Advances in applied microbiology

ADVANCES IN

Applied Microbiology VOLUME 22

CONTRIBUTORS TO THIS VOLUME

R. M. Atlas R. S . Baldwin

R. Bartha Ichiro Chibata

F. S . Chu R. L. Greasham P. H. Hidy Yoshikazu Izumi C. L. Keith John H. Litchfield J. R. McMullen Christoph K. A. Martin Koichi Ogata Tetsuya Tosa J. W. Westley

ADVANCES IN

Applied MicrobioIogy Edited by D. PERLMAN School of Pharmacy The University of Wisconsin Madison, Wisconsin

VOLUME 22

@

1977

ACADEMIC PRESS, New York

San Francisco London

A Subsidiary of Harcourt Brace Jovanovich, Publishers

COPYRIGHT @ 1977, BY ACADEMIC PRESS, INC. ALL RIGHTS RESERVED. NO PART OF THIS PUBLICATION MAY B E REPRODUCED OR TRANSMITTED I N ANY F OR M OR BY ANY MEANS, ELECTRONIC OR MECHANICAL, INCLUDING PHOTOCOPY, RECORDING, OR ANY INFORMATION STORAGE AND RETRIEVAL SYSTEM, WITHOUT PERMISSION I N WRITING F R OM T HE PUBLISHER.

ACADEMIC PRESS, INC.

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

ix

Transformationsof Organic Compounds by Immobilized Microbial Cells

ICHIRO I. 11. 111. IV.

CHIBATA AND

TETSUYA TOSA

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Immobilization Techniques of Microbial Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . Transformations of Organic Compounds by Immobilized Microbial Cells . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

I 2 9 24 25

Microbial Cleavage of Sterol Side Chains

CHRISTOPH K. A. MARTIN Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .... ... Complete Microbial Degradation of Sterols . . . . . .................... Mechanism of Microbial Sterol Metabolism . , , ....................... Selective Side-Chain Cleavage of Sterols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Substrate Addition and Isolation of Fermentation Products . . . . . . . . . . . . VI. Summary and Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . .... References . . . . . . . ..... ................ ....

I. 11. 111. IV. V.

29 30 33 36 52 54 54

Zearalenone and Some Derivatives: Production and Biological Activities

P. H. HIDY, R. S. BALDWIN, R. L. GREASHAM, C. L. KEITH, AND J. R. MCMULLEN I. 11. 111. IV.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . .................... Chemistry . . . . . . . . . . ................................... Fermentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pharmacology . . . . . . . . . . . . . . . . . . . . . . . . .................. References . . . . . . . . . . . . . . . . . . . . . . . . . . . ..................

59 61

63 74 81

Mode of Action of Mycotoxins and Related Compounds

I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Biological Effects of Mycotoxins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

V

83 85

vi

CONTENTS

I11. Modification of Carbohydrate and Lipid Metabolism by Mycotoxins . . . . . . . . . IV . Effect of Mycotoxins on Nucleic Acid and Protein Synthesis . . . . . . . . . . . . . . . . V. Interaction of Mycotoxins with Macromolecules as t h e Mode of Action of Mycotoxins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI . Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

100 107 121 133 135

Some Aspects of the Microbial Production of Biotin

YOSHIKAZU IZUMIAND KOICHI OGATA I . Introduction I1. 111. IV . V. VI . VII .

............................................. ............ ............ Biosynthetic Pathway of Biotin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ............ Regulation of Biotin Biosynthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . .............. Preparation of Biotin and Its Vitamers . . . . . . . . . . . . . Biotin Antimetabolites and Their Actions on Biotin Biosynthesis . . . . . . . . . . . . Biodegradation of Biotin and Its Vitamers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

145 146 155 158 163 170 172 173

Polyether Antibiotics: Versatile Carboxylic Acid lonophores Produced by Streptomyces

J . W . WESTLEY I. I1. I11. IV . \7 . VI . VII . VIII . IX .

Introduction ........................... . . . . . . . . . . .......... Classification .................................. Polyether Antibiotics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .......... Microorganisms That Produce Polyether Antibiotics . . . . . . . . . . . . . . . . . . . . . . . .......... Biosynthesis of Polyether Antibiotic Physical Constants of the Polyether .......... In Vitro Antimicrobial Activity and Antibiotics Coccidiostat Activity of the Polyether Antibiotics . . . . . . . . . . . . . . . .. . . . . . . . . . Antimicrobial and Coccidiostat Activity of Lasalocid Analogs . . . . . . . . . . . . . . . . X . Improved Ruminant Feed Utilization by Addition of Polyethers . . . . . . . . . . . . . .......... XI . Pharmacology of the Polyether Antibiotics . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

177 178 183 189 193 203 203 211 211 215 216 220

The Microbiology of Aquatic Oil Spills

R . BARTHAAND R . M . ATLAS I. I1 . 111. IV .

Sources and Behavior of Oil Pollutants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Effects of Petroleum Hydrocarbons on Microorganisms . . . . . . . . . . . . . . . . . Microbial Emulsification and Degradation of Petroleum Hydrocarbons . . . . Microorganisms and Oil Pollution Abatement . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

226 230 236 253 261

CONTENTS

vii

Comparative Technical and Economic Aspects of Single-Cell Protein Processes JOHN

H . LITCHFIELD

I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I1. End Uses for SCP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. SCP Microorganisms. Processes, and Products . ........................ IV . Economic Aspects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V . Plant and Animal Protein Products Competing with SCP . . . . . . . . . . . . . . . . . . . VI . Nutritional Value and Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VII . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

SUBJECTINDEX . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . CONTENTSOF PREVIOUS VOLUMES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

267 268 268 291 296 299 301 301

307 311

This Page Intentionally Left Blank

LIST OF CONTRIBUTORS Numbers in parentheses indicate the pages on which the authors’ contributions begin.

