stirred tank system on both laboratory and large scale. Some of the advantages for work involving fungi, according to Hesseltine (1972, 1977) are as follows:

1. The medium is relatively simple, for instance whole grain with water just sufficient to moisten the substrate. 2. The space required by the fermentation equipment is small relative to

230

K. E. A I D 0 0 ET AL.

yield of product because less water is used and the substrate is concentrated. The complexity of equipment on a laboratory, pilot-plant, or factory scale is no greater than with conventional fermentation equipment and may be less. 3. Since spores are used directly in the fermentation, neither seed tanks nor preformed inoculum is necessary. 4. The conditions under which the fungus grows are more like the conditions in its natural habitat. 5 . Aeration is easily obtained since there are air spaces between each particle of the substrate. 6. The desired product may be readily extracted from the vessel by addition of solvent directly. Points (l),(2), (S), and (6) would also apply to bacterial fermentations, and point (4) may also be applicable. It would also seem reasonable to argue that energy consumption in operating a slowly rotating drum will be considerably less than that in driving the powerful stirrers used in stirred tank reactor (STR) fermentation. Similar arguments could be applied to aeration. The preparation of genuinely comparable energy balances for production of the same material (e.g., an enzyme) on solid-state and in liquid STR fermentor systems would be an interesting and a potentially very valuable exercise. These advantages, in addition to some of the general characteristics of solid-state fermentation, strongly support the application of solid-state fermentation technique in industrial production of biochemicals and other products. Hesseltine listed the disadvantages of solid state fermentation as

1. The type of organisms are limited to those which can grow at reduced moisture levels, namely fungi, some yeasts, and some bacteria and streptom yce tes . 2 . Heat becomes a problem when large quantities of moist substrates are used. 3. Using monitoring devices to determine moisture, pH, etc., becomes a problem. Also controlling pH, etc., is difficult. 4. Spore inoculum needed is presumably quite large. 5. The substrate may have to be pretreated by pearling, cracking, or dehulling before use. One of the major problems encountered in studying solid-state fermentation systems is the difEculty in the estimation of mycelial biomass. Various workers (Arima and Uozomi, 1967; Ride and Drysdale, 1972; Swift, 1973; Jarvis, 1977) have used chemical methods for estimation of mycelial biomass

SOLID SUBSTRATE FERMENTATIONS

231

in solid-state fermentation by measuring glucosamine (chitin) levels. Wood (1979) showed that laccase activity measured during the mycelial colonization of coinposted wheat straw in a solid-state fermentation system could be used to quantify the mycelial growth of Agaricus bisporus. Bajracharya and Mudgett (1979) estimated fungal biomass production during solid-state fermentation of alfalfa by carrying out experiments in a two-chamber dialysis culture vessel separated by a noncellulosic membrane that restricted d i f i sion of colloidal solids but permitted diffusion of high-molecular-weight soluble proteins.

VI. Future Developments of Solid-state Fermentation Systems Several areas exist for further investigations and developments of the fermentation processes and these include

1. Production of enzymes. Ralph (1976)reported that work on the production of enzymes similar to that of the koji process seemed to have come to an end in the mid-1950s and it is not clear from the literature that comparisons have been made on the relative merits of solid-state fermentations and submerged culture methods for the production of more recently introduced enzymes. Modifications of foods by microbial enzymes produced by solidstate fermentation processes are still being used in many parts of the world, particularly in the developing countries, and methods for improvements are being sought. Hesseltine (1965)pointed out the need for improving strains of microorganisms used in fermenting foods. He stated that the future development of fermenting foods by solid-state fermentation process depends not only on production of hybrids or improved mutants of strains like A. oryzae or Saccharomyces ruwii, but also on isolating or concentrating peculiar or specific selected characteristics. 2. Production of other fungal metabolites such as mycotoxins and antibiotics. Hesseltine (1972) reported on a compound that shows antibiotic activity against certain bacteria and that was obtained by growing the fungus Alternaria hrassicicola on solid substrates. 3. Production of fungal spores. Fungal spores have been shown to have considerable potential as agents for the transformation of organic compounds (Vezina et d.,1968), for food flavors (Hesseltine, 1965; Lawrence, 1966), and as biological control agents for plant pathogens (Rishbeth, 1963). On a dry-weight basis, fungal spores are said to possess between three and ten times the transformation activity of fungal mycelium and would seem to offer interesting possibilities as biochemical catalysts for the production of novel

232

K. E. A I D 0 0 ET AL.

compounds (Ralph, 1976). Published results seem to suggest that they are conveniently produced by solid-state fermentation, but again, there is a need for detailed comparison with their production by other means. 4. Protein enrichment of high starch foodstuff. Solid-state fermentation techniques have been used for protein enrichment of some food items with high starch content such as cassava and wheat (Martinelli and Hesseltine, 1964; Wang and Hesseltine, 1966; Brook et al., 1969; Stanton, 1971; Raimhault et al., 1977; Bajracharya and Mudgett, 1979). At present, solid-state fermentation processes are being actively developed in France in close collaboration with Thailand in the scaling-up of protein enrichment of cassava and other starchy materials (Raimbault et nl., 1977; Senez, 1979). Work is being carried out for large-scale nutritional and toxicological testing on target animals (pigs and poultry), for further optimization of substrate preparation and growth conditions, and for determination of the actual investment and operation costs. It is intended that the experimentation will be extended to the setting-up of experimental production units in tropical Asia and Africa in order to adapt the procedure to local climatic and agroeconomical conditions. 5 . Bioconversion of agricultural and agroindustrial wastes into useful products. 6. The use of solid inert material (e.g., plastic foams), impregnated with suitable media, will give homogenous aerobic conditions throughout the fermentor and contribute no impurities to the fermentation products (e.g., enzymes, organic acids). Meyrath (1965, 1966) reported a high yield of dextrinogenic amylase with A . nryzae on vermiculite impregnated with starch. In a preliminary study with polyurethane ether foam as support material for production of citric acid by A. nigw, Aidoo (1’379) obtained higher yields of citric acid than by submerged- or surface-culture methods. 7. Soil may he used as a solid substrate on which organic biotransformations are ef€ected. “Sludge farming” has been used to degrade oil by means of indigenous microbial population (CONCAWE, 1980). Mixed culture fermentations are currently being used in attempts to degrade oil on railway tracks. Oil accumulates as a result of spillage from locomotives, causing pollution problems. Stimulation of the natural track microflora is being attempted by various methods in the hope of achieving enhanced oil-degradation rates (Smith, 1981).

VII. Conclusions It is evident that solid-state fermentation processes have existed for many, many years and have wide applications in the production of food and enzymes, processing of biological waste materials into useful products, etc.

SOLID SUBSTRATE FERMENTATIONS

233

We are of the opinion that there still exist, particularly for developing countries, areas for further investigations and/or developments of the process, for production of food, animal feed, etc., under controlled conditions, since the process is simple and requires little energy. Aidoo (1979), for instance, found that filamentous fungi belonging to Aspergillus, a genus of fungi widely used in food and pharmaceutical industries, will grow well under conditions that exist in simple solid-state fermentation vessels. ACKSOWLEUCYENTS

We thank the Government of Ghana for their generous support of K. E. Aidoo, and we thank Mrs. T. E. Aidoo for her careful typing of the manuscript.

REFERENCES Adams, S. L., Balankura, B., Andreasen, A. A,, and Stark, W. H . (1947). Znd. Eng. Chem. 39, 1615-1617. Aidoo, K. E. (1979). Ph.D. Thesis, University of Strathclyde, Glasgow. Anthony, W. B. (1971). J. Animal Sci. 32, 799-802. Arima, K. (1964). In “Global lmpact of Applied Microbiology” (M. P. Starr, ed.). Wiley, New York. Arima, K . , and Uozomi, T. (1967). Agric. B i d . Chem. 31, 119-123. Asao, T., Buchi, G . , Abdel-Kadar, M. M., Chang, S. B., Wick, E. L., and Wogan, G. N. (1965).J. Am. C h e m Soc. 87, 882486. Atkinson, R. M. (1881). Proc. R. Soc. (London) 32, 299332. Babel, F. J . (1953). Econ. Bot. 7(1), 2 7 4 2 . Bajracharya, R . , and Mudgett, R. E. (1979). Biotechnol. Bioeng. 21(4), 551-560. Balankura, B., Stewart, D., Scalf, R. E., and Smith, L. A. (1946). J. Bacteriol. 51, 594. Banigo, E. 0. I. (1969). Ph.D. Thesis, University of Leeds, U.K. Batra, L. R., arid Millner, P. D. (1976). Deo. Ind. MierobioZ. 17, 117-128. Beckford, L. D., Kneen, E., and Lewis, K . H. (1945). lnd. Eng. Chem. 37, 692-696. Beckhorn, E. G . (1960). Wallerstein Lab. Cotmnun. 23, 201-210. Brook, E. J . , Stanton, W. R . , and Wallbridge, A. (1969). Biotechnol. Bioeng. 1 1 , 1271-1284. Bush, L. (1959).“Land of the Dragon.” Hale. London. Butler, W. H. (1975). In “The Filamentous Fungi” (J. E. Smith and D. R. Berry, eds.), Vol. 1. Arnold, London. Cahn, F. J. (1935). lnrl. Eng. Chem. 27(2), 201-204. Christian, W. F. K. (1971). Ghana J. Sci. 10, 22. Church, M. B. (1920). J. lad. Eng. Chern. 12, 45-46. Church, M . B. (1923). U . S . Dept. Agric. Bull. No. 1152, pp. 1-26. CONCAWE, (1980). Sludge farming: A technique for the disposal of oily refinery waste. Rep. 3/80. The Hague. Currie, J. N. (1917). J . Biol. Chem. 31, 15-37. Detroy, R. W. and Hesseltine, C. W. (1978). Process Biochern. 13(9), 2-8 and 31. Detroy, R. W., Lillehoj, E. B., and Ciegler, A. (1972). “Microbial Toxins,”Vol. VI. Academic Press, New York.

234

K. E. A I D 0 0 ET AL.

Iljicn, K. S., and Hesseltine, C. W. (1961). Soybecm Dig. 22, 14. Frateur, J. (1950). Cellule 53, 287. Goel, S. K., and Wood, B. J. B. (1978). J. Food Technol. 13, 243-247. Gray, W. D. (1970). Food Techno/. 1(2), 225329. Gray, K. R . , Sherman, K., and Bitldlestone, A. J. (1971). Proccw Biochem. 6(6), 3236; 6(10), 22-28. Gray, K . R . , Biddlestone, A. J., and Clark, R. (1973). Process Biochem. 8(10), 11-15 and 30. Han, Y. W., and Anderson, A. W. (1975). A p p l . Microhid. 30, 930-Y34. Han, Y. W., and Callihan, C. D. (1974). A p p l . Microbiol. 27, 159-165. Han, Y. W., Grant, G. A,, Anderson, A. W., and Lu, P. L. (1Y76). Feedstuff April, pp. 17-20. Hansen, E. C. (1878). C. R . Carlsberg I, 49 and 96. Hao, L. C . , F u h e r , E. I., and Underkofler, I,. A. (1943). Inrl. Kng. Chevr. 35, 814. Hayes, W. A , , and Nair, N . G . (1975). Zn “The Filamentous Fungi” (J. E. Smith and D. R. Berry, eds.), Vol. I. Arnold, London. Hesseltine, C . W. (1965). lllycologia 57, 149-197. Hesseltine, C. W. (1972). Hiotechnol. Bioeng. 14, 517532. Hesseltine, C . W. (1977). Process Biochem. 12(6), 24-27; 12(7), 3 0 3 2 . Heaseltine, C. W., and Wang, H. L. (lY67). Riotechnol. Bioeng. 9, 275-288. Hesseltine, C;. W., Shotwell, 0. L., Ellis, J . J., and Stubblefield, R. D. (1966). Bucteriol. Reu. 30, 7954405. Hesseltine, C. W., Shotwell, 0. L., Smith, M. L., Shannon, 6. M., Vandegraft, E. E., and Goulden, M . L. (1968). Mycolngia 60, 304312. Hesseltine. C. W., Vandegraft, E. E., Fennell, D. I., Smith, M. L., and Shotwell, 0. L., (1972). Mycologia 64(4), 539-550. Hesseltine, C. W., Swain, E. W . , and Wang, H. L. (1976). Deo. Iid. Mia-obiol. 17, 101-115. Horder, L., Dodds, C., and Morgan, T. (1954). “Bread.” Constable, London. Hrubant, G . R. (1975). A p p l . Miwohio1. 30, 113-119. Hrubant, G . R . , Orton, W. L., and Rhodes, R. A. (1976). Am. Soc. Miuobiol. Absh. 182 (as cited in Hesseltine, 1977). Ilieda, Y . , Takahash, E . , Yokogawa, K., and Yoshimure, Y. (1972). 1. Ferment. Technol. 50, 361370. Jarvis, B. (1977). J . Food Technol. 12, 58-591. Jeffreys, G . A. (1948). Food Znd. 20, 688490 and 825. Johnson, M. J. (1Y71). Chela. Technol. June, pp. 338341. Kershaw, M. A. (1968). Process Biochern. 3(5), 53-56. Knapp, A. (1920). “Cocoa and Chocolate. Their History from Plantation to Consumer.” Chapman h Hall, London. Knapp, J. S., and Howell, J. A. (1979). Process Riochem. 14(6), 8-11 and 28. Knapp, J. S., and Howell, J. A. (1980). In “Topics in Enzyme and Fermentation Biotechnology” (A. Wisernan, ed.), Vol. IV. Wiley, New York. Kokke, R. (1977). 1. A p p l . Bucteriol. 43, 303307. Kiitzing, F. (1837). 1. Prakt. Chem. 11, 385409. Lakshminarayana, K., Chaudhary, K., Ethiraj, S . , and Tauro, P . (1975). Biotechtiol. Bioeng. 17(1), 291-293, Lawrence, R. C . (1966). J . Gen. Micmbiol. 44, 393405. Le Mense, E. H., Corman, J . , VanLanen, J. M., and Langlykke, A. F. (1947). J. Bucteriol. 54(2), 149-159. Le Mense, E. H., Sohns, V. E., Corrnan, J., Blom, R. H . , VanLanen, J . M . , and Langlykke, A. F. (1Y49). Ind. Eng. Chein. 41(1), 100-103.

SOLID SUBSTRATE FERMENTATIONS

235

Leisinger, T., and Muller, J. (1967). Process Biochern. 2(4), 10-15. Lewis, G. (1977). M.Sc. Thesis, University of Strathclyde, Glasgow. Lindenfelser, L. A , , and Ciegler, A. (1975). Appl. Micfobiol. 29, 323327. Lockwood, L. B. (1947). So!/hsati Dig. 7, 10-11. Loehr, R. C. (1974). “Agricultural Waste Management: Problems, Processes, and Approaches.” Academic Press, New York. Loehr, R. C. (1977). Proc. S ! / m p . Man. Res. Util. U N E P , FAO, Rome. McCrae, K. C. (1969). Proc. Znst. Civil Eng. 42, 281300. McCullough, E. C. (1945). “Disinfection and Sterilization,” 2nd ed. Lea & Fcbiger, Philadelphia, Pennsylvania. Martinelli, A. F . , and Hesseltine, W. C . (1964). Food Tcchnol. 18(1),167-171. Meyrath, J. (1965). J . Sci. Food Agric. 16, 14-18. Meyrath, J . (1966). Process Biochetn. 1(4), 234-238. Meyrath, J. (1967). Process Riuchern. 2(10), 25-27 and 56. Miall, L. M. (1975). In “The Filamentous Fungi” (J. E. Smith and D. R. Berry, eds.), Vol. I. Arnold, London. Moo-Young, M., Daugulis, A . J., Chahal, D. S., and MacDonald, D. G . (1979). Process Biocheni. 14(10), 3 8 4 0 . Mukhopadhyay, S., and Pathak, A. N. (1973). C h e m Age Zndia 24, 583-587. Muller, H. G . , and Nyarko-Mensah, B. (1972). J. Sci. Food Agric. 23, 544-545. Pasteur, L. (1868). “Etudes sur la Vinaigre” (as cited in Prescott and Dunn, 19.59). Peppler, H. J. (1967). “Microbial Technology.” Van Nostrand-Reinhold, Princeton, New Jersey. Persoon, C. H. (1822). Mycol. Eur. 3 Vols. Erlangen. Platt, B. S. (1962). Spec. Rep. Ser. Med. Res. Counc. Londoti No. 302. Platt, B. S. (1964). Food Technol. 18, 68-76. Pool, E. L., and Underkofler, L. A . (1953). J . Agric. Food Chem. 1(1), 87-90. Prescott, S. C., and Dunn, C. G . (19S9). “Industrial Microbiology.” McGraw-Hill, New York. Prim, J., and Nielsen, T. K. (1970). Process Biochern. 5(5), 3 4 3 5 . Raimbault, M., Deschatnps, F., Meyer, F., and Senez, J. C. (1977). Int. Conf Global Ztnp. Appl. Microbid., 5th, Bangkok. Ralph, B. J (1976). Food Technol. Aust. 28(7), 247-251. Ramstack, J. M., Lancaster, E. B., and Bothast, R. J. (1979). Process Biochetn. Feb., pp. 2 4 , Rao, M. V. R. (1961). Natl. Acad. Sci.-Natl. Res. Counc. Publ. 843, 291-293. Rhodes, R. A,, and Orton, W. L. (1975). Trans. Atti. Soc. Agric. Eng. 18, 728-733. Rhodes, R. A , , Orton, W. L., and Weiner, B. A. (1975). U.S. Patent No. 3, 968,254. Ride, J. P., and Drysdale, R. B. (1972). Physiol. Plant Pathol. 1, 409-420. Rishbert, J . (1963). Ann. A p p l . B i d . 52, 63-77. Roberts, M . , Laufer, S., Stewart, E. D., and Saletan, L. T. (1944). Znd. Eng. Chem. 36, 811-812. Rolz, C . (1978). J. Appl. Chern. Biotechnol. 28, 321339. Sansing, G. A , , and Ciegler, A. (1973). Appl. Mierobiol. 26, 830-831. Sardinas. J. L. (1969). Process. Biochs7n. 4(7), 13-16, 21. Sargeant, K., Sheridan, A., O’Kelley, J., and Carnaghan, R. B. A. (1961). Nature (London) 192, 1096-1097. Scheele, K. (1787). Chem. Ann. Frsunde Naturl. 1, 3-12. Schulze, K. L. (1962). Appl. Mierobiol. 10, 108-122. Scott, R. (1967). Process Biochetn. 2(2), 5-10; 2(3), 23-28; 2(5), 49-53 and 56. Sehgal, S. N., Singh, K . , and Vezina, C. (1966). U.S. Patent No. 3,294,647.

236

K . E. A I D 0 0 ET AL.

Senez, J. C. (1978). Ord. Meet. Soc. Gen. Mimobiol., 8&h, Cardqf, Wales. Sheppard, H . , and Newton, E. (1957). “The Story of Bread,” pp. 15-24, Routledge & Kegan, London. Shibasaki, K., and Hesseltine, C. W. (196la). J. Biochem. Microhiol. Technol. 3, 161-174. Shibasaki, K., and Hesseltine, C. W. (196lb). Deo. Ind. Microbiol. 2, 205-214. Shibasaki, K., and Hesseltine, C. W. (1962). Econ. Bot. 16(3), 180-195. Shotwell, 0.L., Hesseltine, C. W., Stubblefield, R. D., and Sorensen, W. G. (1966). A p p l . Microhid. 4(3), 425-428. Shurtleff, W., and Aoyagi, A. (1976). “The Book of Miso.” Autumn Press, Kanagawa-Ken, Japan. Silman, R. W., Conway, H. F . , Anderson, R. A , , and Bagley, E. B. (1979). Biotechnol. Bioeng. 21(10), 1977-1808. Singh, K . , Sehgal, S. N . , and Vezina, C. (1965). Can. J. Microbiol. 11, 351364. Singh, K . , Sehgal, S. N . , and Vezina, C. (1967). Cun. J. Microbiol. 13, 1271-1281. Singh, K., Sehgal, S. N., and Vezina, C . (1968). A p p l . Microbiol. 16, 393400. Smith, A. D. (1981). Ph.D. Thesis, University of Strathclyde, Glasgow. Smith, L. W. (1973). Am. Soc. Animal Sci. Commun. Animal Nutr. Nut/. Res. Come.-Natl. Acad. Sci. U.S.A. pp. 147-173. Stanton, W. R. (1971). Proc. Int. Symp. Conu. Munuf. Fooclstuffs, Kyoto pp. 133-139. Stanton, W. R. (1978). Process Biochem. 13(12), 6-7. Stanton, W. R., and Wallbridge, A. (1969). Process Biochem. 4(4), 45-51. Stubblefield, R. D., Shotwell, D. L.,Hesseltine, C. W., Smith, M . L., and Hall, H . H. (1967). A p p l . Microhiol. 15, 186-190. Swift, M. J. (1973). Soil Biol. Biochem. 5, 321332. Sylvester, J. C., and Coghill, R. D. (1954). In “Industrial Fermentation” (L. A. Underkofler and R. Hickey, eds), Vol. 11. Chemical Publ., New York. Takamine, J. (1894). U.S. Patent No. 525,820. Takamine, J. (1913). U.S. Patent No. 1,054,324. Takaminc, J. (1914). J. l n d . Eng. Chem. 6, 824-828. Tamiya, H. (1927). Acta Phytochim. u p . )3, 51-173. Terni, G. (1944). “The Sweet Potato Industries.” Kanshokogyo (in Japanese). Terui, G., and Konno, N . (1960). Technol. R e p . Osaka Unio. 38, (29) 889-903. Terni, G . , Shibasaki, I., and Mochizuki, T. (1957). Technol. R e p . Osaka Unio. 7, 215-233. Terui, G., Shihasaki, I., arid Mochizuki, T. (1958). Technol. Rep. Osaka Uniu. 8, 213. Terui, G., Shibasaki, I., and Mochizuki, T. (1959). J . Fermenl. Technol. 37, 479. Terui, G., Shihasaki, I., Mochizuki, T., and Takano, M. (1960). J. Ferment. Technol. 38 (29). Thom, C., and Currie, J. N. (1913). J. Biol. Chetn. 15, 249. Underkofler, L. A. (1939). Ind. Eng. C h e m 31, 734-738. Undcrkofler, L. A. (1954). I n “Industrial Fermentation” (L. A. Undcrkofler and R. Hickey, eds.), pp. 97-121, Chemical Publ., New York. Underkofler, L. A . , Fulmer, E. I . , and Schoene, L. (1939). Ind. Eng. Chem. 31, 734-738. Underkofler, L. A , , Severson, G. M., and Goering, K . J. (1946). Znd. Eng. Chem. 38,980-985. Underkofler, L. A., Severson, G. M . , Goering, K. J . , and Christensen, L. M. (1947). Cereal Che,?l. 24(1), 1-22. VanTieghem, P. E. L. (1867). C. R . Acad. Sci. 65, 1091-1094 Van Veen, A. G . (1957). UN-FAO. Rep. FA0/57/3/1966. Vezina, C., and Singh, K . (197.5). In “The Filamentous Fungi” (J. E. Smith and D. R. Berry, cds.), Vol. 1. Arnold, London. Vtzina, C . , Sehgal, S . N., and Singh, K . (1968). Adu. A p p l . Microbiol. 10, 221-268. Wang, H. H. (1976). Lab. Appl. Microbial. Dept. Agric. Chein. Taiwan, R.O.C.

SOLID SUBSTRATE FERMENTATIONS

237

Wang, H . L., and Hesseltine, C. W. (1966). Cereal Chem. 43,563570, Wehmer, C. (1891). Bot. 2. 49, 233-638. Wehmer, C. (1893). C. R. Acad. des Sciences FranCais 117, 332. White, J. (1966). Process Biochsm. 1(3), 139-144 and 155. Whymper, R. (1921). “Cocoa and Chocolate. Their Chemistry and Manufacture.” Churchill, London. Wood, D. A. (1979). Biotechnol. Lett. I@), 255-260. Yabuta, T. (1912). 1. Coll. Agric. Tokyo Imp. Uniu. 5, 51-58. Yong, F. M . , and Wood B. J. B. (1974). Ado. A p p l . Microbial. 17, 157-194.

This Page Intentionally Left Blank

Microbiology and Biochemistry of Miso (Soy Paste) Fermentation SUMBO H. A B I O S E , ~M . C. ALLAN,

AND

B . J. B . WOOD^

Depurtinent of Applied Microbiology, Univcmity of Strathclyde, Glasgou;, Scotland

I. 11. 111. IV. V. VI.

VII. VIII. IX. X.

XI. XII.

Introduction,. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Fermerited Soy Products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Fermented Rice Products . . . . . . . . . . . . . . . . . . . . History of Miso Production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Types of Miso . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Raw Materials for Miso Production . . . . . . . . . . . . . . . . . . . . . . . A. Soybeans (Gklcine mar) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . €3. Rice (Oryzn scitivn) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Barley (Hurdeutri vulgnrcr or satiuum) . . . . . . . . . . . . . . . . . D. Salt . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ratio of Raw Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Treatment of Raw Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Koji . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Moroini . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chemical Composition of Miso . . . . . . . . . . . . . . . . . . . . . . . . . . . Future Developments in Miso Production . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

239 241 245 247 248 248 250 251 251 251 252 253 255 257 259 261

I. Introduction Fermentation is among the oldest methods of food preservation. Improved flavor and digestibility are some of the reasons for fermenting food (Tanner, 1944). A food is considered fermented if one or more of its constituents has been acted upon by microorganisms so that the chemical composition of the final product is considerably altered (van Veen, 1957). Sometimes these changes are due not only to microbial action, but also to autolytic processes brought about by the enzymes of the material itself. A food fermentation that involves the application of the koji mold, Aspergillus oryzue, is a unique feature of food processing in the Orient (Hesseltine, 1965). The Orient also has a long history of using soybeans in fermented foods. Scarcely a day passes in the average Japanese home without fermented soybeans being served at the table. These soybeaii foods range from miso and natto to seasonings such as soy sauce (shoyu) (Shibasaki and Hesseltine, 1962). Miso, a paste of the consistency of peanut butter, was characterized by Lockwood and Smith (1950-1951) as the most important

'Present address: Department of Food Science, University of Ife, Ile-Ife, Nigeria. 2Author to whom enquiries concerning t b s paper should he addressed. 239 AIIVANCES IN APPLIED MICROBIOLOGY,V O L U M E 28 Copyright 0 19x2 h y Academic Press. Inc. All rights of r e p r n d u c l ~ ~i n~any i form revervcd. ISBN (1-12-00242%7

240

SUMBO H. ABIOSE ET AL.

soybean food product in Japan. Piper and Morse (1923) wrote that “in extent of use, miso is said to surpass all other preparations of the soybean in the Orient.” However, Hesseltine (1965) stated that in terms of volume of production, it was second to soy sauce. According to Lockwood and Smith (1950-1951), miso is made from soybeans, wheat or wheat flour, and rice in a proportion of 2:l:l. Hesseltine and Wang (1967) on the other hand stated that it was made from soybeans and rice. It is now known that there are many types of misos, the main ones being “kome” (made from rice and soybeans), “mugi” (barley and soybeans), and “hatcho” (soybeans only), and the ingredients are used in various proportions. Miso is used primarily as a flavoring material, being added to soups and vegetable dishes. However, Hesseltine and Wang (1967) recommended spreading miso thinly on cucumbers or substituting it for meat sauce on spaghetti. Published information available in the English language on miso is scanty, and most of these papers are by Shibasaki and Hesseltine (1961a,b, 1962), Hesseltine and Shibasaki (1961), Hesseltine (1965),and Hesseltine and Wang (1967). Most of the published information available is in Japanese, and the English abstracts of these do not usually give as much information as one would wish. It is interesting to compare this situation with that of soy sauce. The review of the latter by Yong and Wood (1974) lists 260 references, compared to the 135 of the present review. It is clear (Wood, 1980) that Japanese production of soy sauce (1,434,000 tonnes in 1978)and miso (567,000 tonnes in 1978) are o f a comparable magnitude, and since miso is in the form of a paste with less water and more organic solids than soy sauce, miso must be considered to be an equally important component of the Japanese diet. However, the exports of the two preparations from Japan in 1978 (soy sauce, 6592 tonnes; miso, 1081 tonnes) indicate that on a world scale the two are of rather different importance. The much lesser export importance of miso probably explains the smaller amount of information available to the Occident, since it is difficult to believe that there is such a substantial difference in the research effort devoted to the two preparations in Japan itself. Against this line of reasoning, however, must be set the reports to the present authors by importers of soy sauce and miso. They state that the Japanese miso industry is divided among small- to medium-sized producers compared with the large companies that occupy an important position in the soy sauce industry. Thus, although miso has been prepared for over a thousand years, only a few accounts of this food were available in the West prior to 1961. These were in the form of short essays (Church, 1923; Piper and Morse, 1923; Dyson, 1928; Stahel, 1946; Lockwood and Smith, 1950-1951). Based on the 1978 figures (Wood, 1980) the annual production of miso in Japan alone was equivalent to a daily consumption of about 14 gm per person per day. Besides Japan, miso is also consumed in China, Taiwan, the Phil-

MICROBIOLOGY AND BIOCHEMISTRY OF M I S 0

24 1

ippines, and Korea. In these countries, miso is called by different names, e.g., “taotjo” in the Netherland Indies (Stahel, 1946), “daenjang” in Korea (Kim et al., 1977), and “chiang” in China (Shurtleff and Aoyagi, 1976). Also, the raw materials are sometimes different; for example, in making taotjo, a mixture of boiled soybeans and roasted wheat or glutinous rice is used to make the koji. Essentially miso is made by a two-stage fermentation process. The first stage involves the production of the koji from cooked grains (usually rice, barley, soybeans, or a soybean-wheat mixture) inoculated with Aspergillus oryzae. This is followed by a second fermentation (the moromi) where the koji is mixed with salt, cooked soybeans, and an appropriate inoculum, which is traditionally a portion of an old, good miso in water. There is an increasing amount of miso now being imported to the United Kingdom, mainly from Japan, and sold in “wholefood,” “health food,” and “macrobiotic” shops, following on their popularity in the United States. An understanding of the microbial and biochemical events occurring during the fermentation of miso, therefore, should be of interest. This review is an attempt to inform the Occidental reader of the current state of knowledge concerning this useful and interesting flavoring material. It is based on work by Abiose (1979).

II. Fermented Soy Products Legume seeds, which are among the highest in protein content of all seeds, have been a major source of protein for humans in regions where animal protein is scarce. Examples of foods prepared from different legume seeds in various parts of the world were cited by Altschul (1965). Among these, the soybean, an annual legume native to China, had an outstanding position because of its particularly high protein content (Watanabe et al., 1974). According to Platt (1964), the variety of fermented products obtained from soybean equals, if not exceeds, that obtained from milk. Piper and Morse (1923), in their classical monograph on the soybean, described in detail the traditional soybean foods in both China and Japan. Recently, Hesseltine (1966), Hesseltine and Wang (1967), Gray (1970), Wood and Yong (1974), Goel (1974),and Wood (1977)have given comprehensive accounts of the different kinds of fermented soybean products and the processes involved in making them. Table I, adapted from Hesseltine (1965), gives a list of the different types of fermented foods and also includes the substrate and the organism($ involved. Of these only natto is fermented solely by bacteria-Bacillus subtilis. The most important of these fermented products, in terms of volume of production, is soy sauce. The process of soy sauce production has been well studied, and a multistage fermentation process has been developed by Yong (1971), Wood and Yong (1974), and Goel (1974).

242

SUMBO H. ABIOSE ET AL.

TABLE I FOODFERMENTATION“ Name of food Teinpeh

Sufu (soybean cheese) Miso

Organism(s) used

Substrate

Nature of product

Rhizopus R. 0 l i g o s l ~ o r i r , ~ Actinomucor &guns, Mtrcor sp.

Soybeans

Solid

Indonesia and vicinity

Soybeans

Solid

China, Formosa

Asporgillus. oryzac.,

Rice, barley Soybeans, wheat Soybeans Wheat

Paste

Soybeans

Solid

Japan, China Philippines China, Japan Philippines and other parts of the Orient Japdn

Sacehmrom yce.s rnirxii Soysauce (shoyu) A . orysac Lactobacil1u.s

Liquid

Succhoromyces

Nattn

Area of development

Hansenula Bncillrrs siihtifis

“Adapted from Hesseltine (1965);reproduced with the permission of the New York Botanical Garden.

Miso is considered second to soy sauce according to Hesseltine (1965), although Lockwood and Smith (1950-1951) regarded it as the most important soybean food product in Japan. As the production figures cited above show, soy sauce is the more important product in tonnage terms. However, if one takes a typical composition for soy sauce of 78 gm proteidkg, and for miso values of 115 (kome) to 217 (hatcho) gm protein/kg (Yong and Wood, 1974; Abiose, 1979), then the 1978 production of miso in Japan accounted for about 77,000 tonnes of vegetable protein while that of soy sauce contained about 112,000 tonnes of vegetable protein. According to Dyson (1928), miso occupied the place of porridge on the tables of Japanese children (although its saltiness suggests that it would not be consumed in such great quantities as porridge!), and “no day passed without it being used as the basis of innumerable dishes esteemed as delicacies by the Japanese.”