R. M . ATLAS,Department of Biology, University of Louisville, Louisville, Kentucky (225)

R. S . BALDWIN,ZMC Chemical Group, Inc., Terre Haute, Indiana (59) R. BARTHA,Department of Biochemistry and Microbiology, Rutgers University, New Brunswick, New Jersey (225) ICHIROCHIBATA,Research Laboratory of Applied Biochemistry, Tanabe Seiyaku Co. Ltd., Osaka, Japan (1) F . S . CHU, Food Research Institute and Department of Food Microbiology and Toxicology, University of Wisconsin, Madison, Wisconsin (83)

R. L. GREASHAM, ZMC Chemical Group, Inc., Terre Haute, Zndiana (59) P . H . HIDY,IMC Chemical Group, l n c . , Terre Haute, Indiana (59) YOSHIKAZU IZUMI,Department of Agricultural Chemistry, Kyoto University, Kyoto, Japan (145) C. L. KEITH, IMC Chemical Group, lnc., Terre Haute, Zndiana (59) JOHN

H. LITCHFIELD,Battelle Columbus Laboratories, Columbus, Ohio

J. R. MCMULLEN,IMC Chemical Group, Znc., Terre Haute, Indiana (59)

CHFUSTOPHK. A. MARTIN,Gesellschaft fur Biotechnologische Fmschung mbH, Braunschweig-Stockheivn, West Germany (29) KOICHI OGATA,Department of Agricultural Chemistry, Kyoto University, Kyoto, Japan (145)

TETSUYATOSA, Research Laboratory of Applied Biochemistry, Tanabe Seiyaku Co. Ltd., Osaka, Japan (1) J. W. WESTLEY,Hoffmann-La Roche Inc., Nutley, New Jersey (177)

ix

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Transformat ions of Organ ic Compounds by Immobilized Microbial Cells ICHIRO CHIBATA AND

TETSUYA TOSA

Research Laboratory of Applied Biochemistry, Tanabe Seiyaku Co. Ltd., Osaka, Japan I. Introduction . . . . ... 11. Immobilization T .............. 111. Transformations of Organic Compounds by Immobilized Microbial Cells ...................... A. Production of ~-AsparticAcid from A B. Production of L-Malic Acid from Fumaric Acid C. Production of L-Citrulline from L-Arginine . . . D. Production of Urocanic Acid from L-Histidine E. Production of 6-Aminopenicillanic Acid from Penicillin . , F. Other Production of Useful Compounds . . . . . . . . . . . . . . . IV. Conclusion . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1 2

9 9 17 19 20

22 23 24 25

1. Introduction Over the past ten years, the immobilization of enzymes has been the subject of increased interest, and a number of papers on transformations of organic compounds by immobilized enzymes have been published. Among these many studies, in 1969 we (Tosa et al., 1966, 1967; Chibata et al., 1972) first succeeded in the industrial application of immobilized enzyme, i.e., immobilized aminoacylase, for continuous production of L-amino acids from acyl-DL-amino acids. Succeeding to this industrialization, productions of 6-aminopenicillanic acid (6-APA) by immobilized penicillin amidase and fructose by immobilized glucose isomerase are said to be industrially operated in the United States, Europe, and Japan. Further, very recently a number of papers have been published on the immobilization of microbial cells for the purpose of transformations of organic compounds. In this field, we also studied the immobilization of whole microbial cells-Escherichia coli having high aspartase activity-and in 1973 succeeded in industrial application of these immobilized cells for continuous production of L-aspartic acid from ammonium fumarate (Chibata et al., 1974e; Tosa et al., 1974). This is also the first industrial application of immobilized microbial cells in the world. Further, in 1974 we (Chibata et al., 1 9 7 5 ~Yamamoto ; et al., 1976, 1977) succeeded in industrial production of L-malic acid from fumarate using immobilized Breuibacteriurn arnrnoniagenes having high fumarase activity. In this review, these two industrial examples using immobilized microbial cells are described as the focus and some of other current and potential applications are reviewed. 1

2

lCHlRO CHIBATA A N D TETSUYA TOSA

II. Immobilization Techniques of Microbial Cells At present many papers on immobilization of microbial cells have been published, as shown in Tables I and 11. There are mainly three general methods for immobilization of microbial cells as follows.

1. Microbial cells can be immobilized by ionic binding to water-insoluble ion exchangers. Hattori and Furusaka (1960, 1961) bound E. coli cells and Azotobacter agile cells to Dowex-1 resin (C1 form), and revealed that these immobilized cells showed oxidation activity for succinic acid. After these studies, Johnson and Ciegler (1969) bound spores of molds to ion-exchange cellulose derivatives and detected invertase activity of these immobilized spores. However, it is considered that this method is not advantageous, because an enzyme may easily leak out from cells owing to autolysis of cells during continuous enzyme reaction. 2. Microbial cells can be immobilized by cross-linking each other with bifunctional reagents. We (Chibata et al., 1974e) could immobilize E. coli cells by glutaraldehyde as a bifunctional reagent. These immobilized cells showed aspartase activity corresponding to 34.2% of that of intact cells. This is the first paper on immobilization of cells by cross-linking method. Further, we attempted the immobilization of E. coli cells by cross-linking with toluene diisocyanate, but active immobilized cells could not be obtained. 3. Microbial cells can be immobilized by entrapping them into a polymer matrix where they are physically restrained. In this method, the following matrixes are employed: (i)polyacrylamide, (ii) collagen, (iii) cellulose triacetate, (iv) agar, (v) alginate, and (vi) polystyrene. Among these gels, polyacrylamide has been extensively employed as shown in Tables I and 11. This technique was applied at first to immobilization of a lichen, Umbilicaria pustulata, by Mosbach and Mosbach (1966). Further, Franks (1971) immobilized Streptococcus faecalis cells into polyacrylamide gel lattice and investigated catabolism of L-arginine in this microbial cells. Succeeding in these studies on the immobilization of lichen and Streptococcus cells, we (Chibata et al., 1974e) attempted the immobilization of bacterial cells by this polyacrylamide gel method for industrial applications. To prepare the most efficient immobilized microbial cells, we investigated the type and concentration of bifbnctional reagents and the concentration of acrylamide monomer. Table I11 shows the results of immobilization of E. coli using various bifunctional reagents. The aspartase activity of the obtained immobilized E. coli cells is almost the same except the cases with ethylene urea bisacrylamide and 1,3,5-triacryloylhexahydro-s-triazine.Thus, N,N I methylenebisacrylamide (Bis) was chosen, because it is commercially available at low cost. The concentration of acrylamide monomer and Bis, and the

TABLE I PRODUCTION OF ORGANIC COMPOUNDS BY IMMOBILIZED MICROBIAL CELLS Enzyme system

Product

Microorganism

Immobilization method

Reference

Sorbose dehydrogenase

L-Sorbosone

Glucono bacter melanogenus

Polyacrylamide

Martin and Perlman (1976)

Steroid Up-hydroxylase

Cortisol

Curvularia lunata

Polyacrylamide

Mosbach and Larsson (1970)

NAD kinase

NADP

Achroinobacter aceris

Polyacrylamide

Chibata et u1. (1975a)

Glucose phosphatase

Glucose 6-phosphate and glucnw 1phosphate

Escherichia freundii

Polyacrylamide

Saif et al. (1975)

P-Galactosidase

D-Glucose and D-galactose

Lactobacillus hulgaricus

Agar

Miyata et al. (1975)

Saccharomyces lactis

Cellulose triacetate

Dinelli (1972)

Fungal spores

Ion-exchange celluloses

Johnson and Ciegler (1969)

Saccharoin yces pastorianus

Agar

Toda and Shoda (1975)

Yeast

Lyophilization

Nanba and Matsuo (1970)

Escherichia coli

Cellulose triacetate

Dinelli (1972)

Pol yacr ylamide

Sato et al. (1976)

Invertase

Penicillin amidase

D-Glucose and D-fructose

6-Aminopenicillanic acid

(continued)

Enzyme system Penicillin amidase (synthesis)

Product Penicillin

Microorgan is in

Immobilization method

Reference

Bacillus meguterium Achromobacter sp.