Ill. Fermented Rice Products The two main fermented rice products reported in the literature, apart from miso, are saki: and idli. Of these, sakk is better known and more related to miso in that its production also involves a koji fermentation. Saki: is an important traditional alcoholic drink in Japan. It is believed to have been made at least since the eighth century (Miall, 1974), and its production reached a high degree of perfection during the seventeenth to nineteenth centuries (Uemura, 1971). The history of sake brewing and the method of producing it have been reviewed by Kodama (1970) and Kodama and Yoshizawa (1977). According to these workers, the first stage in the production of saki: is the preparation of rice koji, as described for miso. In the

MICROBIOLOGY AND BIOCHEMISTRY OF M I S 0

243

second (moto) stage, rice koji, steamed rice, yeast, and water are added together. The mot0 stage acts as a yeast starter for the moromi stage. According to Kodama (1970) and Kodama and Yoshizawa (1977), in the conventional method for mot0 preparation (kimoto), steamed rice (120 kg) is mixed with rice koji (60 kg); then water (200 liters) is added. The mixture is kept at a temperature of 13-14°C for 3 to 4 days with intermittent stirring. During this period the rice grains are partially degraded and saccharified by the rice koji enzymes. The mot0 mash is then gradually warmed to a temperature of 15°C over a period of 15 days. Many microorganisms are known to be involved during the mot0 stage. These include Leuconostoc inesenteroides var. sakd, Lactobacillus sakB, and Saccharomyces sakB. A well-defined floral change is known to occur. The lactic acid bacteria which grow first give way to the sake yeasts. At this stage, the mash is cooled to prevent weakening or death of the yeast due to the high concentrations of alcohol and acid. The mot0 mash is thus rested for a period of 5-7 days before it is used for the main fermentation, called the moromi. Sometimes, the mot0 is acidified by adding lactic acid at the beginning of the process rather than allowing the lactic acid bacteria to develop and produce the acid. This type of mot0 is called “sokujo-moto.” The next stage, which is the moromi, takes a period of about 25 days. For the moromi stage, steamed rice, rice koji, and water are added to the mot0 in three batches. By careful control of the process a highly alcoholic beverage is produced. Figure 1 presents a flow sheet for sake production, permitting comparison with that for miso production (see Fig. 2). Idli is a fermented food consumed as a staple in many parts of India (Batra and Millner, 1976). According to van Veen et al. (1967), Steinkraus et al. (1967), van Veen and Steinkraus (1970), and Batra and Millner (1976), idli is made by fermenting a mixture of soaked and milled parboiled rice and dehulled black gram (Phaseolus mungo). The ingredients are mixed in various proportions and fermented overnight. The final product is steamed before consumption as a form of spongy pancake. Because parboiled rice is used, the fermenting microorganisms are thought to be derived from the black gram (van Veen et al. (1967). The role which these organisms play was reported by Mukherjee et al. (1965) and Batra and Millner (1976). Fermentation occurs by the joint action of Torulopsis candida and Trichosporon pullulan. However, the predominant microorganism responsible for souring as well as for gas production (leavening) was found to be Leuconostoc mesenteroides. This was the first record of a leavening action produced exclusively by the activity of lactic acid bacteria. Other microorganisms such as Pediococcus cerevisae were also found. Idli is said to have nutritive properties that are superior to those of the rice and black gram. Methionine content is said to increase by as much as 18%. Choline and folic acid contents were also increased (Rao, 1961)

I

WASHING

I

STEEPING IN WATER for 1-20 HR

1

EXCESS WATER DRAINED OFF

1

STEAMING 30-60 MIN

1 1

COOLING STEAMED RICE A. oryzae

SAKE YEAST tACTIC ACID MOROMI

1

FERMENTATION SOLIDS FILTRATION

-I

I 1

FRESH SAKE SETTLING SE DIMENT+ FILTRATI 0N

1 1 STORAGE 1 BLENDING

PASTEURIZATION

1 1 BOTTLING i PASTEuR IZATION

DILUTION-WATER

-

SAKC

FIG. 1. Flow sheet of sake brewing process.

MICROBIOLOGY AND BIOCHEMISTRY OF MISO

245

SOY BEANS

POLISHED RICE

I I SOAKED IN WATER OVERNIGHT I I EXCESS WATER DRAINED OFF EXCESS WATER DRAINED OFF I 1 STEAMED 60 MIN BOILED UNTIL SOFT I I COOLED COOLED I INOCULATED WITH “TANE” KOJl I SPREAD ON WOODEN TRAYS TO SOAKED IN WATER FOR 17 HR

A DEPTH OF 1-2 INCHES

I

INCUBATED FOR 50 HR AT 30°C \MIXING

I

i’

INOCULUM

SALT

PACKED IN WOODEN KEGS

1 1 RIPENING

FERMENTAT1 0N

I

MISO FIG2. Flow sheet for miso fermentation.

Two minor fermented rice products in Japan are “mirin” and “amazake.”

The former is prepared by aging a mixture of “shochu” (distilled ethanolic liquor), rice koji, and steamed glutinous rice. It is said to contain 13-22% alcohol and 2548% sugar and is used as a food seasoning (Murakami, 1971). Amazake is a sweet beverage made by standing a mixture of boiled rice and rice koji at 60°C overnight. Tape-ketan is a sweet fermented rice from Indonesia (Hedger, personal communication).

IV. History of Miso Production The earliest known ancestor of miso was the Chinese food called “chiang,” thought to have originated before the Chau dynasty (722-481 BC). Accord-

246

SUMBO H. ABIOSE ET AL.

ing to Shurtleff and Aoyagi (1976) the earliest varieties of chiang were made with fish, shellfish, and game. The present day miso did not come into use until sometime between the second century BC and fourth century AD. It is thought to have been introduced into Japan shortly before the introduction of Buddhism (540-552 AD). The first written records of miso date from the Nara period (710-784 AD), and the earliest Japanese document to contain information about the production of miso was “Engi Shiki,” written about 927 AD. In it we are told that “miso was a fermented food with soybeans as its main ingredient, but also containing rice, rice koji, wheat, salt, and saki.” (Shurtleff and Aoyagi, 1976). According to these writers, the first westerner to study the miso-making process was a German named Otto Kellner who wrote a detailed article on the subject in 1893. Piper and Morse (1923) later wrote their classical work, “The Soybean,” which gives useful information relating to miso. They write that “in extent of use, miso is said to surpass all other preparations of the soybean in the Orient.” There is evidence (Watanabe et al., 1974; Aihara and Aihara, 1975; Shurtleff and Aoyagi, 1976) to suggest that the miso fermentation predates the production of soy sauce. According to these workers, soy sauce was first called tainari miso. Tamari literally means a liquid drip and is the name given to the liquid that drips to the bottom of a miso keg while miso is aging. Thus, tamari, as the original form of soy sauce in Japan, was made from miso. Miso is in fact claimed to be the oldest fermented food of Japan (Aihara and Aihara, 1975). Production of miso developed as a widespread household industry, and much of the miso consumed in the Orient is still produced on a small scale. Therefore many modifications of the miso production have occurred to produce many types and varieties. Although the Japanese consume more protein in the form of miso than as soy sauce, the latter is presently better known and more widely used in the West. The reason for this is thought to lie in the difference in structure between the miso and soy sauce industries (Shurtleff and Aoyagi, 1976). Most Japanese soy sauce is produced by a few large companies which had the capacity and foresight to engage in international advertising and commerce. By comparison, the largest miso companies are quite small and have only a few decades of modern business experience. Shibasaki and Hesseltine (1962) showed that domestic production in the early 1960s was roughly two-thirds the size of factory production. There are many substantial miso factories now in Japan. Shurtleff and Aoyagi (1976) gave a list of 24 factories which produce miso in Japan. Outside the Orient, ‘many groups are setting up centers for iniso production, for example, Lima in Belgium (Shurtleff and Aoyagi, 1976), but most of them are in the United States. These Occidental centers have one thing in common-the promotion of consumption of natural foods with no added preservatives.

MICROBIOLOGY AND BIOCHEMISTRY OF M I S 0

247

V. Types of Miso There are numerous varieties of miso (Church, 1923; Dyson, 1928; Lockwood and Smith, 1950-51; Shibasaki and Hesseltine 1961a,b, 1962; Hesseltine, 1965; Hesseltine and Wang, 1967; Watanabe et al., 1974; Aihara and Aihara, 1975; Shurtleff and Aoyagi, 1976). There is, however, some confusion regarding the nomenclature of this food product. Lockwood and Smith (1950-51) refer to white, red, and black miso. Nakano (1959) classified rniso into three types, mentioned previously, viz., kome (rice-based) miso, mugi (barley-based) miso, and mame or hatcho (soybean only) miso. These three basic types of rniso were also recognized by Hesseltine and Wang (1967) and Watanabe et al. (1974). For the production of kome and mugi misos, the koji is prepared from polished rice and barley, respectively. Mame or hatcho miso, on the other hand is made from soybeans or soybean-wheat mixture koji. In Japan the ratio of volume of production of the three major types, rice to barley to soybean miso is 80:11:9 (Watanbe et al., 1974). In the preparation of miso, the koji is mixed with an appropriate mixture of cooked soybeans, salt, and inoculum. The precise proportion of beans and grain used and also the extent to which the beans are cooked depends on the type of miso being made. Thus the rice-based miso is further subdivided by Shibasaki and Hesseltine (1962) into four types, namely, white, edo, sendai, and shinshu, based on the relative proportions of raw materials used, the color of the miso, the length of time of fermentation, and the locality in which they are preferred. Aihara and Aihara (1975) reported that the different kinds of miso developed through local traditions because of the availability of ingredients and/or the taste desired. Thus there is a variety of different misos, and a variety of different names has been applied to this material. To the user no two varieties of iniso taste the same, for miso’s range of flavors and colors, textures, and aromas is as varied as that of the world’s fine cheeses and wines (Shurtleff and Aoyagi, 1976). The color of miso is determined mainly by the cooking pressure and length of the cooking period of soybeans. A low cooking pressure (5-7 psi) for a period of 30 minutes to 1 hour gives a light colored miso; high cooking pressure (10-15 psi) for 1 to 2 hours results in dark colored miso. Darkening can b e further intensified by allowing the cooked beans to stand for a few hours or overnight. Thus, for example, a low-salt miso can be made dark or light colored. Since the color of the iniso is based on the treatment of the beans prior to fermentation, the proportion of the beans in the raw materials also affects the color of the product. This can be illustrated by referring to edo and sendai misos. These are both kome misos, which are reddish brown in color. According to

248

SUMBO H. ABIOSE ET AL.

Shibasaki and Hesseltine (1962),the raw materials for edo are soybeans, rice, and salt, in quantities of 10,8-10, and 0.25-0.4parts by volume, respectively. Sendai on the other hand has its ingredients in the proportion 10, 4, and 0.4-0.5parts by volume for soybeans, rice, and salt, respectively. It is therefore obvious that the matter of the nomenclature of miso needs some attention. Again, soybean miso is usually referred to as hatcho or mame miso. There is however a special kind of soybean miso called "miso-dama" believed to have come to Japan from Korea. This miso differs from the ordinary soybean miso in the way that it is prepared. In the preparation of miso-dama, cooked soybeans are mashed, shaped into balls, inoculated with mold spores to form the koji, and then incubated for 50 hours at 30°C. The balls are then crushed, mixed with salt and water, and fermented in crocks to give the final product. Barley miso, referred to as mugi miso, is usually classified as sweet or salty varieties.

VI. Raw Materials for Miso Production The raw materials used for miso production are soybeans, rice, barley, salt, and sometimes wheat. All of these raw materials are relatively inexpensive and easily available. The amount of miso produced in Japan in 1956 and the amounts of the raw materials used are given in Table 11.

A. SOYBEANS(Glycine max) The soybean, also known as the soja bean, the soyabean, Chinese pea, or Manchurian bean in some countries, is an annual leguminous plant native to eastern Asia and extensively cultured in China and Japan long before re-

MISO PRODUCTION Type of miso

Factory made Rice miso Barley miso Soybean miso TOTAL Home made Miso of' all typcc GRAND TOTAL

TABLE I1 MATERIALSUSED

AND RAW

Annual production

379,000 146,000

Soybeans

583,000

134,000 52,000 32,000 218,000

391,000 974,000

143,000 361,000

58,000

IN JAPAN I N

Rice

1956",b Barley

Salt

-

30,000

72,000

30,000

58,000 22,000 9000 89,000

28,000 58,000

70,000 159,000

72,000

43,000

lL5,oOO

-

-

"Adapted from Shibasaki and Hesseltine (1962), with permission of Economic Botany. "Amounts are given in metric t o m e s .

MICROBIOLOGY AND BIOCHEMISTRY OF M I S 0

249

TABLE I11

COMPOSITION OF SOYBEANSFROM VARIOUSSOURCES(a). Country

Water content

Total nitrogen

Crude protein

Crude fat

Invert sugars

Japan China U. S. A.

13.0 11.0 10.0

6.0 6.0 6.0

38.1 38.1 38.1

16.0 19.0 20.0

15.0 19.0 17.0

uAdapted from Yokotsuka (1960).

corded history. Most authors, according to Scott and Aldrich (1970), credit the earliest writings on soybeans to Emperor Shennung who is said to have ruled in China in 2838 BC. According to Aihara and Aihara (1975), the soybean crop was considered the most important cultivated legume and one of the five sacred grains essential to the existence of Chinese civilization. In a Chinese dictionary dating from about the beginning of the Christian era, the soybean is called “sou,” which very probably was the source of the names “ >> “ soi, soy, and “soja.” The chemical composition of soybeans from different countries differs slightly as shown in Table III. According to Wolf and Cowan (1971), commercial soybeans consist of approximately 8% hull, 90% cotyledon, and 2% hypocotyl and plumule. Oil and protein make up about 60%of the bean, but about one-third consists of carbohydrates including the oligosaccharides, stachyose (3.8%),raffinose (1.I%), and sucrose (5%)(Wolf and Cowan, 1971). The protein content of the whole seed (approximately 40% of the seed weight) is the highest of any commercially important crop (Circle and Smith, 1975). Liener (1972)has reviewed the nutritional value of soybean protein and found that the limiting amino acids, on the basis of the chemical score, are the sulphur-containing compounds methionine and cystine. Soybeans, however, contain more lysine than most plant proteins. They therefore complement the amino acids of most cereal grains. It is a generally recognized fact that the soybean is one of the best and cheapest sources of food energy in terms of calories per unit cost of production, if consumed directly as a human food rather than after conversion to meat in farm animals, which is the usual practice in the Occident (Noyes, 1969). For the production of miso, whole soybeans are normally used. Shibasaki and Hesseltine (1961b)and Smith et al. (1961),however, found soybean grits to be satisfactory for the purpose of miso making, especially of white or light yellow miso. They pointed out that the grits must not be too fine. The color of the soybean is important for the production of miso. Green colored beans are unsuitable, because the color of the miso becomes dark and dirty resulting in a loss of appeal to the consumer (Shibasaki and Hesseltine, 1962). In general, miso manufacturers prefer large beans of uniform size with a very ?>

250

SUMBO EX. ABIOSE ET AL.

light yellow color and a white hiliuni. Large beans are preferred because they are inore easily cooked and have a smaller amount of seed coat. Thus, not all types of soybeans are suitable for making miso. Japanese soybeans are found to be better than imported soybeans from the United States.

B. RICE (Oryza sativa) Rice is the principal food crop of about one half of the population of the world. About 92% or more of the world rice crop is produced and consumed in Monsoon Asia (Leonard and Martin, 1963). According to these authors, the cultivated rice plant probably originated somewhere in the area between Southern India and Indochina. Rice was introduced into Japan from China as early as 100 BC. In China, the sowing of rice was an important religious ceremony nearly 5000 years ago. The origin and groups of cultivated rice, mainly 0. sativa var. indica are grown largely in the tropical zones and 0. sativa var. japonica is confined to the more northern subtropical conditions. The indica group consists mostly of long grain varieties and includes all of the cultivated rices of India, Indochina, and Southern China. The japonica group are short grain varieties, and comprises all of the cultivated rice indigenous to Japan, Korea, and Northern China. The proximate composition of rice as given by Leonard and Martin (1963) is shown in Table IV. Most of the protein in rice is thought to consist of glutelins. The important B vitamins in rice are thiamin, riboflavin, and niacin. Modern milling removes most of these vitamins. For the production of miso-koji, polished glutinous rice is used and this preferably should be short grained. According to Shibasaki and Hesseltine (1962), brown rice is unsuitable for production of miso-koji because the surface texture is hard and contains wax and thus is unsuitable for mold growth. TABLE IV PROXIMATE COMPOSITION OF SOYBEANS, RICE, A N D BARLEY O N DRYW E I G H T BASIS (%)" Protein Whole soybean cotyledon

hull Brown rice Milled rice Barley

40 43

8.8 9.8 8.6 14.1

Fat 21 23

1 2.2 0.3

2.5

C-Hyd

Ash

34 29 86 85.9 90.5 90.2

4.9 5.0 4.3 2.1 0.5 3.2

"Adapted from Wolf and Cowan (1971); Leonard and Martin (1963), with permission of C. R. C. Press, Inc. and Macinillan Publishing Co., Inc.

MICROBIOLOGY AND BIOCHEMISTRY OF M I S 0

251

C. BARLEY(Horrleum vulgare or sativum) Barley is a cultivated grass and belongs to the tribe Triticeae (Brouk, 1975). According to Leonard and Martin (1963), barley is grown in nearly all cultivated areas of the temperate zones, in many subtropical areas, and in high altitude sections of the torrid zones of both hemispheres. Barley probably was introduced into Japan from Korea at the dawn of Japanese history (100 BC). The chemical composition of barley is given in Table IV. The protein content of barley varieties may vary from 7.5 to 15% of the dry weight of the grain, while the starch content varies between 50 and 60%. The moisture content when threshed may vary from 10 to 20%. Pearled barley is used for human food. While it is clear from figures cited by Shibasaki and Hesseltine (1962) that barley miso is fairly important, accounting for about a quarter of the total annual miso production, no published information is available in the West on this variety of miso.

D. SALT Salt was used in preservation of food either alone or in combination with drying even before recorded history (Joslyn and Timmons, 1964). It acts as a preservative and exerts a selective action on the microorganisms which grow in the fermentation (Yong and Wood, 1974). It is clear (Yong, 1971; Goel, 1974) that the salt is necessary to permit the exclusive development of the flavor and aroma-forming yeasts and lactic acid bacteria. Commercial, not chemically pure, sodium chloride is usually used for making miso. The tendency, however, is for small-scale miso producers to use sea salt. The latter is believed to give a miso with better flavor and nutritional value (Shurtleff and Aoyagi, 1976)and is strongly favored by Western companies trading in miso.

VII. Ratio of Raw Materials The different types of miso are made by mixing different amounts of raw materials. The precise proportions of the raw materials used depend on the type of miso being made (see Table V). Shibasaki and Hesseltine (1962)listed five types of miso and gave the figures for the ratio of raw materials for each type. Shurtleff and Aoyagi (1976) gave the proportions by weight of basic ingredients for various homemade misos. It seems that there are more variations in the ratio of ingredients of the rice-based Kome misos than for the other two types. “Virtually all barley miso calls for equal parts by weight of soybeans and barley” (Shurtleff and Aoyagi, 1976).

252

SUMBO H. ABIOSE E T AL.

TABLE V CONDITIONS FOR PRODUCING DIFFERENT TYPESOF MISO”

Type White Edo Sendai Shinshu Rarlcy Mellow harlcy

Mame

Color

Nature

-

Sweet Sweet

Pale red brown Red brown Yellowish brown -

Deep red brown

salty Salty Sweet Salty

Ratio of beans: grain:salt(w/w) 10 : 10 to 20 : 2 . 5 3 . 5 10 : 8 to 10 : 0 . 2 . 5 4 . 4 10 : 4 : 0 . 5 10 : 5 : 4 . 5 10 : 15 : 5 10 : 10 : 5 10 : - : 2.5

“Compiled from various sources.

VIII. Treatment of Raw Materials

The preparation of raw materials for fermentation is an important part of the process for making miso. Shibasaki and Hesseltine (1961a) discussed the preparation of soybeans for miso fermentation. They examined 28 American soybean varieties, four Japanese varieties, and one Chinese variety. Soaking and cooking conditions were investigated. In the manufacture of miso, whole soybeans are soaked in water prior to steaming. The time necessary for soaking varies according to the properties of the soybean, the kind of product desired, and the temperature and hardness of the water. When sendai (rice-based) miso, for example, is made in Japan, soybeans are soaked for 15 to 18 hours in water at a temperature of 12 to 15°C. The purpose of soaking is to soften the soybean so that subsequent cooking is rapid. Uniform soaking is considered important, as irregular water absorption is said to lead to irregular cooking and to irregular fermentation. The difference in water absarption by various varieties of soybeans was attributed to the original moisture content ofthe beans. After soaking is complete, the beans are cooked by steaming. Before the introduction of pressure cooking, beans were cooked in large open pans until soft enough to be easily pressed between thumb and finger. The pressure of steam cooking and the length of cooking time depend on the variety of miso being made. Thus, when sendai miso (dark brown) is prepared, the desired softness and dark color can be obtained by autoclaving at 10 to 15 psi for 1 to 2 hours and then allowing the beans to stand for a few hours or overnight (Shibasaki and Hesseltine, 1962). The beans for shinshu miso, which is yellowish brown, are autoclaved at 7 psi for 1 hour, according to Shibasaki and Hesseltine (1961b). The information available in the Western literature on the preparation of rice and barley for miso is not as detailed as for soybeans. Essentially, the

MICROBIOLOGY AND BIOCHEMISTRY OF M I S 0

253

rice needs to be polished to facilitate the attack by the koji mold. For the same reason, pearled barley is used. The polished rice or pearled barley is cleaned and soaked. Whereas the rice is soaked for 12 to 17 hours, soaking time for barley is said to be 2 to 3 hours (Shurtleff and Aoyagi, 1976). After soaking, the grain is then cooked by steaming. Boiling the rice is not recommended as this “process creates a wet texture which encourages the growth of undesirable bacteria” (Shurtleff and Aoyagi, 1976).

IX. Koji The word “koji” is an abbreviation of “kabi-tachi,” meaning “bloom of mold” (Tamiya, 1958). Production of a koji is the initial step in almost all Oriental food fermentations, and is accomplished by inducing the growth of molds belonging to the Aspergillus flavus-oryzae group over soaked and cooked soybeans or grains. The growing mold releases numerous hydrolytic enzymes. Such enzymes break down proteins and polysaccharides present in the seeds to monomers and soluble oligomers which are then fermented by yeasts and bacteria in subsequent anaerobic stages of the process concerned (Hesseltine et al., 1976). An account of koji and the development of the Japanese fermentation industry can be found in a paper by Sakaguchi (1972). The use of koji may be considered as analogous to that of malted barley in the Occident (Yong and Wood, 1974). A “tane-koji” (mold seed or mold starter) is used to inoculate a production koji. It is prepared by a process similar to that used for the main koji. Polished rice is steeped in water overnight, drained, and then cooked by steaming for about 1 hour. The ash of twigs and leaves of oak, chestnut, camellia, and other woody plants are added at a level of 2% based on the amount of rice, and the mixture is inoculated with A . oryzae. The ash is believed to furnish phosphorus and potassium salts that are necessary for spore formation (Shibasaki and Hesseltine, 1962). The wood ash was found to increase the viability and to increase the number of conidia produced by the mold (Matsuura and Nakano, 1964b,c). The same investigators (1964a) observed that the addition of ash had no influence on the amylase or protease activity of the koji mold. Conventionally, the inoculated rice is spread out in trays in a layer approximately one-half inch (13-14 mm) thick and covered with damp cloths to favor the growth of mold mycelium. Incubation of the rice is carried out at about 28°C for 5 days or until maximum sporulation occurs. After harvesting, the conidia are dried first at 45°C for 15hours, then at 25°C until the moisture level is 6% (Hesseltine et al., 1976). They are then packaged and stored at 10°C until sold. Shibasaki and Hesseltine (1962) and Peppler (1967) report that each gram of dry tane-koji contains 2.5 x 108 active spores of A. oryzae.

254

SUMBO H. ABIOSE ET AL.

According to Kodama (l970), rice koji can be classified into many types, but in general two main types can be distinguished by particular features of the mycelial development on the rice grains. These are “sohaze koji” and “tsukuhaze-koji.” The former type is described as having a white layer of mycelial growth covering the whole surface of the grains. The latter type is said to have several white spots of fungal colonies on the surface of the grains with abundant mycelia invading deeply into the interior of the grains. Both types of rice koji are used for saki. brewing, though “tsukuhaze-koji” is said to give sake of a superior quality but is more difficult to prepare. There is no information in the literature to indicate which of these two types of koji is more suited to miso fermentation. However, Shibasaki and Hesseltine (1962) wrote “the miso koji at its best looks like a white felt of mycelium or a solid cake of mould rice.” This could be interpreted to mean that the koji for miso is probably “sohaze-koji” type. According to Miall (1974), the introduction of the koji process to the western world is due chiefly to the work of Takamine, which started about 1891. Takamine developed a process for making a fungal diastase that could be operated successfully on a large scale. This involved the growth of A . w y z a e on wheat bran, making a preparation called Taka-koji. The scientific name for the koji mold, A . uryzae, dates back to Ahlburg and Matsubara (1878), but it was not until the work of Wehmer (1895) was published that this mold was described in detail. Later, mycological studies revealed that it is a member of the large group of A . flauus-myzae, including many strains in which there are slight graded variations in morphological and physiological properties from strain to strain (Thom and Church, 1926). However, Murakami (1971) made a taxonomic study of 406 strains of koji mold and found all of them to belong to the A. oryzae group. It is also worth noting that no aflatoxin-producing strains were found among the Oriental industrial strains of koji mold (Murakami et al., 1967, 1968; Matsuura, 1970). Rice koji is known to contain various enzymes, mainly amylases and proteases (Morita et al., 1966; Kodama and Yoshizawa, 1977). Okazaki (1950) as quoted by Kodama and Yoshizawa (1977) indicated that sometimes maltase and isomaltase are present in rice koji in addition to a- and @-amylases. There is evidence for the production of sucrase, cellulase, and lipase by the koji mold (Yong, 1971; Yong and Wood, l977a; Goel and Wood, 1978). Suzuki et al. (1956) studied the effect of incubation time and temperature on the enzymes. Judging from the literature (Mogi et a l . , 1956; Sakaguchi, 1958; Bergkvist, 1963a,b; Nunokawa, 1965; Hayashi et al., 1967; Tagami and Sugawara, 1977), three kinds of proteases exist in crude enzyme preparations of A. myzae. These are acid, neutral, and alkaline proteases. Although a lot of work has been done on the isolation and purification of

MICROBIOLOGY AND BIOCHEMISTRY OF M I S 0

255

the proteases and amylases of A . oryzae, there is little work on the levels of these enzymes in the koji and moromi throughout the whole period of fermentation. The koji for a production run is prepared by steeping and cooking the appropriate grain and then inoculating it with tane koji at a level of 1gm/kg of raw material. The inoculated material is spread into trays and incubated in a humid atmosphere maintained at 30°C. Incubation is for 40 to 50 hours, in contrast to soy sauce where a 72-hour incubation is favored. During incubation the koji must be stirred at least twice in order to prevent overheating and to maintain uniform temperature, moisture, and aeration through the mass. “The miso koji at its best looks like a white felt of mycelium or a solid cake of mould rice” (Shibasaki and Hesseltine, 1961a,b, 1962; Hesseltine, 1965; Hesseltine and Wang, 1967). Concurrently with the preparation of the koji, whole soybeans are washed, steeped in water overnight, drained, and cooked by steaming. During soaking the water is changed at least once to prevent undesirable fermentation due to spore-forming bacilli (Shibasaki and Hesseltine, 1962). X. Moromi The cooked beans are mixed with salt and koji in proportions appropriate to the type of miso being made. A suitable inoculum, traditionally a portion of a previous good miso, is also incorporated into the mixture. The mixture, called a “green” miso at this stage (Watanabe et al., 1974), is packed into a wooden tub or concrete tank (Sakurai, 1965) the interior of which has been thoroughly scrubbed and then sprinkled or rubbed with salt (Shurtleff and Aoyagi, 1976). The container is covered with a weighted lid to ensure that it is well-sealed during fermentation, and it is left to ferment for an appropriate time. According to Shurtleff and Aoyagi (1976), when miso is fermented in the traditional, natural way it takes place in the open in an area protected from direct sunlight so that the inoromi may “experience the rhythmic temperature changes of the days and seasons.” In modern fermentation at controlled temperatures the moromi is moved from a lower to a higher, then finally back to a lower, temperature during the process. According to Shibasaki and Hesseltine (1962), during the fermentation period components of rice and soybeans are digested by enzymes from the koji. The rice starch is broken down by amylase and maltase to dextrin, maltose, and glucose. Soybean protein is degraded by protease to peptides and amino acids. The enzymatically digested and fermented products of koji and soybeans react with each other chemically. For example, esters are produced from organic acids by reaction with ethyl and higher alcohols. Some pigments are produced by the browning reaction of amino acids with

256

SUMBO H. ABIOSE ET A L .

sugar. This is the so-called soya inelanine and, in part, gives the color of miso (Shihasaki and Hesseltine, 1962). Amadori compounds, important factors contributing to oxidative browning of soy sauce, were isolated and identified froin miso (Hashiba, 1978). This suggests that amino-carbonyl reactions are involved in oxidative browning of miso. After fermentation, miso is aged or ripened at room temperature for a few weeks to months. Some chemical reactions that occur during fermentation continue during agjng. Thus the length of time of aging depends on the variety of iniso being prepared. The fermentation of white miso requires a few weeks, whereas that of the brown miso requires months. Some miso manufacturers sometimes add caramel to improve the color of miso (Shurtleff and Aoyagi, 1976). An alcoholic yeast fermentation is believed to take place during rniso fermentation. These yeasts contribute to the development of miso flavor (Sakurai, 1965). According to Onishi (1957~)and Yoshii and Hayakawa (1968), most of the salt-tolerant yeasts isolated from miso belong to Succlzaroinycrs rouxii Boutroux, a heterothallic yeast. This yeast has been extensively studied (Onishi, 1957a,b; Matsumoto and Imain, 1973; Yong et a l . , 1978) and reviewed by Yong and Wood (1974). It is now recognized that these yeasts vary in their sugar and salt tolerance from strain to strain and that these two qualities are linked with the pH value of the medium in which the yeast is cultivated. In their studies, Hesseltine and Shibasaki (1961) found S. rouxii as the dominant type of organism in miso. The yeast was extensively studied by Wickerham and Burton (1960) and proved to be S. rouxii Boutroux. Synonyms of this species are S . soya Saito, Zygosaccharomyces soya Saito, Z . japonicus var. soya, and Zygopichia japonica (Saito) Klocker (Lodder and Van Rij, 1967). Apart from S. rouxii, other yeasts have been isolated from miso. A halophilic, ester-producing yeast was isolated from a salty red miso and was identified as Hunsenula subpelliculosa Bedford (Matsumoto and Imai, 1977a). The same workers (197713) also isolated Torulopsis versatilis and T . etchellsii from miso. They made miso with these yeasts and S. rouxii as inoculnm. An earlier report (Matsumoto and Imai, 1974) dealt with the effects of adding S. rouxii. Many bacteria have been found in miso; however, it has never been clearly proven whether bacteria are useful in the miso fermentation (Shibasaki and Hesseltine, 1962). From 60 kinds of iniso, 161 strains of aerobic bacteria were isolated. Of these, 65 strains were Bacillus mesentericus, 7 were B . subtilis, and 2 were B. ineguterium (Mogi and Nakajima, 1942). Many lactic acid bacteria are said to develop in miso, according to Shibasaki and Hesseltine (1962), although they made excellent miso without the use of lactic acid bacteria. Mochizuki (1978) has discussed the role of

257

MICROBIOLOGY AND BIOCHEMISTRY OF M I S 0

microbes during miso maturation, and earlier studies were carried out by Mogi (1940, 1942).