DEAE-cellulose

Fujii et ul. (1973)

Escherichia coli

Cellulose triacetate

Marconi et al. (1975)

Cephalosporin amidase (synthesis)

Cephalosporin

Achromobacter sp.

Ion-exchange celluloses

Fujii et al. (1974)

Aminoacylase

L-Methionine

Aspergillus ochraceus

Egg albumin and glutaraldehyde

Hirano et al. (1975)

L-Menthol ester hydrolase

L-Menthol

Alginomonas nonfermentas

Polyacrylamide

Nonomura et al. (1976)

Diaminopimelic acid decarboxylase

L-Lysine

Microbacteriuin ammonia p hilum

Pol yacr ylamide

Kanemitsu (1975)

Orsellinic acid decarboxylase

Orcinol and orcinol methyl ester

Lichen

Polyacrylamide

Mosbach and Mosbach (1966)

Aspartate 4-decarhoxylwe

L- Alanine

Pseudomonas dacunhae

Pol yacr ylamide

Chibata et ul. (1975b)

P-Tyrosinase

L-Dopa

Erwinia herbicola

Collagen and dialdehyde starch

Kumagaya et al. (1976)

L-Tyrosine

Erwinia herbicolu

Collagen and dialdehyde starch

Yamada et a1 (1975)

L-Malic acid

Breoibacteriuin aminoniagenes

Pol yacrylamide

Yamamoto et a/. (1976)

P

Fumarase

Aspartase

L-Aspartic acid

Escherichia coli

Glutaraldehyde, polyacr ylamide

Chibata et a1 (1974e)

Tryptophan synthetase

t-Tryptophan

b;scherichia coli

Pol yacrylamide

Chibata et al. (197413)

5-Hydroxy-~tryptophan

Escherichia coli

Polyacrylamide

Chibata et a1 (1974a)

L-Histidine ammonia lyase

Urocanic acid

Achromobacter liquidum

Polyacrylamide

Yamamoto et al. (1974b)

L-Arginine deiminase

t-Citrulline

Pseudoinonas putida

Polyacrylamide

Yamamoto et a1 (1974a)

Glucose isomerase

D-Fructose

Actinomycetes

Anion exchange resin

Ishimatsu et al. (1976)

Gelatin and glutaraldehyde

Yuta et al. (1975)

Heat treatment

Takasaki and Kanbayashi (1969)

P-Ray irradiation

Tsumura (1969)

Drying after dipping in organic acid

Tsumura et al (1976)

Bacillus coagulans

Glutaraldehyde

Novo Industri (1976a,b)

S h eptom yces

Polyacr ylamide

Chibata et a1 (1974d)

Cellulose triacetate

Kolarik et a1 (1974)

griseus Streptom yces phaeochromogenes

(continued)

TABLE I (continued) Enzyme sy5tt.m

Product

Microorganism

Glucose isomerase (continued)

Immobilization method

Reference

Collagen

Vieth et a1 (1973)

Streptomyces venezuelae

Collagen

Venkatasubramanian et al. (1974)

Coenzyme A

Brevibacterium ammoniagenes

Polyacrylamide

Shimizu et aZ. (1975)

L-Glutamic acid

Cmy nebacterium glutamicuin

Polyacrylamide

Slowinski and Charm (1973)

Collagen

Brownstein et (11. (1974)

Polyacrylamide

Yagi et al. (1976)

Wang et al. (1973)

Multienzyme system

Lactic acid

Arthrobacter oxyduns

7

TRANSFORMATIONS BY IMMOBILIZED MICROBIAL CELLS

TABLE I1 IMMOBILIZED MICROBIALCELLS AND THEIROTHER APPLICATIONS

Microorganism

Immobilization method

Application

Reference

Azotobacter agile Escherichia coli

Dowex 1

Oxidation of glucose and succinic acid

Hattori and Furusaka (1960, 1961)

Tetrahymena pyriformis

Pol yacr ylamide

Survival of microorganism in polymer lattice

Updike et al. (1969)

Streptococcus faecalis

Polyacrylamide

Catabolism of L-arginine

Franks (1971)

Pseudomonus inephitica var. lipolytica

Heat treatment

Hydrolysis of triacetin and tributyrin

Kosugi and Suzuki (1973)

Baker’s yeast

Alkaline metal salts of alginic acid

Bread making

Chibata et al. (1974~)

Achromobacter

Pol yacr ylamide

Hydrolysis of 6-aminocaproic acid cyclic dimer

Kinoshita et al (1975)

Candida tropicalis

Aluminum alginate, polystyrene, polyacr ylamide

Degradation of phenol

Hackel et al. (1975)

Clostridium butyricum

Polyacrylamide

Production of hydrogen from glucose (hydrogen battery)

Karube et al. (1975)

Mold

Glutaraldehyde

Hydrolysis of r a t h o s e interfering with sugar crystallization

Nishimaru et al. (1975)

Micrococcus denitrifkans

Liquid membrane

Reduction of nitrate and nitrite

Mohan and Li (1975)

guttatus

amount of cells to be entrapped were investigated, and following optimum conditions for immobilization of E . coli cells were decided. E . coli cells (10 gm, wet weight) are suspended in 40 ml of physiological saline. To this suspension are added 7.5 gm of acrylamide, 0.4 gm of Bis, 5 ml of 5% P-dimethylaminopropionitrile, and 5 ml of 2.5%potassium persulfate. The

8

ICHIRO CHIBATA A N D TETSUYA TOSA

TABLE I11 IMMOBILIZATION OF Escherichia Cali CELLSBY USINGVARIOUS BIFUNCTIONALREAGENTS~ ~~

~

~

Bifunctional reagents

Aspartase activity (Wmoleslhr)

Yield of activity (%)

N , N '-Methylenebisacrylamide N , N '-Propylenebisacrylamide Diacrylamide dimethyl ether l,&-Diacrylamide ethyleneglycol N,N '-DiaUyl tartardiamide Ethylene urea bisacrylamide 1,3,S-Triacryloyl hexahydro-s-triazine

1220 1104 1048 1136 1320 128 128

67.0 60.7 57.6 62.4 72.5 7.0 7 .0

"One gram of intact cells (packed wet cells; 1820 gmolesihr) was used. Immobilization was carried out as described in the text except for using various bifunctional reagents (Chibata et al., 1974e).

mixture is allowed to stand at below 40°C for 10-15 minutes, and the resulting stiff gel is made 2 3 mm cubes. After entrapping into polyacrylamide gel, the immobilized cells are thoroughly washed and used for enzyme reaction. In this reaction mixture, any unreacted acrylamide monomer can not be detected by gas chromatographic analysis. This immobilized microbial cells have been industrially employed at Tanabe Seiyaku Co. Ltd. since 1973. Immobilization of microbial cells by collagen was carried out by Vieth et al. (1973). This technique is suitable for preparation of a membrane type of immobilized cells. Immobilization of microbial cells into cellulose fiber was presented by Dinelli (1972), and enzyme activities of these immobilized cells are said to be very stable. Further, there have been reports on immobilizations of microbial cells by agar (Miyata et al., 1975), alginate and polystyrene (Hackel et al., 1975). However, their detail immobilization conditions and characteristics of immobilized cells have not been clarified. Recently, Mohan and Li (1975) reported the immobilization of cells by the liquid membrane. In this method, a buffer solution containing the cells is dispersed in an oil phase composed of surfactants, various additives, and hydrocarbon solvent to form an emulsion. The cell suspension is carefully added dropwise into this emulsion, and cells are encapsulated by the liquid membrane. As shown in Tables I and 11, other immobilization methods have been published. Takasaki and Kanbayashi (1969)immobilized Streptomyces sp. by

TRANSFORMATIONS BY IMMOBILIZED MICROBIAL CELLS

9

heat treatment and investigated the conditions for conversion of D-glucose to D-fructose by this immobilized cells; i.e., when the cells are heated at 60"-85"C for 10 minutes, glucose isomerase is fixed inside the cells and does not leak out from the cells, even if the cells are incubated under the conditions for enzyme reaction. This immobilized cells are used for industrial production of D-fructose. Tsumura (1969)also fixed glucose isomerase inside Streptomyces phaeochromogenes cells by irradiation of P-ray (1-10 mrad).

Ill. Transformations of Organic Compounds by Immobilized Microbial Cells As described in Tables I and 11, a number of papers on immobilization of microbial cells and their application for transformation of organic compounds have been published. In this section, we mainly describe our studies on the continuous production of useful compounds by immobilized microbial cells. Some of them have been industrially operated at Tanabe Seiyaku Co. Ltd., Osaka, Japan. A. PRODUCTIONOF L-ASPARTICACID

AMMONIUM

FROM

FUMARATE As described above, we (Chibata et al., 1972) succeeded in the industrial application of immobilized aminoacylase for continuous production of L-amino acids from acyl-DL-amino acids in 1969. Succeeding to this technique, we studied continuous production of L-aspartic acid which is widely used as medicines and food additives. The acid has been industrially produced by fermentative and enzymic methods from fumaric acid and ammonia by the action of aspartase as follows. HOOCCH=CHCOOH

+ NH3'HOOCCH2CHCOOH apariaase

Fumaric acid

I

N H2 L-Aspartic acid

As this reaction has been carried out in batch process, there had been some disadvantages for industrial use. Thus, to overcome this disadvantages we (Tosa et al., 1973) investigated the immobilization of aspartase. The active immobilized aspartase was obtained by entrapment into a polyacrylamide gel lattice, but this immobilized enzyme was relatively unstable, i.e., its half-life was 27 days at 37°C. Further, it was necessary to extract the enzyme from microbial cells before immobilization. Therefore, this immobilized aspartase was considered not to be satisfactory for industrial purpose. Thus, we considered that if the microbial cells having the enzyme activity can be immobilized, these disadvantages might be overcome. From

10

ICHIRO CHIBATA A N D TETSUYA TOSA

these points of view, we studied the immobilization of whole microbial cells, and succeeded in industrialization of this technique in 1973 (Chibata et al., 1973, 1974e; Tosa et al., 1974; Sato et al., 1975). Around 1970, only a few papers have been published on the immobilization of microbial cells and little is known about the suitable conditions for the immobilization and the enzymic properties of the immobilized cells. Thus, we have extensively studied the immobilization of E. coli having aspartase activity and its application for continuous enzyme reaction. Eschaichia coli cells were immobilized by the following methods and their aspartase activities were measured, i. e., they were (i) entrapped into a polyacrylamide gel lattice by using acrylamide monomer and N , N ‘-methylenebisacrylamide, (ii) cross-linked by a bifunctional reagent, such as glutaraldehyde or 2,Ctoluene diisocyanate, and (iii) encapsulated by polyurea produced from 2,ctoluene diisocyanate and hexamethylenediamine. Among these methods, active immobilized E. coli cells were obtained by entrapping the cells into a polyacrylamide gel lattice and by cross-linkingthe cells with glutaraldehyde. The more active preparation was obtained in the former case, and the aspartase activity of this immobilized cells obtained under the standard conditions is in general 1310 pmoleshrlper gram of wet cells. In these immobilized cells, an interesting phenomenon was observed. When the freshly prepared immobilized E . coli cells were suspended at 37°C for 2.4-48 hr in 1M ammonium hmarate solution, pH 8.5, containing 1 mM M 8 + , the activity increased about 10 times. This activation is caused by an increase of the membrane permeability for substrate and/or product due to autolysis of E . coli cells in the gel lattice. Of course, even if lysis of the cells occurs, the aspartase does not leak out from the gel lattice, though the substrate and the product easily pass through the gel lattice. The enzyme activities of native aspartase, immobilized aspartase, E. coli cells, and immobilized E. coli cells are summarized in Fig. 1. It is clear that the immobilized E. coli cells are superior to the immobilized aspartase in respect of enzyme activity. Further, we compared the stability of aspartase activity of immobilized cells and intact cells, i.e., not immobilized cells, and the results are shown in Fig. 2. The initial activities of both preparations are almost same, but the activity of intact cells rapidly decrease and its half-life is around 11days. On the other hand, the activity of immobilized cells is very stable, and its half-life is 120 days as shown in Fig. 3. These results indicate that the immobilized cells are more advantageous for production of L-aspartic acid than the intact cells and the immobilized enzyme. As to the mechanism of this stabilization, we investigated in detail the use of the information obtained for stabilization of enzyme in other microbial cells (Tosa et al., 1975).

TRANSFORMATIONS BY IMMOBILIZED MICROBIAL CELLS

11

Intact cells (1700 pnoles/hr)

Crude aspartase

Autoiyzed cell suspension

Immobilized cells -

(2160 pmoles/hr)

(11,290 pnoles/hr)

(1310 pnoles/hr)

Immobilized aspartase (620 pmoles/hr)

Homogenized s*c (12,780 pmoles/hr)

Activated immobilized cells

Homogenized immobilized cells

(12,200 pnoles/hr)

(11,000 pnoles/hr)

FIG. 1. Comparison of aspartase activity of various enzyme preparations per unit of intact cells. One gram (packed wet weight) of intact cells corresponds to 0.2 gm of dried cells. Numerical values in parenthesis are aspartase activities obtained from 1 gm of intact cells.