XI. Chemical Composition of Miso According to Shibasaki and Hesseltine (1962), miso contains 48-52% moisture. Miso containing 45% moisture is too hard, and if it contains 55% moisture, then it is too soft. To obtain the proper moisture level, about 10% water is added with the miso inoculum at the second stage of fermentation. Table VI gives the chemical composition of some miso varieties, according to Watanabe et ul. (1974), whose figures for moisture content are lower than those cited above. The predominant carbohydrate in miso is glucose, although nine other sugars have been reported as present (Mochizuki et al., 1972). The relative amounts of starch and sugar in miso depend on the type of miso. The larger part of the starch is hydrolyzed to glucose. Matsushita (1970) working with barley miso measured the free sugars extracted by 80% ethanol and found fiuctose, glucose, sucrose, maltose, rafhose, stachyose, and verbascose. During the maturation process the amount of sucrose decreased from 3.15% to trace amounts, and the amount of glucose increased from 1.82 to 19.93%. At the last stage of the maturation process, glucose was found to be the major component of the free sugars. Almost all types of miso contain about 9-15% crude protein except hatcho miso which contains 1540%. A large part of the protein is digested by proteases to peptides and amino acids. According to various workers, (Ouchi and Mochizuki, 1968; Hondo and Mochizuki, 1968; Hesseltine and Shibasaki, 1962), glutamic acid is the major amino acid present in miso, followed by aspartic acid, glycine, valine, threonine, and serine in that orTABLE V1 COMPOSITION OF SEVERALKINDS OF MISO (%)" Variety of miso

Rice misos White Edo sweet Salty light yellow Salty red Barley sweet Barley salty Soybean

Moisture

Protein

Reducing sugar

Fat

Sodium chloride

44 46

8

33 20

2

10

49 50 46 48 47

11 12 11 12 19

I3 14 15 11 2

5 6 12 13 11 12 10

"Adapted from Watanabe et a1 (1974)

4 5 6 5 5 10

258

SUMBO H . ABIOSE ET AL.

der. The ratio of these acids changes markedly at the initial stage of fermentation, becoming constant after fermentation for 35 days (Hondo et al., 1969). The total free amino acid content shows a rapid increase in the first 10 days of fermentation and then a slow increase in the latter stages of the process. However, free glutamic acid, aspartic acid, and proline increase almost linearly with the progress of the fermentation up to 50 days (Ouchi and Mochizuki, 1968). The cystine content of miso, like that of soybeans, is relatively low. Tamura et al. (1952) determined the amino acid content of three types of miso by microbiological means and found them to compare favorably with the amino acid contents of casein and soybean meal. Gray (1970) and Takahashi (1976) also discuss amino acid patterns in miso. Miso contains about 2 to 10% fat (Table VI). The change in the lipid content of miso-dama (see above) was investigated by Kiuchi et al. (1975). The triglyceride content was found to be 88%in soybean and 70.6% in misodama. Free fatty acid was, however, higher in iniso than in the soybean. Few vitamins are present in miso (Hesseltine and Shibasaki, 1962). Mogi et al. (1951) found that one-third of vitamin B , present in the rice was lost by soaking in water and by steam cooking. The amount of vitamin B2in the koji was increased by using a mutant strain of A. oryzae (Mogi et al., 1952). They claimed that for increased vitamin Be in miso, the protein content is more important than the starch. Shibasaki and Hesseltine (1961b) assayed two samples of miso for vitamin B,, and found them to contain less than 0.02 p g per gm. The main organic acids present in some varieties of miso are given in Table VII. Teramoto et al. (1962) determined the content of organic acids in Japanese fermented food products by partition column chromatography. They observed an increase in lactic, succinic, and acetic acids during the fermentation process. They suggested that it might be possible to evaluate the quality of samples and recognize the method by which they were produced by the identification and estimation of organic acids present. Miso that TABLE VII ORGANIC ACIDS I N Miso" Samples

Lactic

Acetic

Citric

30.7 101.5 14.8 102.4

87.1 70.6 17.8 60.0

Pyroghtamic

Succinic

Pyruvic

81.2 91.2 6.5 54.5

17.8 22.9 4.1 4.3

~

Hatcho Mugi Shimb Shinshu* ~~~

165.2 345.4 22.1 64.1

109.9 66.2 7.4 5.0

~

NAclaptedfrom Terarnoto et ul. (1962), with permission of Cordon & Breach Science Publishers, Ltd. Units are mg per 100 gm. 'Kome miso varieties.

MICROBIOLOGY AND BIOCHEMISTRY OF M I S 0

259

had been brewed for long periods appeared to have higher amounts of total acids and of lactic acid than miso brewed for short times. The amount of citric acid in miso was found to be higher than in sake and soy sauce. The ethyl alcohol content of the different types of misos quoted in the literature varies between 976 and 1557 mg% (Sato et al., 1975, 1976; Ohsawa et al . , 1976; Sato and Mochizuki, 1977). Mochizuki et al. (1972) attributed chemical changes occurring in miso during maturation to (1) the presence of Pediococcus halophilus which produces lactic acid, (2) Saccharomyces rouxii which produces ethyl alcohol and higher alcohols, (3) viable Aspergillus oryzae in the koji which produces aromatic ethyl esters, and (4)the proteinase, lipase, and amylase in the koji. Shibasaki and Iwabuchi (1970) found that removal of carbonyl compounds from the volatile fraction of miso resulted in a loss of “miso aroma,” indicating that these compounds contribute to miso aroma. The distribution of tetramethylpyrazine in fermented foodstuffs was studied by Kosugi et al. (1971). They thought the high amounts of this compound found in miso, natto, and soy sauce suggest that alkyl pyrazines play an important role in the flavor of these foods. Matsumoto and Imai (1973)attributed flavor in miso to the alcohols produced during fermentation. Amano and Namba (1972) examined the composition of an aroma concentrate produced from soybean miso by gas chromatography and by thin layer chromatography; of 72 gas chromatograph peaks obtained, 7 were alcohols, 8 were esters, 16 were organic acids, and the largest peak was due to acetic acid. Iwabuchi and Shibasaki (1973) also prepared an aroma concentrate from miso and identified the compounds in the fractions as ethyl alcohol, f u h r a l , phenylacetaldehyde, isobutyric acid, and isovaleric acid. They concluded that the aroma of miso was composed of many components in different proportions. Finally, Ina (1973) studied hatcho miso and reported that methyl esters of higher fatty acids were the main components that contribute to its flavor. Takahashi (1977) has reported on saccharide, acid, and alcohol patterns in miso.

XII. Future Developments in Miso Production Although koji preparation, indispensable for making miso, has been simplified in this decade by the use of rotary cookers, the process still requires a well-trained operator. During koji making there is a loss of 10% of the raw material as nutrients consumed by the mold (Watanabe et al., 1974). Therefore, much effort has been devoted to the substitution of koji by commercially available enzyme preparations. Work on miso (Kitaoka, 1972) suggests that the entire replacement of koji by enzymes, particularly for rice and barley kojis, would result in an unsatisfactory product with an inferior flavor. This may result in part from our incomplete knowledge of the en-

260

SUMBO H. ABIOSE ET AL.

zyines involved. However, it also suggests that the mold may make contributions, other than the production of enzymes, to the quality of the final product. On the other hand, a combination of koji and enzymes could be beneficial (Saito et al., 1973; Takeuchi and Yokoo, 1974). Various other Japanese workers (Mogi and Nakajima, 1972a,b,c; Hosokawa and Yoshii, 1973; Harayama et al., 1976; Ikebe et a l . , 1976) have made miso by use of enzyme preparations, mainly proteases, and decreased amounts of koji. In general, this was found to bring about a considerable reduction in aging time. An A . oryzae enzyme preparation was found to give miso with the best flavor (Hosokawa et al., 1973a,b). Other enzyme preparations, for example, papain (Mogi and Nakajima, 1972a), takadiastase (Mogi and Nakajima, 1972b), and bacterial proteinases (Harayama et aZ., 1976) have been tried, but these gave less satisfactory results. Also, more success is being achieved with soybean miso than with rice and barley misos (Watanabe et al., 1974). The next stage of development is the microbiological control of the miso process. This should accelerate the production and also enhance the quality of the final product (Shibasaki and Hesseltine, 1962). There is a lot of work being done on the substitution of other grains and legumes in miso production. This is an attractive venture for countries like the United Kingdom, where soybeans cannot be grown commercially. The use of indigenous crops is attractive both in the potential saving on imports and in the chance to upgrade materials of marginal utility in human nutrition. Kao (1974) and Robinson and Kao (1977) produced miso from chick pea (Cicer arietinum) and horse bean (Viciafaba) by substituting these for soybeans. Miso-type products were produced by substituting commercially available defatted soybean flakes for whole soybeans and substituting wheat, corn, sorghum, oats, potatoes, sweet potatoes, or bananas for rice in koji (Ilany-Feigenbaum et al., 1969). Fukunaga (1973) used waste liquor from boiled soybeans in the manufacture of miso. The pH of the liquor was adjusted to 4.0 with hydrochloric acid, a mixture of cellulase and amylase at 0.1% was added, and the medium was held at 45°C for 3 hours. He suggested the use of the concentrated waste liquor as a sweetening agent in the manufacture of miso or for a yeast inoculum. Oonishi (1976) made miso koji from cereal starch and concentrated soybean cooking juice inoculated with A . oryzae; the resulting koji had a high alkaline protease activity, and the protease that was produced had high heat stability. Miso made from this koji was superior in flavor and matured in a comparatively short time. A number of modern miso-type products have been developed. These include dehydrated, low-salt, and high-protein varieties. Ito et al. (1965) produced a new type of low-salt and high-protein miso by mixing enzymatically hydrolyzed, defatted soybeans with ordinary salty red miso, followed

MICROBIOLOGY AND BIOCHEMISTRY OF M I S 0

261

by short-term ripening. Such miso contains 53% moisture, 6.3% sodium chloride, and 17.6% protein. Dehydration of miso has been developed and patented by some Japanese companies (Ajinomoto Inc. 1972; Nisshin Oil Mills Ltd., 1977). In the former process, dried miso is prepared by allowing a liquid enzyme derived from A. myzae to act on steam-cooked soybeans. The mixture is then salted and subjected to the action of yeast and lactic acid bacteria prior to drying. In the latter process, miso is dried to a moisture content of less than 30%, then crushed and blended with fats, emulsifying agents, condiments, and an antioxidant, and then molded at a temperature of less than 80°C to yield a solid product with good water solubility and storage life. According to Watanabe et al. (1974), dehydrated miso is used principally for making an instant miso soup, and the amount produced annually in Japan is less than 10,000 tonnes though its production may increase in the future. Imai and Matsumato (1977) reported on low-salt miso. Miso is still a rather rare item on the United Kingdom market and probably on the European market. Its primary incursions seem to be through the “health f o o d and “whole food” trades, and there is no reason why it should not progress to a wider market in the future. ACKSOWLEUCMESTS Sumbo H. Ahiose wishes to acknowledge the generous support given to her by the Government of Nigeria. We thank Mrs. Jean Winter for her careful typing of the manuscript.

REFERENCES Abiose, S. H. (1979). Ph.D. Thesis, University of Strathclyde, Glasgow. Ahlhurg, H . , and Matsubara, S. (1878). Tokyo Iji Shinshi 24, 12 (cited in Tamiya, 1958). Aihara, H . , and Aihara, C . (1975). “Soybean Diet.” George Ohsawa Macrobiotic Foundation, Oroville, California. Ajinomoto Inc. (1972).“Dried Miso Preparation.” Japanese Patent 26 719/72 (cited in Food Sci. Technol. Ahstr. 1973, No. 1JBO). Altschul, A. M. (1965). “Proteins, their Chemistry and Politics.” Basic Books, New York. Amano, T., and Namha, T. (1972). Aichi Ken Shokuhin Kogyo Shikensho Nemp. 13, 43-51 [cited in Chem. Abstr. (1976), 84, 29388~1. Arima, K. (1957). Saki. and Similar Yeasts. I n “Yeasts” (W. Roman, ed.), pp. 143-154. Junk Publ., The Hague. Batra, L. R., and Millner, P. D. (1976). Dew. Ind. Microbial. 17, 117-128. Bergkvist, R. (1963a). Acta Chim. Scund. 17, 1521-1540. Aergkvist, R. (1963h). Acla Chim. Scand. 17, 154-1551. Brouk, B. (1975). “Plants Consumed by Man.” Academic Press, New York. Church, M. B. (1923). U . S . Dept. Agric. Bull. 1152, 1-26. Circle, S. J., and Smith, A. K . (1975). Soybeans: Processing and Products. In “Food Protein Sources. International Biological Programme 4” (N. W. Pirie, ed.). Cambridge Univ. Press, London and New York.

262

SUMBO H. ABIOSE ET AL.

Dyson, G. M. (1928). Pharm. J. 121, 375377. Fukunaga, Y. (1973). Miso No Kagaku T o Gigutsu 237,2730 [cited in Chem. Abstr. (1975), 82, 16902711. Goel, S. K. (1974). Ph.D. Thesis, University of Strathclyde, Glasgow. Goel, S . K . , and Wood, B. J. B. (1978). J. Food Technol. 13, 243-247. Gray, W. D. (1970). C.R.C. Crit. Reo. Food Technol. 1, 225-329. Grist, D. H. (1970). “Rice,” 4th ed. Longman, London. Harayama, F., Rokukawa, K., and Mochizuki, T. (1976). Miso Nu Kagaku To Gijuutsu 268, 2 5 3 3 [cited in Chein. Ahstr. (1977), 86, 28495x1. Hashiba, H. (1978). Agric. B i d . Chem. 42, 1727-1731. Hayashi, K., Fukushima, D., and Mogi, K. (1967). A @ . Biol. Chem. 31, 1171-1178. Hayashida, M., Ueda, R., and Teramoto, S . (1968). J. Ferment. Technol. (Osaka) 46, 77-91 (cited in Kodama, 1970). Hesseltine, C. W. (1965). Mycologia 57, 149-197. Hesseltine, C . W. (1966). Fermented products miso, sufu, tempeh. Proc. Znt. Conf. Soybean Protein Foods, Peuriu, I l l . pp. 170-179. Hesseltine, C . W . , and Shibasaki, K. (1961). Appl. Microbiol. 9, 515-518. Hesseltine, C. W., and Wang, H. L. (1967). Biotechnol. Bioeng. 9, 275-288. Hesseltine, C. W., Swain, E. W., and Wang, H. L. (1976). Dew. Znd. Microbiol. 17, 101-115. Hoggerheide, J. C . (1954). In “Industrial Fermentations” (L. A. Underkofler and R. J. Hickey, eds.), Vol. 11, pp. 122-154. Chemical Publ., New York. Hondo, S . , and Mochizuki, T. (1968).J. Food Sci. Technol. (Jpn.) 15,414417 [cited in Food. Sci. Technol. A b s h . (1969), 7G270] Hondo, S., Ouchi, I., and Mochizuki, T. (1969). Food. Sci. Technul. (Tokyo) 16, 155-157 [cited in Food. Sci. Technol. Abstr. (1971), 2Hi891. Hosokawa, N . , and Yoshii, H. (1973). J. Soc. Brew. J p n . 68, 195-198 [cited in Food Sci. Technol. Absh-. (1974), 3J4101. Hosokawa, N., Takeuchi, T., Yoshii, H., Yoshuda, M., Nanba, T., and Amano, T. (1973a). Aichi Ken Shokuhin Kogyo Shikensho Nenipo 14,50-59 (cited in Chem. Abstr. 84, 15853). Hosakawa, N., Takeuchi, T., Yoshii, H , , Yoshida, M., Nanba, T., and Amano, T. (1973b). Aichi Keu Shokuhin Kogyo Shikensho Nsinpo 14, 59-67 (cited in Chem. Ahsh. 84, 15854g). Ikebe, H. M., Ishida, K., and Yunogami, M . (1976). Kuinamoto Ken Kogyo Shikenjo Kenkyu Hokoku 51, 2 6 4 3 [cited in Chern. Absh. (1979). 90, 4685hl. Ilany-Feigenbaum, J . , Diamant, J., Laxer, S . , and Pinsky, A. (1969). Food. Technol. 23, 554-556. Imai, S., and Matsumoto, I. (1975).J. Soc. Brew. Jpn. 70, 413415 [cited in Food. Sci. Technol. Ahstr. (1976), 7J11491. Imai, S., and Matsumoto, I. (1977).J . Soc. Brew. J p n , 72,879-900 [cited in Food. Sci. Technol. Absh. (1978), 8H 10911. h a , K. (1973). Nippon Shokuhin Kogyo Crakkaishi 20, 71-74 [cited in Chem. Alntr. (1976), 84, 42185dl. Ito, E., Ebine, H., and Nakano, M. (1965). Rep. Food. Res. Inst. (Tokyo) 20, 127 (cited in Watanabe et a l . , 1974.) Iwabuchi, S., and Shibasaki, K. (1973). Nippon Shokuhin Kogyo Gakkai Shi 20,48-53 [cited in Chem. A b s h . (1976), 84, 42183b3. Joslyn, M. A,, and Timmons, A. (1964). In “Food Processing Operations” (M. A. Joslyn and J. L. Heid, eds.), Vol. 111, pp. 50-77. Avi Pub., Westport, Connecticut. Kao, C . (1974). Dissertution Abstr. Intwimt. B, 35 2250. Kim, Y . , Hwangbo, J., and Lee, S. (1977). Hanguk Sikpuin Kwahakhoe Chi 9, 73-80 [cited in Chem. Abstr. (1977), 87, 150378ql.