We considered that the aspartase of immobilized cells may be stable owing to the binding of some particles or membranes in cells. To confirm this assumption, we carried out the following experiments. After sonication or autolysis of intact cells, both precipitate and supernatant fractions were obtained by centrifugation (27,000g, 20 minutes), and they were immobilized separately by polyacrylamide method. The stabilities of aspartase activity of immobilized fractions were investigated, and it was found that the activity of immobilized precipitate fraction is very stable, but that of immobilized supernatant fraction is very unstable (Table IV). Further, after intact cells were preliminarily treated with deoxycholate or Triton X, which is used as a solubilizing agent for membrane-binding enzyme, or were autolyzed, they were centrifuged (27,000 g, 20 minutes). Both precipitate and supernatant fractions were immobilized separately by polyacrylamide method, and the stabilities of aspartase activity of these preparations were investigated. The stabilities of these preparations were found to be low and almost the same in both, as shown in Table n7; i.e., the results indicate that the aspartase becomes unstable owing to solubilization. In other words, the binding of aspartase to membranes and particles plays an important role in the stabilization of the enzyme. For further confirmation, the following experiments were carried out. The partially purified aspartase was immobilized by binding it to water-insoluble carriers via covalent binding, ionic binding, and hydrophobic binding, and then these immobilized aspartases were entrapped by polyacrylamide gel. The enzyme stabilities of these preparations were investigated and compared with immobilized aspartase prepared by the polyacrylamide method.

12

ICHIRO CHIBATA A N D TETSUYA TOSA

I

I

I

1

I 20

I

I

30

40

8

d!

10

I

1

Incubation time at 3 7 O C (day)

FIG. 2. Stability of enzyme activities of intact and immobilized cells. In the case of Escherichia coli, intact cells and immobilized cells stood in 1 M ammonium fumarate (PH 8.5, containing 1 mM Mg+) at 37°C. In the case of Brewibacterium ammoniagenes, bile extracttreated cells and bile extract-treated immobilized cells stood in 1 M sodium fumarate (PH 7.0) at 37°C. At the indicated intervals, their remaining enzyme activities were determined, tlIz indiB . ammoniagenes fumarase cates half-life of enzyme activity. ( 0 ) E. coli aspartase activity. activity.

I.)

tn .d

c

.rl

20-

rb

5

a I

I

I

I

I

I

I

I

I I0

FIG. 3. Stability of immobilized Escherichio coli column. A solution of 1 M ammonium fumarate (PH 8.5, containing 1 mM M g + ) was applied to immobilized E. coli column (total volume: 1044 ml) at 37°C at the flow rate of 0.666 x lo-* m/min for the complete conversion of ammonium fumarate to L-aspartic acid, and sampling of reaction mixture was carried out at indicated times (Sato et ol., 1975).

13

TRANSFORMATIONS BY IMMOBILIZED MICROBIAL CELLS

TABLE IV OPERATIONAL STABILITY OF ASPARTASEACTIVITY OF VARIOUS IMMOBILIZED hEPARATION5'

Immobilized preparationb Cells Sonicated cells Whole cells Precipitate Supernatant Deoxycholate-treated cells Whole cells Precipitate Supernatant Immobilized aspartases Covalent binding to CNBr-activated Sepharose Ionic binding to TEAE-cellulose Hydrophobic binding to 1,5-diaminopentaneSepharose

Stability (half-life, days) 120

38 76 6

9' 13' 8c

>30 >30 >30

Except in the case of deoxycholate-treated cells, stabilities of immobilized preparations were determined by continuously passing a substrate solution through columns packed with these preparations at 37°C (Tosa et al., 1975). *Immobilization was carried out by polyacrylamide gel method. rData for standing at 25°C.

As shown in Table IV, all immobilized preparations prepared by binding native aspartase to water-insoluble carriers were very stable than the aspartase entrapped into polyacrylamide gel lattice. Therefore, the above assumption is considered to be proper. To find out the most suitable conditions for continuous production of L-aspartic acid from ammonium fumarate by a column packed with the immobilized E . coli cells, we investigated the enzymic properties of the immobilized cells and obtained the results shown in Table V. The native aspartase is activated by Mn2+,but intact and immobilized cells are not activated by this metal ion. On the other hand, bivalent metal ions such as Caz+, Mg2+,and Mn2+show the protective effect against heat inactivation of aspartase activities of intact and immobilized cells. Further, these bivalent metal ions have stabilizing effect to aspartase activity of the inimobilized cells during the continuous enzyme reaction. The reaction mechanism and decay behavior of aspartase activity for immobilized E. coli cells were investigated by using a sectional packed column (Sato et a l . , 1975). Reaction within the immobilized cell column proceeded

TABLE V COMPARISON OF ENZYMIC PROPERTIES OF INTACTAND IMMOBILIZED MICROBIAL CELLS ~~

Enzyme:

Aspartase

Fumarase

L-Arginine deiminase

L-Histidine ammonia-lyase

Penicillin amidase

Microorganism:

Escherichia coli

Brevibacterium ummoniagenes

Pseudomonas putida

Achromobacter liquidum

Escherichia coli

10.5 8.5

7.5' 7.0

5.5-6.0" 5.5-6.0'

9. Ob 9.0b

8.5 8.5

50" 50"

60"

37"'

mob

60"

55""

60°b

40" 40"

50"C, 30 min

55"C, 60 min

60"C, 60 min

-

6OoC, 30 min

49

63 65

42 63

-

58

-

10 25

Chibata et ul. (1974e)

Yamamoto et al. (1976)

Yamamoto et al. (1974a)

Yamamoto et al. (1974b)

Sato et ul (1976)

Optimum p H Intact Immobilized Optimum temperature

E

("C) Intact Immobilized Heat stability (relative remaining activity (8)) conditions Intact Immobilized References

"In the presence of 0.05% cetyltrimethylammonium bromide. "In the presence of 0.025% cetyltrimethylammonium bromide. "Data for native enzyme.

TABLE VI CONDITIONS FOR CONTINUOUS PRODUCTIONOF ORGANICCOMPOUNDS BY IMMOBILIZED MICROBIALCELLS

Enzyme:

Aspartase

Fumarase

L-Arginine deiminase

L-Histidine ammonia-lyase

Penicillin amidase

Microorganism:

Escherichia colt

Brevibacteriuin aininoniagenes

Pseudoinonas putida

Achromobacter liquiduin

Escherichia coli

1.0 M Ammonium fumarate 8.5 Caz+, M g ' or MnZ+

1.0 A4 Sodium fumarate 7.0

0.9 M L-Arginine 6.0

0.05 M 0.25 M L-Histidine Penicillin G 9.0 8.5 Caz+, Coz+, M$+ or Znz+-

37"

37"

37"

37"

40"

0.80 t-as partic acid

0.23 L-Malic acid

0.26 L-Citrulline

0.06 Urocanic acid

0.24 6-Aminopenicillanic acid

95

70

96

91

80

120 (37") Sato et a1 (1975)

52.5 (37") Y amamoto et al. (1976)

140 180 17, 42 (37") (40") (309 (37") Yamamoto Yamamoto Sato et al. et a/. (1974a) et a / . (1974b) (1976)

Substrate PH Stabilizer Temperature ("C) Flow rate" (SV, hr-') Product Yield of product (%) Operational stability (half-life, days) References

=Data show flow rate for complete conversion of substrate to product. SV: space velocity.