MICROBIOLOGY AND BIOCHEMISTRY OF M I S 0

263

Kitaoka, K. (1972). Miso N o Kagaktr To Cijutsu 218, 19-24 [cited in Chern. Abstr. (1974), 80, 8109Ofl. Kiuchi, K., Ohta, T., and Ebine, H. (1975). Hokko Kogaku Znsshi 53, 869-874 [cited in C h m i . Abstr. (1976), 85, 57557aI. Kodama, K. (1970). In “The Yeasts” (A. H. Hose and J. S. Harrison, eds.), Vol. 3, pp. 225-282. Academic Press, New York. Kodama, K., and Yoshizawa, K. (1977). I n “Economic Microbiology” (A. H . Rose, ed.), Vol. 1. Academic Press, New York. Kosugi, T., Zenda, H . , Tsuji, K., Yamamoto, T., and Narita, H. (1971). A p i c . B i d . Cheiu. 35, 693-696 (cited in Food. Sci. Technol. Abstr. (1971), 12HI8521. Leonard, W. H . , and Martin, J. H. (1963). “Cereal Crops.” Collier-MacMillan, London. Liener, I. E. (1972). I n “Soybeans: Chemistry and Technology” (A. K. Smith and G . T. Circle, eds.), Vol. I , pp. 158-202. Avi Puhl., Westport, Connecticut. Lockwood, L. B., and Smith, A. K. (1950-51). Yearb. Agric. (U.S. Dept. Agric.) pp. 357361. Lodder, J . , and Kreger-van Rij, N. J. W. (1967). “The Yeasts-A Taxonomic Study”. NorthHolland Publ., Amsterdam. Matsumoto, i . ,and Imai, S. (1973).J . Food Sci. TeciznoZ. ( / p n . ) 20,513-518 [cited in Food. Sci. Technol. Abstr. (1975), 212491. Matsumoto, I . , and Imai, S. (1974). Nippon Jozo K!yokai Zasshi 69, 590-594 [cited in Chetn. Ahstr. (1975), 82, 123564nl. Matsumoto, I., and Imai, S. (1977a). /. Sue. Brew. j p n . 72(2), 147-150 [cited in Food Sci. TechnoI. Abstr. (1978), 13361. Matsumoto, I., and Imai, S. (1977b). J . soc. Brew. J p n . 72, 74-77 [cited in Food Sci. Technol. Ahstr. (1978), 1T321. hfatsushita, A. (1970). /. Agric. Chern. Soc. Jpn. 44, 51-54 [cited in Food Sci. Technul. Abstr. (1970), 7H8931. Matsuura, S. (1970). Jpn. Agric. Res. Q. 5, 46-51 [cited in Food Sci. Technol. Abstr-. (1971), 4C661. Matsuura, S., and Nakano, M. (1964a). Rep. Food Res. Inst. Jpn. 18, 115-121 (cited in Hesseltine et al., 1976). Matsuura, S . , and Nakano, M. (196411). Hep. Food Res. Inst. J p n . 18, 122-126 (cited in Hesseltine et u / . , 1976). Matsuura, S., and Nakano, M . (1964~).Rep. Food Res. Inst. J p n . 18, 127-134 (cited in Hesseltine et d . , 1976). Miall, L. M. (1974). I n “The Filainentous Fungi” (J. E. Smith and D. R. Berry, eds.), Vol. 1, pp 104-121. Academic Press, New York. Moehizuki, T. (1978). Hnkko Kogakn Kubhi 56, 630-644 [cited in Chem. Abstr. (1979), 90, 4465ml. Mochizuki, T., Yasuhira, H., Hondo, S., Ouchi, I., Rokugawa, K . , and Itoga, L. (1972). Proc. Itit. F m n e n t . Syinp., 4th pp. 663468 [cited in Chein. Abstr. (1976), 84, 29405fl. Mogi, h4. (1940). J. Agric. Cher~i.Soc. Jpn. 16, 7-17. Mogi, M .(1942). J . Agric. C h o n . Sue. J p n . 18, Fv3-554. Mogi, M . , and Nakajiina, S. (1942). J. Fertnent. Technol. Jpn. 20, 107-113. Mogi, M . , and Nakajima. S. (1972a). h’ipponJozo Ktyokni Znsshi 67,262-264 [cited in Food Sci. Technol. Ahstr. (1973), 35372). Mogi, M., and Nakajirni, S. (1972b). Nipporl]fmJKyokai Zusshi 67, 265-268 [cited in Food Sci. Trchiiol. Abstr. (1973), 333731. Mogi, M . , and Nakajima, S. (1972~).J . Brew. J p n . 67, 359361 [cited in Food Sci. Techno/. Ahstr. (1973), 7J9431. Mogi, M . , Nakajima, S . , Iguchi, N . , and Yoshida, F. (3951). ,/. Ferment. Technol. J p n . 29, 302310 [cited in Chein. A k s h . (1954),48 2981el.

264

SUMBO H. ABIOSE ET AL.

hlogi, M . , Nakajiina, S., Iguchi, N . , and Yoshida, F. (1952). J . Fenuerit. T e c h r d . J p u . 30, 363369 [citrd in Chrrn. Abstr. (1954), 48, 2981el. Vogi, Xl., Igiichi, N . , Yoshida, F., Yamainoto, K., Mizununia, T., and Nagasaina, M. (1956). H n k k o K ~ J ~ Znsshi u ~ u 35, 150-156 [cited in C h i . A h t r . (1957), 61, 17080gl. Morita, Y., Shiinizu, K., Ogha, \I.* and Koreiiaga, T. (1966). Agric. Rid. Chenr. 30, 114-121. Xfukherjee, S . K . , Alhury, N . N . , Pedcrson, C . S., van Veen, A . G., and Steinkraus. K . 1%. (19G5). Appl. Jlicrobiol. 13, 227-231. Murakaini, 11. (1971).J . Gen. Appl. M i c 7 0 b i o / . 17, 281309. Murakaini, H., Takas?, S., and Ishii, 1‘.(19G7). J . Cen. Appl. Microbid. 13, 323334. Miirakaini, H . , Sugawa, H , , and Takase, S. (1968).J . C e n . Appl. hlicrobiol. 14, 251-262. Nakano, W . (1959).Regional Seminar on Food Technology for Asia and the Far East. Food Res. Inst., \fin. of Agric. and Forestry, Japan. Nisshin Oil Mills Lttl. (1977).Miso-soybean paste prodiict. Japanese Patent 5,243,920 [cited in Foot/ Sci. Teclztiol. Abstr. (1978), G3MN)I. Noyes, R. (1969).“Protein Food Supplements-Food Processing,” Rrview No. 3, pp. 116-231. Noyes Dev. Corp., New Jersey. Nunokawa, Y. (1965).Agric. B i d . Chevr. 29, 687-692. Ohsawa, Y.,Sato, T . , and Mochizuki, T. (1976). Shinshu Miso Kenkyusho Kenkyu Hokokri. 17, 48-,55 [citcd in Chem. A h t r . (1978), 88, 49213~1. Okaxaki, H. (1950).J . Agric. C h e m Soc. J p n . 24, 201-215 (cited in Kodama, 1970). Onishi, H. (1957a). Bull. Agric. Chem. Soc. J p n . 21, 137-142. Onishi, H. (19571)). Bull. Agric. Chem. Soc. .Jpn. 21, 143-150. Onishi, H. (1957~).Bull. Agric. C h i n . Soc. J p n . 21, 151-156. Oonishi, K . (1976).J p n . Kokai 76, 889 [cited in C h e w . Abstr. (1976), 85, 4086kJ. Ouchi, I., and Mochizuki, T. (1968). J . Food Sci. 7’echnol. Tokyo 15, 418421 [cited in Food Sci. T P C ~ W JAhsfr. /. (1969), 762901. Pepplcr, H. J. (1967).“Microbial Technology,” Van Nostrand-Reinhold, Princeton, New Jersey. Piprr, C. V., and Morse, W. J. (1923). “The Soybean.” McCraw-Hill, New York. Platt, B. S. (1964). Food Techno/. 18, 68-76. Fiao, R. (1961). Natl. Acud. Sci. India Natl. Res. Counc. Pub/. 843, 291. Hobiiison, R. J . , arid Kao, C. (1977). Cereal Chew. 54, 1192-1197. Saito, Y., Ito, K., Kawano, K., and Endo, H. (1973). Miso No Kaguku T o Gijutsu 236, 20-30 [cited iii C h e m A h t r . (197S), 82, 169030p]. Sakaguchi, K . (1958). Bull. A@. Chem. Soc. J p n . 22, 345352. Sakaguchi, K. (1972). Proc., Int. S!ymp. Convers. dManufact. Foorlstufis Miwowg., Kyoto, pp. 7-10. Sakurai, Y . (1965). I n “Food Tcchnology the World Over” (M.S. Person and D. K. l‘ressler, eds.), Vol. 2, pp. 374394. Avi Pnbl., Westport, Connecticut. Sato, T., and Mochizuki, T. (1977). Shinshu Miso Kenk!yusho Kenkyu Ifokoku 18, 35-38 [cited in Chem. Abstr. (1978), 88, 61251~1. Sato, T., Ohsawa, Y . , and Mochizuki, T. (1975). Shinshu Miso Kelzk!yushoKenkyu Hokoku 16, 25-29 [cited in C h e m Abstr. (1978), 88, 49217g1. Sato, T. Ohsawa, Y . . and Mochizuki, T. (1976). Shinshu Miso Kedcyushu Kenkyu Hokoku 17, 4447 [cited in Chem Abstr. (1978), 88, 49212b]. Scott, W. O. , and Aldrich, S. R. (1970).“Modern Soybean Production.” S . &A. Publ., Chicago, Illinois. Shibasaki, K . , and Hesseltinc, C . W. (1961a). Biotechnol. Bioeng. 3, 161-174. Shibasaki, K., and Hesseltine, C. W. (lN1b). Proc. Gen. Meet. Soc. Ind. Mimobiol., 7th 2, 205-214. Shibasaki, K., and Hesseltine, C. W. (1962). E C ~ ~ ( J But. ? I Z . 16, 180-195.

MICROBIOLOGY AND BIOCHEMISTRY OF M I S 0

265

Shibasaki, K., and Iwabuchi, S. (1970).J . Food Sci. Technol. Jpn. 17, 193-200 [cited in Food Sci. Techriol. Abstr. (1971), 4J3841. Shurtleff, W., and Aoyagi, A. (1976). “The Book of Miso.” Autumn Press, Kanagawa-Ken, Japan. Smith, A. K., Hesseltine, C. W., and Shibasaki, K. (1961). U . S . Patent No. 2,967,108. Stallel, G. (1946). J . N . Y . Bat. Garden 47, 261-267. Stcinkraus, K. H., vanVeen, A. G., andThiebeau, D. B. (1967). Food Technol. 21, 110-113. Suzuki, M., Nunokawa, Y., Imajuku, I . , Teruchi, Y., and Uruma, M. (1956). Nippon Jozo Kyokai Zasshi 51, 318322 [cited in C h e m Abstr. (1957), 51, 1708Ogl. Tagami, H., and Sugawara, S . (1977). Nippon Shoyu Kenkyusho Zasshi 3, 111-15 [cited in Chein. Abstr. (1977), 87, 166166el. Takahashi, S. (1976). Miyagi Ken Nog!lo Tanki Daigaku Gakujut.w Hokoku 23, 13-17 [cited in Chem. Abstr. (1977), 86, 3731al Takahashi, S. (1977). Miyagi Ken Nogyo Tanki llaigaku Gakujutsu Hokoku 24, 19-23 [cited in Chem. Abstr. (1978), 88, 20778el. Takeuchi, T., and Yokoo, Y. (1974). Hakko Kogaku Zasshi 52, 58-61 [cited in Chetn. Abstr. (1974), 80, 81093jl. Tamiya, H. (1958). Proc. Int. S y m p . Enzyme Chem., Jpn. pp. 21-24. Tamura, G . , Kirirnura, J., Hara, H . , and Sugimura, K. (1952). 1.Agric. Chen,. Soc. Jpn. 26, 483. Tanner, F. W. (1944). “The Microbiology of Foods,” 2nd ed. Garrard, Champaign, Illinois. Teramoto, S . , Taguchi, H . , and Ueda, R. (1962). Proc. Znt. Conf. Food Sci. Technol. l s t , London 11, 549556. Thom, C., and Church, M. (1926).“The Aspergilli.” Williams & Wilkins, Baltimore, Maryland. Uemura, T. (1971). In “Biochemical and Industrial Aspects of Fermentation” (K. Sakaguchi, T. Uemura, and S . Kinoshita, eds.), pp. 1-8. Kodansha, Tokyo. van Veen, A. G. (1957). “Fermented Protein-rich Foods.” F A 0 Report No. FA01571311966. van Veen, A. G . , and Steinkraus, K. H. (1970). J . Agric. Food Chem. 18, 576-578. van Veen, A. G., Hackler, L. R., Steinkraus, K. H., and Mukherjee, S. K. (1967).J . Food Sci. 32, 339341. Watanabe, T., Ebine, H., and Okada, M. (1974). In ”New Protein Foods” (A. M . Altshul, ed.), Vol. l A , pp. 414453. Academic Press, New York. Wehmer, C . (1895). Zentralb. Bakteriol. Parasitenk. Infektionskr. Hyg. Abt. 1, 150. (cited in Kodaina and Yoshizawa, 1977). Wickerham, L. J., and Burton, K. A. (1960). J . Bacterial. 68, 594597, Wolf, W. J., and Cowan, J. C. (1971). “Soybeans as a Food Source.” Buttenvorths, London. Wood, B. J. B. (1977). In “Genetics and Physiology of Aspergillus” (J. E. Smith and J. A. Pateman, eds.), p. 481. Academic Press, New York. Wood, B. J. B. (1980). In “Economic Microbiology’’(A. H. Rose, ed.), Vol. 5. (In press.) Wood, B. J. B., and Yong, F. M. (1974). In “The Filamentous Fungi” (J. E. Smith and D. R. Berry, eds.), Vol. 1, pp. 241-280. Arnold, London. Yokotsuka, T. (1960). Adu. Food Res. 10, 75-134. Yong, F. M. (1971). “Studies on Soy-Sauce Fermentation”. MSc. Thesis, University of Strathclyde, Glasgow. Yong, F. M., and Wood, B. J. B. (1974). Ado. Appl. Microbiol. 17, 157-194. Yong, F. M., and Wood, B. J. B. (19773).J . Food Tschnol. 12, 163-175. Ynng, F. M., and Wood, B. J. B. (1977b).J . Food Technol. 12, 263-273. Yong, F. M., Lee, K. H., and Wong, H. A. (1978). J . Food Technol. 13, 385. Yoshii, H., and Hayakawa, H. (1968). Annu. Rep. Food Res. Znst. Aichi Prefect. J p n . 9, 47-53 [cited in Food Sci. Technol. Abstr. (1973), TJ9451.