16

ICHIRO CHIBATA A N D TETSUYA TOSA

at zero order on substrate solutions ranging in concentration from 0.1 to 1.0 M, and the initial reaction rate was found to be 1.556 x lo-' moles/minutes/ liter of immobilized cells. The effect of temperature on the reaction rate constant was investigated. The Arrhenius plot was a straight line at temperature below 43"C, and the activation energy for immobilized cells was calculated to be 12.36 kcal/mole. As shown in Fig. 3 decay of enzyme activity has been shown to be exponential with the time on stream rather than the volume on stream of substrate solution. Aspartase activity decayed in parallel in all sections of the column. The figure shows that this column is very stable and its half-life is approximately 120 days at 37°C. Of course, if the operation is carried out at lower temperature, stability should be increased. For purposes of reactor design, it is necessary to clarify the effect of column dimensions on the rate of formation of L-aspartic acid. In the case of immobilized aspartase prepared by entrapping the isolated enzyme into polyacrylamide gel lattice, we found previously that the rate of formation of L-aspartic acid increases with an increase of column length (Tosa et al., 1973). However, in the case of immobilized cells, the formation of L-aspartic acid proceeded at the same rate regardless of the column dimension. This

100

-

Labor,

80

fl

others

+J

P r o d u c t i o n of c a t a l y s t s and enzyme reaction

60 a,

>

.d

c,

2

40

a

M a t e r i a l s and substrates 20

0

l -

tional (batch)

Imnnobilized ( c o n ti nuous)

FIG.4. Comparison of relative cost for industrial production of L-aspartic acid

TRANSFORMATIONS BY IMMOBILIZED MICROBIAL CELLS

17

behavior is the same as that observed for the DEAE-Sephadex-aminoacylase column previously presented (Tosa et al., 1969). On the basis of this information, we designed a most efficient reactor system for continuous production of L-aspartic acid using immobilized E . coli cells. This new system has been operating industrially since 1973 in Tanabe Seiyaku Co. Ltd. A comparison of the costs for production of L-aspartic acid by the conventional batch process using intact cells and the continuous process using this immobilized cells is shown in Fig. 4. As a result, the overall production cost of this system is reduced to about 60%of the conventional batch process using intact cells because of the remarkable reduction in cost for the preparation of catalysts and of the reduction of labor cost by automation. This is considered to be the first industrial application of immobilized microbial cells in the world.

B . PRODUCTION OF L-MALIC ACID FROM FUMARICACID L-Mdic acid is used in pharmaceutical field as an antidote for hyperammoniemia and a component of amino acid infusion. This acid is industrially produced from fumaric acid by the action of fumarase in a batch process using microbial broth as follows. HOOC-CH4H-COOH

+ HzO--HOOC-CH-CHz-COOH fbmarase

OH Fumaric acid

L-Malic acid

For the continuous production of L-malic acid, we studied, by the polyacrylamide gel method, the immobilization of several microorganisms having high fumarase activity and found that Brevibacterium ammoniagenes was Yamamoto most active before and after immobilization (Chibata et al., 1975~;

et al., 1976, 1977). However, these immobilized cells formed succinic acid as a by-product, the separation of which from L-malic acid is very difficult. Therefore, the successful industrial production of pure L-malic acid rests on prevention of succinic acid formation during enzyme reaction. Thus, various treatments of intact or immobilized cells were carried out, and the treatment with detergents, such as deoxycholic acid, bile acid, and bile extract, was found to be very effective for suppression of succinic acid formation. The results are shown in Table VII. These detergents also strikingly enhanced the formation of L-malic acid by the immobilized cells. Readily available bile extract is considered to be the most suitable for industrial use. The most effective conditions were studied and determined as follows. The immobilized cells are allowed to stand in 1 M sodium fumarate (PH 7.5) containing 0.3%bile extract at 37°C for 20 hours.

18

ICHIRO CHIBATA AND TETSUYA TOSA

TABLE VII EFFECTOF DETERGENT TREATMENTS ON FORMATIONS OF L-MALIC ACID AND SUCCINICACID BY IMMOBILIZED Breoibacterium ammoniagenep

Treatmentb Control‘ No addition

CPC, 0.02% CPC, 0.16 SLS, 0.02 SL-10, 0.02 Triton X-100,0.20 Bile extract, 0.20 Bile acid, 0.20 Deoxycholic acid, 0.20

L-Malic acid (mmole/hour/gm of cells)

Succinic acid (mole % to Lmalic acid)d

0.49 0.99 4.57 3.07 6.05 1.22 5.36 7.48 6.57 7.38

2.5-5.0 2.5-5.0 1.0-2.5 1.0-2.5 2.5-5.0 >5.0 <0.2 <0.2 <0.2

“Chibataet al. (1975~); Yamamoto et al. (1976). *Stood at 37°C for 20 hours in 1 M sodium fumarate (pH 7.5) containing detergent. ‘Immediately after preparation of the gel. dEstimatedby ppc after the reaction reached equilibrium.

To confirm the superiority of immobilized cells to intact cells, we compared the stability of fumarase activity of immobilized cells and intact cells. The results are shown in Fig. 2. As in the case of E. coli aspartase, the fumarase activity of immobilized cells is more stable than that of intact ones. Therefore, immobilized cells are considered to be more advantageous than intact cells for industrial production of L-malic acid. Thus, to find out the most suitable conditions for continuous production of L-malic acid from fumaric acid by using the immobilized B . ammoniagenes, we investigated its enzymic properties. The results are shown in Table V. No difference was observed between optimum pH, optimum temperature, and heat stability of the intact and immobilized cells. Optimum conditions for the continuous production of L-malic acid were investigated in detail and were decided as shown in Table VI. When 1 M sodium fumarate solution (PH 7 . 0 )was passed through the column packed with bile extract-treated immobilized cells at 37°C at a space velocity of 0.23, the reaction reached to equilibrium with about 80% conversion of fumaric acid to L-malic acid. From the effluent, furmaric acid was removed by acidification, and L-malic acid was separated in about 70% yield from raw material, fumaric acid. The stability of fumarase activity of immobilized cell column at continuous enzyme reaction was investigated at various temperatures. From the graph

19

TRANSFORMATIONS BY IMMOBILIZED MICROBIAL CELLS

8

1.0

. 0.9

&

0

$

0.8

rp

u

$.I

0.7

4J

.rl

.% 0.6 4J

s

0.5

0

10 20 Operation period (day)

30

FIG. 5. Effect of temperature on stability of immobilized Brewibaderium ammoniugenes column. A solution of 1 M sodium fumarate (PH 7.0) was applied to immobilized B . ammoniagenes column at indicated temperature. Other conditions are given in the text.

of semilog plots of reaction rate and operational period, the decay of enzyme activity was found to be well expressed by the exponential relationship, 4 = exp (-&, 0), where 4 is the ratio of decay of activity, & is a deactivation rate constant, and 0 is time of operation. The & value of each temperature was estimated graphically, and a straight line was obtained between log & and 1/T, indicating that & is expressed in an Arrhenius-type equation, & = Ad * exp (-E,IRT), where Ad is a frequency factor, R is the gas constant, and Ed is a deactivation energy. From the least-square regression method the values o f & and E d were calculated to be 3788lday and 7741 cd/mole, respectively. The deactivation rate constant of the indicated temperature was calculated from the equation & = -3788 exp (-7741/RT), and the calculated decay lines of each temperature were drawn in Fig. 5. The plots of experiment were well coincident with the calculated line at each temperature. The half-life of fumarase activity of immobilized cell column was calculated to be 52.5 days at 37°C. It is concluded that 15.4 metric tons of theoretical yield of L-malic acid are produced for 1 month by using a 1000-liter volume column under the conditions described in Table VI. This production system has been industrially operated in Tanabe Seiyaku Co. Ltd. since 1974. We are satisfied with this system in both points of economical efficiency and the quality of the product.

c. PRODUCTION OF L-CITRULLINE FROM L-ARGININE L-Citrulline is used for medicines and is industrially produced from L-arginine by the action of microbial L-arginine deiminase in batch process as follows.

20

ICHIRO CHIBATA A N D TETSUYA TOSA

L-H,NCNHCH,CH,CH,CHCOOH I

It

+ H,O-L-H,NCONHCH~CH~CH~CHCOOH + NH3 ! L- Argmne

NHZ

NH


L-Ar@nine

N Hz L-Citrullme

In most cases, however, a part of L-citrulline formed is further converted to L-ornithine by the action of ornithine transcarbamylase. On the other hand, we found that Pseudomonus putida has a higher activity of L-arginine deiminase and no activity of ornithine transcarbamylase. The microorganism has been industrially used for the production of L-citrulline at Tanabe Seiyaku Co. Ltd. (Kakimoto et al., 1971). These procedures have been carried out in a batch process by incubating a mixture of substrate and fermentated broth. In order to produce L-citrulline more advantageously, the immobilization of P . putida was investigated, and its immobilization was carried out using the polyacrylamide gel method as in the case of the E . coli cells. Some of the enzymic properties of the immobilized cell L-arginine deiminase were investigated and compared with those of the intact cells (Table V). A marked difference was observed between the permeability of substrate or product through the cell wall for the intact and immobilized cells; i.e., formation of L-citrulline by the intact cells was scarcely observed in the absence of surfactant, cetyltrimethylammonium bromide, whereas formation occurred by the immobilized cells without the reagent. This phenomenon indicated that the cell wall of intact cells was a barrier for L-arginine or L-citrulline, and that the immobilization process of the cells possibly removed this barrier. As shown in Table V, the heat stability of L-arginine deiminase activity of P . putida cells was considerably increased by immobilization. The conditions for the continuous production of L-citrulline by using a column packed with the immobilized P . putida cells were investigated (Yamamotoet al., 1974a). As shown in Table VI, the reaction was found to be completed when 0.5 M L-arginine hydrochloride solution (PH 6.0) was passed through the column at 37°C at a space velocity of 0.26. From the effluent of the column, L-citrulline was easily obtained in good yield. This column is very stable, and the half-life is estimated to be 140 days at 37°C as shown in Table VI. This technique is considered to be more advantageous for the mass production of L-citrulline than the batch method using microbial broth.

D. PRODUCTION

OF

UROCANIC ACID FROM L-HISTIDINE

Urocanic acid is used as a sun-screening agent in the pharmaceutical and cosmetic fields; it is produced from L-histidine by the action of microbial L-histidine ammonia-lyase as follows:

21

TRANSFORMATIONS BY IMMOBILIZED MICROBIAL CELLS

H

c=c/ CH, CHCOOH I I I NHZ .NU L-

L-histidine ammonia-

-

HC=C

Histidine

,CH=CHCOOH

+

NH,

Urocanic acid

/

,CHZCH,COOH

Ho\C=C

I

urocanase

1

N+ ,NH C H Imidazolone propionic a c i d

The enzyme is widely distributed in many bacteria, and Achrornobacter liquidurn was found to be one of the most suitable enzyme sources for industrial production of urocanic acid (Shibatani et al., 1974). This enzymic process also has some disadvantages for industrial production of the acid, since the procedure is carried out by batch incubation of a mixture of L-histidine and intact cells. To develop a more efficient method, the continuous production of urocanic acid was investigated using immobilized microbial cells having high L-histidine ammonia-lyase activity. Several microorganisms having high enzyme activity were immobilized in a polyacrylamide gel lattice. Achrornobacter liquidurn was found to show the highest activity after immobilization. Although the organism had urocanase activity converting urocanic acid to imidazolone propionic acid, this undesired activity was removed by a simple heat treatment at 70°C for 30 minutes before immobilization of the cells (Shibatani et al., 1974). Enzymic properties of the immobilized A . liquidurn cells were compared with those of the intact cells, and the results are shown in Table V. Besides the results described in Table V, the permeability of substrate or product through the cell wall was found to be increased by immobilization of the cells as in the case of immobilized P . putida for the production of L-citrulline from L-arginine. By using a column packed with the immobilized A . liquidurn cells, the conditions for the continuous production of urocanic acid were investigated (Yamamotoet al., 1974b). As shown in Table VI, when an aqueous solution of 0.25 M L-histidine (PH 9.0) containing 1 mM M$+ was passed through the column at a space velocity of 0.06, L-histidine was completely converted to urocanic acid. From the column effluent pure urocanic acid was crystallized by merely adjusting the pH to 4.7 in good yield. The enzyme activity of the column is very stable in the presence of M$+, and its half-life is about 180 days at 37°C.