This Page Intentionally Left Blank

INDEX

A Acetate polymalonate, pathway for, 60-63 Actinorhodin, genetics of biosynthesis of, 41-42 Ajmalicine, biosynthesis of, on immobilized plant cells, 19, 20 Albumin, partition affinity ligand studies on, 129 Amino acids, as secondary metabolite precursors, 65-73 Anhydrotetracycline hydratase, properties of, 34 Antibiotics, similar subunit structures in, 33 Autotoxicity, of secondary metabolites, regulation of, 86-94

B Barley, as miso raw material, 251 Biocatalysts, immobilized, 1-3 Biotransformations, by immobilized plant cells, 14-22 Bromophos, metabolism in microorganisms, 173

Cells, partition affinity ligand studies on, 133-145 Cephalosporium acremonium, enzyme induction in, 35 Cheesemaking, as solid-state fermentation, 204-205 Chlorpyrifos, effects on microorganisms, 181-182 Chorismate, pathway for, 6 3 6 5 Chromosomal mapping, in secondary metabolism, 4 1 4 2 Citric acid, production by solid state fermentations, 213-215 Composting, as solid substrate fermentation, 224 Concanavalin A, partition affinity ligand assay of, 129-132 Curing agents, in antibiotic production, 4446

D

c Catabolism, biotransformation and regulation of, 90-94 Catabolite repression in secondary metabolism, 77-81 Cathenamine, biosynthesis of, on immobilized plant cells, 19 Catharanthus roseus, immobilized cells of ajmalicine biosynthesis on, 18-20, 22, 23

267

DDVP, effect on microorganisms, 182-183 Diazinon effects on microorganisms, 179-180 metabolism in microorganisms, 161-163, 172 DichlorvoslDDVP, metabolism in microorganisms, 166 Digitoxin, biotransformation of, on immobilized plant cells, 14-18 Digoxin, partition affnity ligand assay of, 127-129 Dimethoate, effect on microorganisms, 182 Disulfoton, effects on microorganisms, 182 DNA, extrachromosomal, role in secondary metabolism, 46-48

268

INDEX

Drugs derived from higher plants, 4 cultured plant cells, 5 I)yfoiiate/fonofc,s, metabolism in microorganisms, 167-168

E Energ! charge, in secondary metabolism, 85-86 Etiiiiatiti, biosynthesis of, 37 Environment, effect on sccondary metabolisin, 38-39 Eiizymes immobilized, preparative methods, 2 modification of, insecondary metabolism, 81 organophosphorus insecticide effects on, 189-191 prod~ictionby solid-state fermentation, 208-2 13 of secondary metaholism, 32-39 cotnpartmentalization, 35-36 inducible, 34-35 specificity, 36-38

F Factors, in secondary metabolism, 82 Feedback inhibition, in secondary metabolism, 74-76 Feuitrotlion, effects on microorganisms, 180 Frnsulfothion, efl'ects on microorganisins, 182 Fenthion, en'ect on microorganisms, 183 Fcrtnentation, on solid substrates, 201-237 Fermented foods, from solid substrates, 216-217 Fish preservation, by solid-state fermentations, 206-207 Fonofos effect on microorganisms, 183 iiictabolisin in ~nirroorgatiisins,173 Fungal spores, organic compound transformation hy, 217-218

G Gallic acid, from solid-state fermentation. 207-208

Gardona, effects on microorganisms, 182-183 Genetic engineering, of secondary nietabolites, 48-51 Gcnctics, of secondary metabolism, 27-115 Genome, illegitamate sequences of, 31-32 Glutaniine synthetase, in secondary metabolism, 82-85 Glycosides, bintransformation of, with immobilized plant cells, 15

I Immobilization, of plant cells, 1-26 Idiophase, definition of, 55 Iron, effect on secondarv metabolism. 39 Isoprene unit, inevalonate D S precursor of, 65

K Koji process, 253-255 solid-state fermentation in, 205-207 Kojic acid, production by solid-state fermentations. 215-216

L P-Lactam antibiotics, biosynthesis of, 66-68 P-Lactamases, occurrence of, 32 Lysirre elrect, in secondary metabolism, 68-69

M Malathion effects on microorganisms, 180-181 metabolism in microorganisms, 164-166, 171-172 Metabolites, secondary, see Secondary metabolites Methionine, in antibiotic biosynthesis, 69-73 Methyl parathion, effects on microorganisms, 179 6-Methylsalicylic acid synthetase, induction of, 35 Mevalonate, secondary metabolites from, 65 Microorganisms, organophosphorus insecticide effects on. 149-200

269

INDEX

Miso, 239-265 from barley, 251 :hemica1 composition of, 257-259 fermentation flow sheet, 245 fiiture developments in, 259-261 history of, 245-246 raw materials for, 248-251 ratio of, 251-252 treatment of, 252-253 from rice, 250 from soybeans, 248-250 types of, 247-248 Mold enzymes, production by solid-state fermentation, 208-213 Morinda citrifolia, immobilized cells of, anthraquinone biosynthesis on, 21-22 Moromi, production of, 255-257 Mutagenesis, secondary metabolism and, 52 Mutagens, organophosphorus insecticides as, 187-191 Mycotoxins, from solid-state fermentations, 218-220

N p-Nitrophenol, metabolism in microorganisms, 171 Nutrition, effect on secondary metabolism, 38

0 Organic acids, production by solid-state fermentations, 213-216 Organophosphorus insecticides accumulation of, 152-153 effects on metabolic pathways, 189-191 microorganisms, 149-200 photosynthesis, 186-187 entry into microbial environments, 151-152 hydrolytic reactions of, 153-154 metabolism of, 153-177 enzymes, 168-177 pathways, 155-169 as mutagens, 187-189 oxidative reactions of, 155 reductive reactions of, 154-155 structures of, 150

P Parathion effects on microorganisms, 177-179 metabolism in microorganisms, 157, 170171 Partition affinity ligand assay (PALA), 117-147 albumin-dye studies by, 129 of cells, 133-145 charged polymer effects on, 123-124 of concanavalin A, 129-132 of digoxin, 127-129 hydrophobicity role in, 124-126 partition modification in, 121-122 phase systems in, 120 principle of, 118-120 of Saccharoinyces cerevisiae, 138-141 salt effects on, 122-123 separator molecules in, 126-127 of Staphylococcus aureus, 134-138 of Streptococcus B1, 141-145 of triiodothyronine, 129 Penicillin history of, 29-31 synthesis of, 3 6 3 7 Peptide(s), antibiotics, biosynthesis of, 65-66 Permeabilization, of plant cdlls, 22-24 pH, effect on secondary metabolism, 39 Phase partitioning, in biochemical preparation, 118 Phorate effect on microorganisms, 183-184 metabolism in microorganisms, 166-167, 173 Photosynthesis, organophosphorus insecticide effects on, 186-187 Plant cells cultures of, 3 4 drugs from, 5 immobilized, 1-26 hiosynthetic capacity, 14-28 preparative techniques, 6-10 reactors for, 12-14 viability, 10-12 permeabilized, 22-24 Plasmids, role in antibiotic synthesis, 42 Propionate kinase, as enzyme of secondary metabolism, 35 Protoplasts, immobilization of, 24

270

INDEX

R Rice, as miso raw material, 250 Rice products, fermented, 242-245

S Sacchuroiiiyces cereuisiue partition affinity ligand assay, 138-141 Sake, brewing flow sheet for, 244 Secondary metabolism, 27-115 catabolite repression in, 77-81 control of, 53-86 energy charge in, 8 5 4 6 environmental factors in, 38-39 enzymes of, 32-39 modification, 81 factors in, 82 feedback inhibition in, 74-76 gene isolation in, 51 genetics of, 39-53 chromosomal mapping, 41-42 ext racliromosomal elements, 42 -48 glutamine synthetase in, 82-85 growth-linked suppression in, 55-58 history of, 29-30 nutrition factors in, 38 pH effects on, 39 plasinid role in, 42-43 primary metabolism relationship to, 59 reciprocal genetics of, 51-53 role of, 97-101 sporulation, exoenzyme formation, and, 94 -97 Secondary metabolites autotoxicity of, 86-04 examples of, 29 genetic engineering of, 48-51 multivalent induction of, by precursors, 58-74 Shikimic acid, pathway for, 6345 Solid substrate fermentations, 201-237 advantages and disadvantages of, 229-231 characteristics of, 226-227 equipment for, 224-226 examples of, 203 future developments in, 231-232 history of, 202-224 physiological asnects of, 227-229

Soy paste, see Miso Soy products, fermented, 24-242 Soybeans, as miso raw material, 248-250 Sporulation, secondary metdbolisni and, 94-97 Stuphylococcus aurws, partition affinity ligand assay of, 134-138 Streptococcus B1, partition affinity ligand assay Of, 141-145 Streptoinyces, enzymes of secondary metabolism in, 35 Streptomycetes, genetics of secondary metabolism in, 40-41 Sugar derivatives, i n secondary metabolism, 73-74 Snmithion/fenitrothion, metabolism in inicroorganisms, 163-166, 172-173

T Trace metals, effect on secondary metabolism, 38 -39 Trichlorofon/dipterex effect on microorganisms, 184 metabolism in microorganisms, 168, 173 Triiodothyronine, partition affinity ligand assay of, 129 Trophophase, definition of, 55

V Valine, in antibiotic biosynthesis, 69 Vicia faba, ininiohilized protoplasts of, 24 Vinegar production, by solid-state frrinentation, 207

W Wastes, solid substrate frrmentation of, 220-224

2

Zinc, effect on secondary metabolism, 38-39

CONTENTS OF PREVIOUS VOLUMES

Volume 1

Volume 2

Protected Fermentation

Newer Aspects of Waste Treatment

Nandor Porges

Miloi Herold and Jan Netasek The Mechanism of Penicillin Biosynthesis

Aerosol Samplers

Harold W . Batchelor

Arnold L. Demain Preservation of Foods and Drugs by Ionizing Radiations

A Commentary on Microhiological Assaying F . Ka va nagh

W . Dexter Bellamy Application of Membrane Filters

Richard Ehrlich

The State of Antibiotics in Plant Disease Control

Microbial Control Methods in the Brewer).

David Pramer

Gerhard J . H a s Microbial Synthesis of Cobamides

D. Perlman Factors Affecting the Antimicrobial Activity of Phenols E . 0. Bennett Gemfree Animal Techniques and Their Applications Arthur W . Phillips andlames E . Smith

Newer Development in Vinegar Manufactures Rudolph J . Allgeier and Frank M . Hilde-

brandt The Microbiological Transformation of Steroids

T. H . Stoudt Biological Transformation of Solar Energy

William J . Golueke

Insect Microbiology S . R. Dutky

Oswald and Clarence G .

SYMPOSIUMON ENGINEERING ADVANCESIN FERMENTATION PRACTICE

The Production of Amino Acids by Fermentation Processes

Shukuo Kinoshita

Rheological Broths

Continuous Industrial Fermentations

Properties

of

Fermentation

Fred H . Deindoerfer and John M . West

Philip Gerhardt and M . C . Bartlett Fluid Mixing in Fermentation Process

1. Y. Oldrhue

The Large-Scale Growth of Higher Fungi Radclgfe F . Robinson and R. S. Davidson

Scale-Up of Submerged Fermentations AUTHOR INDEX-SUBJECT

W . H . Bartholemew

INDEX

27 1

272

CONTENTS OF PREVIOUS VOLUMES

The Metabolism of Cardiac Lactones by Microorganisms Elwood Titus

Air Sterilization Arthur E . Humphrey Sterilization of Media for Biochemical Processes Lloyd L. Kempe Fermentation Kinetics and Model Processes Fred H . Deindoerfer Continuous Fermentation W. D . Maxon

Intermediary Metabolism and Antibiotic Synthesis J . D . Bu'Lock Methods for the Determination of Organic Acids A. C.Huline AUTHOR INDEX-SUBJECT

Control Applications in Fermentation George]. Fuld AUTHOR INDEX-SUBJECT

INDEX

Volume 3 Preservation of Bacteria by Lyophilization Robert 1. Heckly

Sphaerotilus, Its Nature and Emnornic Significance Nonnan C. Dondero

INDEX

Volume 4 Induced Mutagenesis in the Selection of Microorganisms S. 1. Alikhunian The Importance of Bacterial Viruses in Industrial Processes, Especially in the Dairy Industry F . J. Babel Applied Microbiology in Animal Nutrition Harlow €1. Hall

Large-Scale Use of Animd Cell Cultures Donald 1. Merchant and C. Richard Eiduni

Biological Aspects of Continuous Cultivation of Microorganisms

Protection against Infection in the Microbiological Laboratory: Devices and Procedures Mark A . Chatigny

Maintenance and Loss in Tissue Culture of Specific Cell Characteristics Charles C . M m i s

Oxidation of Aromatic Compounds by Bacteria Martin H . Rogofl

T. H o l w

Submerged Growth of Plant Cells L . G . Nickell AUTHOR INDEX-SUBJECT

Screening for the Biological Characterizations of Antitumor Agents Using Microorganisms Frank M . Schabel, Jr., and Robert F . Pittillo The Classification of Actinomycetes in Relation to Their Antibiotic Activity Elw Baldacci

INDEX

Volume 5 Correlations between Microbiological Morphology and the Chemistry of Biocides Adricln Albert Generations of Electricity by Microbial Action 1. B . Dauid

273

CONTENTS OF PREVIOUS VOLUMES

Microorganisms and the Molecular Biology of Cancer

G . F . Cause Rapid Microbiological Determinations with Radioisotopes Gilbert V. Levin The Present Status of the 2,3-Butylene Glycol Fermentation Sterling K . Long and Roger Patrick Aeration in the Laboratory W . R . Lockhnrt and R . W . Squires

Microbial Formation and Degradation of Minerals

Melvin P. Silverman and Henry L. Ehrlich Enzymes and Their Applications

Zrwin W . Sizer A Discussion of the Training of Applied Microbiologists B . W . Kofi and Wayne W . Umbreit AUTHOR INDEX-SUBJECT

INDEX

Volume 7 Microhial Carotenogenesis

Stability and Degeneration of Microhial Cultures on Repeated Transfer Fritz Reusser Microbiology of Paint Films

Alex Ciegbr Biodegradation: Problems of Molecular Recalcitrance and Microbial Fallibility

M . Alexander

Richard T . Ross Cold Sterilization Techniques

]ohn B . Opfell and Curtis E . Miller

The Actinomycetes and Their Antihiotics

Selman A . Waksman Microbial Production of Metal-Organic Compounds and Complexes

Fuse1 Oil

A. Dinsnioor Webb andlohn L. Ingraham AUTHOR INDEX-SUBJECT

Development of Coding Schemes for Microbial Taxonomy S. T . Cowan

INDEX

Volume 6 Global Impacts of Applied Microbiology: An Appraisal Carl-G*an He&n and Mnrtimer P. Stam Microbial Processes for Preparation Radioactive Compounds

D. Perlman

of

D. Perlman, Aris P. Bayan, and Nancy A Giufie

Secondary Factors in Fermentation Processes P. Margalith

Effects of Microbes on Germfree Animals

Thomas D. Luckey Uses and Products of Yeasts and Yeast-Like Fungi Walter I . Nickerson and Robert G. Brown Microbial Amylases

Walter W . Windish and Nagesh S. Mhatre The Microbiology of Freeze-Dried Foods

Gerald J . Silverman and Samuel A. Goldhlith

Nonmedical Uses of Antibiotics

Herhert S. Goldherg

Low-Temperature Microbiology

/udith Farrell and A. H. Rose Microbial Aspects of Water Pollution Control

K . Wuhrmann

AUTHOR INDEX-SUBJECT

INDEX

274

CONTENTS OF PREVIOUS VOLUMES

Volume 8 Industrial Fermentations and Their Relations to Regulatory Mechanisms Arnold L. Demain

Antiserum Production in Experimental Animals Richard H. Hyde Microbial Models of Tumor Metabolism G. F . Gause

Genetics in Applied Microbiology S . G . Bradley

Cellulose and Cellulolysis Brigitta Nwkrans

Microbial Ecology and Applied Microbiology Thomus D. Brock

Microbiological Aspects of the Formation and Degradation of Cellulose Fibers L. Jurubek, J. Ross Coluin, and D . R . Whitaker

The Ecological Approach to the Study of Activated Sludge Wesley 0. Pipes Control of Bacteria in Nondomestic Water Supplies Cecil W. Chnmhws and N m u n A. Clarke The Presence of Human Enteric Viruses in Sewage and Their Removal by Conventional Sewage Treatment Methods Stephen Alan Kollins

Bulking of Activated Sludge Wesley 0 . Pipes Malo-Lactic Fermentation Ralph E . Kunkee AUTHOR INDEX-SUBJECT

Oral Microbiology Heiner Hofftnan

INDEX

Volume 10

Media and Methods for Isolation and Enurneration of the Enterococci Paul A . Hartman, George W . Reinhold, and Devi S. Saraswat Crystal-Forming Bacteria as Insect Pathogens Martin H . R0goj-T Mycotoxins ,in Feeds and Foods Emnuel Borker. Nino F . Insalata, Colette P . h v i , and John S . Witzeman AUTHOR INDEX-SUBJECT

The Biotransformation of Lignin to HumusFacts and Postulates R. T. Ogleshy, R . F . Christman, and C . H . Driver

INDEX

Volume 9 The Inclusion of Antimicrobial Agents in Pharmaceutical Products A . D. Russell, Junelenkins, and 1. H . Harrison

Detection of Lit? in Soil on Earth and Other Planets, Introductory Remarks Robert L. Starkey For What Shall We Search? Allan H . Brown Relevance of Soil Microbiology to Search for Life on Other Planets G. Stotzky Experiments and Instrumentation for Extraterrestrial Life Detection Gilbert V. Levin Halophilic Bacteria D . J . Kushner Applied Significance of Polyvalent Bacteriophages

S. G. Bradley

275

CONTENTS OF PREVIOUS VOLUMES

Proteins and Enzymes as Taxonomic Took

Edward D. Garber and John W . Rippon

Ergot Alkaloid Fermentations

William J . Kelleher

My cotoxins Alex Ciegler and Eioind B. Lillehoj

The Microbiology of the Hen’s Egg

Transformation of Organic Compounds by Fungal Spores

Training for the Biochemical Industries

Claude Vizina, S. N . Sehgal, and Kamar Singh Microbial Interactions in Continuous Culture

R. G . Board I . L. Hepner AUTHOR INDEX-SUBJECT

INDEX

volume 12

Henry R . Bungay, 111 and Mary Lou BunWY

Chemical Sterilizers (Chemosterilizers)

History of the Development of a School of Biochemistry in the Faculty of Technology, University of Manchester

Thomas Kennedy Walker

Paul M . Borick Antibiotics in the Control of Plant Pathogens

M . J. Thirumalachar

Fermentation Processes Employed in Vitamin C Synthesis

Mild Kulha nek AUTHOR INDEX-SUBJECT

INDEX

Flavor and Microorganisms

CUMULATIVE AUTHOR INDEX-CUMULATIVE TITLE INDEX

P. Margalith and Y. S c h m r t z

Volume 11

Mechanisms of Thermal Injury in Nonsporulating Bacteria M . C. Allwood and A . D. Russell

Successes and Failures in the Search for Antibiotics

Collection of Microbial Cells

Selmnn A . Waksman

Daniel 1. C . Wang and Anthony J . Sinskey Fermentor Design

Structure-Activity Relationships of Semisynthetic Penicillins

K . E . Price Resistance to Antimicrobial Agents J. S . Kiser, G . 0. Gale, and G . A. Kemp

Micromonospora Taxonomy George Luedemann Dental Caries and Periodontal Disease Considered as Infectious Diseases

William Gold The Recovery and Purification of Biochemicals

Victor H . Edwards

R. Steel and T . L. Miller The Occurrence, Chemistry and Toxicology of the Microbial Peptide-Lactones

A. Taylor Microbial Metabolites as Potentially Useful Pharmacologically Active Agents I). Perlman and G. P. Pmzzotti AUTHOR INDEX-SUBJECT

INDEX

Volume 13 Chemotaxonomic Relationships Among the Basidiomycetes

Robert G . Benedict

276

CONTENTS OF PREVIOUS VOLUMES

Proton Magnetic Resonance SpectroswpyAn Aid in Identification and Chemotaxonomy of Yeasts P. A. J . Gwin and J . F. T . Spencer

Mathematical Models for Fermentation Processes A . G. Frederickson, R. D.Megee, 111, and

Large-Scale Cultivation of Mammalian Cells R. C . Telling and P. J . Radlett

AUTHOR INDEX-SUBJECT

H. M . Tsuchija INDEX

Volume 14 Large-Scale Bacteriophage Production

K . Sargent Microorganisms as Potential Sources of Food

Jnanendra K . Bhattacharjee Structure-Activity Relationships among Semisynthetic Cephalosporins

M . L. Sassiuer and Arthur Lewis Structure-Activity Relationships in the Tetracycline Series Rob& K . Blackwood and Arthur R . En-

glish Microbial Production of Phenazines ]. M . lngram and A. C . Blackwood The Gihberellin Fermentation

E . G . Jeflre!ps Metabolism of Acylanilide Herbicides

Richard Bartha and David Pramer

Development of the Fermentation Industries in Great Britain

J o h n ] . H. Hastings Chemical Composition as a Criterion in the Classification of Actinomycetes H. A. Lecheualier, M a r y P. Lecheoalier, and Nancy N. Gerher Prevalence and Distribution of AntibioticProducing Actinomycetes John N . Porter Biochemical Activities of Nocardia R . L. Raymond and V . W . Jamison Microbial Transformations of Antibiotics

Oldrich K . Sehek and D . Perlnwn In Viuo Evaluation o f Antibacterial Chemotherapeutic Substances A. Kathrine Miller Modification of Lincomycin

Therapeutic Dentrifrices J . K . Peterson

Barney J . Magerlein Fermentation Equipment

Some Contributions of the U.S. Department of Agriculture to the Fermentation Industry

George E. Ward Microbiological Patents in International Litigation John V. Whittenburg Industrial Applications of Continuous Culture: Pharmaceutical Products and Other Products and Processes

R. C . Righelato and R. Elswwth

G. L. Solomons The Extracellular Accumulation of Metabolic Products by Hydrocarbon-Degrading Microorganisms

Bernard]. Ahbott and William E . Gledhill AUTHOR INDEX-SUBJECT

INDEX

Volume 15 Medical Applications of Microbial Enzymes

Irwin W . Sizer

277

CONTENTS OF PREVIOUS VOLUMES

Immobilized Enzymes K. L. Smiley and 6. W . Strandberg

Intestinal Microbial Flora of the Pig

Microbial Rennets

Antimycin A,, a Piscicidal Antibiotic Robert E. Lennon and Claude Ve'zina

Joseph L. Sardinas Volatile Aroma Components of Wines and Other Fermented Beverages A. Dinsmow Webb and C a s h . J . Muller

Correlative Microbiological Assays

h d i s l a v 1. Harika

R. K e n w d h y

Ochratoxins

Kenneth L. Applegate and John R . Chipley Cultivation of Animal Cells in Chemically Defined Media, A Review

Kiyoshi Higuchi Insert Tissue Culture

W . F . Hink

Genetic and Phenetic Classification of Bacteria

Metabolites from Animal and Plant Cell Culture Irving S. Johnson and George B. Bodw Structure-Activity Relationships in Coumermycins John C . Godfrey and Kenneth E . Price Chloramphenicol

R. R . Coltuell Mutation and the Production of Secondary Metabolites

Arnold L. Demain Structure-Activity Relationships in the Actinomycins Johannes Meienhofm and Eric Atherton

Vetlpal S. Malik Microbial Utilization of Methanol

Charles L. Cooney and David W . Levine Modeling of Growth Processes with Two Liquid Phases: A Review of Drop Phenomena, Mixing and Growth

P. S . Shah, L. T. Fan, I . C . Kao, and L. R. Erickson Microbiology and Fermentations in the Prairie Regional Laboratory of the National Research Council of Canada 1946-1971

R. H . Haskins AUTHOR INDEX-SUBJECT

INDEX

Volume 16 Public Health Significance of Feeding L o w Levels of Antibiotics to Animals

Thorns H . Jukes

Development of Applied Microbiology at the University of Wisconsin

William B . Sarles AUTHOR INDEX-SUBJECT

INDEX

Volume 17 Education and Training in Applied Microbiol-

om Wayne W . Umbreit Antimetabolites from Microorganisms

David L. Pruess andlames P. Scannell Lipid Composition as a Guide to the Classification of Bacteria

Norman Shaw Fungal Sterols and the Mode of Action of the Polyene Antibiotics

J . M . T. Hamilton-Miller

278

CONTENTS OF PREVIOUS VOLUMES

Methods of Numerical Taxonomy for Various Genera of Yeasts 1. Campbell

Recent Developments of Antibiotic Research and Classification of Antibiotics According to Chemical Structure

Janos B k d y Microbiology and Biochemistry of Soy Sauce Fermentation F . M . Young and B . J . €3. Wood

SUBJECT INDEX

Volume 19 Contemporary Thoughts on Aspects Applied Microbiology P. S . S . Dawson and K . L. Phillips

of

Some Thoughts on the Microbiological Aspects of Brewing and Other Industries Utilizing Yeast

G . G . Stewart Linear Alkylbenzene Sulfonate: Bicdegradation and Aquatic Interactions

William E . Gledhill The Story of the American Type Culture Collection-Itc History and Development (1899-1973)

Culture Collections and Patent Depositions T . C. Pridhatn and C . W . Hesseltine Production of the Same Antibiotics by Members of Different Genera of Microorganisms Hubert A . L*.chevalier Antibiotic-Prodncing Fungi: Current Status of Nomenclature C . W . He.weltine a n d ] . I . Ellis Significance of Nucleic Acid Hybridization to Systematics of Actinomycetes

S . C . Bradley

William A. Clark and Dorothy H . Geary Current Status of Nomenclature of AntihioticProducing Bacteria

Microbial Penicillin Acylases

E

I . Vandamme and J P

Vorts

SUBJECT INDEX

Erwin F . Lssel Microorganisms in Patent Disclosures

Irving Marcus

Volume 18 Microbiological Control of Plant Pathogens Microbial Foundation of Environmental Pollutants

Martin AlexandaMicrobial Transformation of Pesticides

lean-Marc Bollag Taxonomic Criteria for Mycobacteria and Nocardiae S . G . Bradley a n d ] . S . Bond Effect of Structural Modifcations on the Biological Properties of Aminoglycoside Antibiotics Containing 2-Deoxystreptamine

Kenneth E . Price, John C . Godfrey, and Hiroshi Kawaguchi

Y . Henis and 1. Chet Microbiology of Municipal Solid Waste Composting Meloin S. Finrtein and Merry L. Morris Nitrification and Dentrification Processes Related to Waste Water Treatment D. D . Focht and A. C . Chang The Fermentation Pilot Plant and Its Aims D. J . D. Hockenhull The Microbial Production of Nucleic AcidRelated Compounds

Koichi Ogata

CONTENTS OF PREVIOUS VOLUMES Synthesis of t-Tyrosine-Related Amino Acids by P-Tyrosinase

Hideaki Yamada and Hidehiko Kumagai Effects of Toxicants on the Morphology and Fine Structure of Fungi Donald V . Richmond SUBJECT INDEX

Volume 20

279

Cytotoxic and Antitumor Antibiotics Produced by Microorganisms

J . Fuska and B. Proksa SUBJECT INDEX

Volume 21 Production of Polyene Macrolide Antibiotics

Juan F. Martin and Lloyd E . McDaniel Use of Antibiotics in Agriculture

The Current Status of Pertussis Vaccine: An Overview

Tomomasa Misato, Keido KO. and Isamu Yamaguchi

Charles R . Manclark Biologically Active Components and Properties of Bordetella pertussis

Enzymes Involved in P-Lactam Antibiotic Riosynthesis

E. J . Vandamme

Stephen I , Morse Role of the Genetics and Physiology of BWdetella pertussis in the Production of Vaccine and the Study of Host-Party Relationships in Pertussis

Charlotte Parker

Information Control in Fermentation Development

D . 1.D. Hockenhull Single-Cell Protein Production by Photosynthetic Bacteria

R. H . Shipman, L. T . Fan, and I . C . Kao Problems Associated with the Development and Clinical Testing of an Improved Pertussis Vaccine

George R. Anderson Problems Associated with the Control Testing of Pertussis Vaccine

Jack Canwron

Environmental Transformation of Alkylated and Inorganic Forms of Certain Metals

Jitendra Sarena and Philip H. Howard Bacterial Neuraminidase and Altered Immunological Behavior of Treated Mammalian Cells

Prasanta K. Ray Vinegar: Its History and Development Hubert A. Conner and Rudolph J .

Allgeier Microbial Rennets

Pharmacologically Active Compounds from Microbial Origin Hewitt W. Matthews and Barbara Fritche

Wade

M . Sternberg SUBJECT INDEX

Biosynthesis of Cephalosporins

Toshihiko Kanzaki and Yukio Fujisawa Preparation of Pharmaceutical Compounds by Immobilized Enzymes and Cells

Bernard J. Abbott

Volume 22 Transformation of Organic Compounds by Immobilized Microbial Cells

lchiro Chibata and Tetsuya Tosa

280

CONTENTS OF PREVIOUS VOLUMES

Microhial Cleavage of Sterol Side Chains

Christoph K . A. Martin Zearalenone and Some Derivatives: Production and Biological Activities

P. H . Hidy, R. S . Baldsin, R. L. Greashain. C . L. Keith, and J. R. McMulbn Mode of Action of Mycotoxins and Related Compounds F . s. Chu Some Aspects of the Microbial Production of Biotin

Yoshikazu lzumi and Koichi Ogata Polyether Antibiotics: Versatile Carhoxylic Acid Ionophores Produced by Streptomyccs J. W . Weatley The Microbiology of Aquatic Oil Spills

Introduction to Injury and Repair of Microbial Cells F . F . Busta Injury and Recovery of Yeasts and Mold

K . E. Stetjenson and T. R . Graunilich Injnry and Repair of Gram-Negative Bacteria, with Special Consideration of the Involvement of the Cytoplasmic Membrane

L. R . Bruchat Heat Injury of Bacterial Spores

Donid M . Adorns The Involvement of Nucleic Acids in Bactcrial Injury M . D. Pier.wn, R . F . Goirwz, and S . E .

Mnrtin SUHJECT INDEX

R . Burtha and R . M . Atlas Volume 24 Comparative Tcchnical and Erunomic Aspects of Single-Cell Protein Processes

John H . Litchfield SUHJECT INDEX

Volume 23 Biology of Bacillus popilliae Lee A. Bulla, Jr., Ralph N . Costilou;, and

Eugene S. Sharpe

Preservation of Microorganism

Rohclt J. Heckly

Streptococcus mutana Drxtranwcraw: A Review

Thomus J . Montvilk, Charles L. CoonetJ and Anthony J . Sinskey Microhiology of Activated Sludge Bulking

Wesley 0 . Pipes Production of Microbial Polysaccharides M . E . Slodki and M . C . Cadmus Effects of Cadmium on the Biota: Influence of Environmental Factors

H . Babich and G . Stotzky Microbial Utilization of Straw (A Review)

Youn W . Han The Slow-Growing Pigmented Water Bacteria: Problems and Sources

Lloyd G . Herman The Bicdegration of Polyethylene Glycols

Donald P. Cox

Mixed Cultures in Industrial Fermentation Processes David E . F . Harrison Utilization of Methanol by Yeasts

Yoshiki Tani, Nohuo Kato, and Hideaki Yamada Recent Chemical Studies on Peptide Antihiotics

Jun'ichi Shoji The CBS Funrrus Collection J. A. Von Arx and M . A. A. Schipper

CONTENTS OF PREVIOUS VOLUMES

Microbiology and Biochemistry of Oil-Palm Wine

Ndukn Okafor

281

Volume 26 Microbial Oxidation of Gaseous Hydrocarbons

Ching-Tsung Hou Bacterial-Amylases

M . B 1n&nnd R

J Ericksorr

SUBJECT INDEX

Volume 25 Introduction to Extracellular Enzymes: From the Rihosome to the Market Place Rudy J . Wodzinski

Ecology and Diversity of Methylotrophic Organisms R. S. Hnnson Epoxidation and Ketone Formation by C , Utilizing Microbes Ching-T.wng Hou, Roinesh N . Potel, and

Allen I . Luskin

Applications of Microbial Enzymes in Food Systems and in Biotechnolom Mutthew 1. Tnylrn- and Toni Richurdson

Oxidation of Hydrocarhons by Methane Monooxygenases from a Variety of Microbes

Molecular Biology o f Extracellular Enzymes Robert F . Rainaley

Propane Utilization of Microorganisms ]eroiii~J . Perry

Increasing Yields of Extracellular Enzymes Douglas E . Evelrigh and Bland S

Production of Intracellular and Extracellular Protein from n-Butane by Fseudomonas Iiutcrnocorci 5p. nov.

Montenecourt

Howard Dalton

Joji

Regulation of Chorismate-Derived Antibiotic Production

Vedpul S. Mnlik

T nku hns h i

Effects of Microwave Irradiation on Microorganisms

John R . Chipkey Structure-Activity Relationships in Fusidic Acid-Type Antibiotics W. oon Daehne, W. 0. Godifredsen, and

P. R. Rasniussen

Ethanol Production by Fermentation: An Alternative Liquid Fuel N . Kosuric, D.C . M . N g , 1. Russell. nnd G.

Antibiotic Tolerance in Producer Organisms Leo C . Vining Microbial Models for Drug Metabolism John P. Ro.pazza and Rohert V . Smith

c. Stewc17-t

Surface-Active Compounds from Microorganisms

D. C. Cooper a n d ] . E . Znjic INDEX

Plant Cell Cultures, a Potential Source of Pharmaceuticals W . G. W . Kurz and F . Constahel

Volume 27 Recombinant DNA Technology

Bacteriophages of the Genus Clostridium.

Vedpal Singh Malik

Seiya Ogata and Mvtoyoshi Hongo SUBJECT INDEX

Nisin A. Hurst

282

CONTENTS OF PREVIOUS VOLUMES

The Coumerrnycins: DevelopmenG in the Late 1970s John C. Corlfiey Instrumentation for Process Control in Cell Culture

Rohert J. Fleischaker, James C. Weaver, and Anthony J. Sinskey

Rapid Counting Methods for Coliform Bacteria A. M . Cundell Training in Microbiology at Indiana ~ ~ i ~ ~ ~ ~ L. S. McClung INDEX

i

~

-