22

ICHIRO CHIBATA AND TETSUYA TOSA

This process is also more advantageous for the industrial production of urocanic acid than the batch process using extracted enzyme or microbial broth. E. PRODUCTION

OF 6-AMINOPENICILLANIC

ACID FROM

PENICILLIN 6-Aminopenicillanic acid (6-APA) is used as a starting materials for the synthetic penicillin, and industrially produced from penicillin by the action of penicillin amidase in batch method using microbial cells, extracted enzyme, or immobilized enzyme as follows. RCONHCH-~/S\C/CH’ I I I‘CH, CO-NC, H CmH Penicillin

+

H

H S H,NCH-C’ ,ccCH3 penicillin , O Z 1 I I CH, CO-N-C, H COOH

+

RCOOH

6-APA

Recently, many studies have been done on the continuous production of 6-APA by using a column packed with immobilized penicillin amidase, and some of the methods are said to be industrialized in the United States and Europe. However, in this case, it is necessary to extract the enzyme from microbial cells before the immobilization process, and in general the extracted penicillin amidase is unstable. Thus, to find out the more advantageous method, we investigated immobilization of E . coli cells having penicillin amidase activity by the polyacrylamide gel method. Enzymic properties of both intact and immobilized cells are shown in Table V. The results show that the heat stability of the enzyme increases by immobilization. By using a column packed with the immobilized E . coli cells, continuous production of 6-APA from penicillin G was investigated (Sato et al., 1976). These microbial cells contain penicillinase activity that decomposes both penicillin and 6-APA, and specific inactivation of penicillinase activity is very difficult. However, penicillinase activity is much lower than penicillin amidase activity. Therefore, optimum conditions for the continuous production of 6-APA without removing the penicillinase activity can be chosen as shown in Table VI; i.e., when 0.05 M penicillin G solution (PH 8.5) was passed through the immobilized E . coli cell column at 40°C at a space velocity of 0.24, 6-APA was efficiently produced. From the column effluent 6-APA was obtained in about 80% yield. Therefore, this technique is considered to be useful for the industrial production of 6-APA as well as the continuous method using immobilized penicillin amidase.

TRANSFORMATIONS BY IMMOBILIZED MICROBIAL CELLS

23

F. OTHERPRODUCTION OF USEFULCOMPOUNDS Besides the studies described above, as shown in Table I we have further studied the continuous production of useful compounds by employing immobilized microbial cells, and we have applied for patents on these studies as follows. l n these studies, microbial cells can be effectively immobilized by the polyacrylamide method as in the case of E. coli cells having aspartase activity. Immobilized S . griseus cells having glucose isomerase activity are used for continuous conversion of D-glucose to D-fructose (Chibata et al., 1974d). In this system, M?+ is necessary for the activation of enzyme reaction and Co2+ is useful for the stabilization of enzyme activity. Immobilized E . coli cells having tryptophan synthetase activity are used for productions of L-tryptophan from indole and DL-serine, and of 5-hydroxy-~-tryptophan from 5-hydroxyindole and DL-Serine, respectively (Chibata et al., 1974a,b). Immobilized Achromobacter aceris cells having NAD kinase activity are used for continuous production of NADP &om NAD and ATP in the presence of Mn2+ or M$+ as an enzyme activator (Chibata et al., 1975a). As a last example, immobilized Pseudomonas dacunhae cells containing L-aspartate 4-decarboxylase activity are used for continuous production of L-alanine from L-aspartic acid (Chibata et al., 1975b). In this system, additions of pyridoxal phosphate as a cofactor for enzyme reaction and of Co2+as an enzyme stabilizer to substrate solution are effective. Some of these techniques are expected to be industrialized in the near future. In addition to these studies, a number of interesting papers and patents have been published for the production of useful compounds as shown in Table I. Among these papers, most have been published on the isomerization of D-glucose to D-fructose by immobilized microbial cells having glucose isomerase activity. As the industrial isomerization of D-glucose to D-fructose by immobilized enzyme has been carried out in Japan, in the United States, and in Europe, this immobilized microbial cell system can be expected to compete with the immobilized enzyme system in the near future. As advantageous production of penicillin and cephalosporin is greatly wanted in the world, the application of immobilized microbial cells having penicillin amidase activity is useful for industrial purposes. Already a few papers on this have been published, as shown in Table I. This system also will compete with the immobilized enzyme system. Immobilized penicillin amidase has been industrially used for production of 6-aminopenicillanic acid. Regarding the production of L-amino acids, Yamada et al. (1975) and Kumagaya et al. (1976) reported the production of L-tyrosine from phenol, pyruvate, and ammonia, and of d o p a from pyrocatechol, pyruvate, and

24

ICHIRO CHIBATA A N D TETSUYA TOSA

ammonia, respectively, by the action of immobilized Erwinia herbicola cells having P-tyrosinase activity. As described in first section of this review, we succeeded in the industrial application of immobilized aminoacylase for continuous production of L-amino acids from acyl-DL-amino acids. Instead of this system, Hirano et al. (1975) immobilized Aspergillus ochraceus cells having aminoacylase activity by cross-linking with egg albumin and glutaraldehyde, and tested the continuous optical resolution of acetyl-DL-methionine by using this immobilized mold. A number of papers on the immobilization of P-galactosidase and its applications have been published. As microbial cells having this enzyme activity are also immobilized as shown in Table I, this system also will compete with immobilized enzyme system. Recently, Martin and Perlman (1976) immobilized Gluconobacter melanugenus cells by the polyacrylamide gel method and applied it for continuous conversion of L-sorbose to L-sorbosone. In this study, they observed a very interesting phenomenon: the addition of antibiotics such as neomycin, ampicillin, chloramphenicol, or tetracycline increases the stability of the enzyme activity. Current and potential industrial applications described above for continuous enzyme reaction using immobilized microbial cells are mainly carried out by the action of a single enzyme. However, many useful compounds are usually produced, especially in fermentative methods, by the action of several kinds of enzymes. The immobilization of microbial cells for multienzyme reaction has been attempted, as shown at the bottom of Table I. AS described above, if the immobilization of microbial cells can be developed, energy generation and oxidation-reduction reactions can be eEciently and easily carried out. Immobilized microbial systems are expected to become highly advantageous bioreactors or catalysts for the industrial production of many useful chemical compounds, such as steroids, antibiotics, peptides, nucleic acids, and coenzymes.

IV. Conclusion On the basis of our experience in industrializing one immobilized enzyme system and two immobilized microbial cell systems, we consider that the utilization of immobilized microbial cells for continuous enzyme reaction has some advantages as follows. 1. Processes for extraction andlor purification of enzyme are not necessary. 2. Yield of enzyme activity on immobilization is high. 3. Operational stability of immobilized cells is generally high. 4. Cost of enzyme is low. Further, we consider that the enzyme reactions by immobilized microbial cells are promising and advantageous in the following cases: (a) when en-

TRANSFORMATIONS BY IMMOBILIZED MICROBIAL CELL5

25

zymes are intracellular; (b) when enzymes extracted from the cells are unstable; (c) when enzymes are unstable during and after immobilization; (d) when the microorganism contains no interfering enzymes; (e)when interfering enzymes are readily inactivated or removed; (f) when substrates and products are not higher molecular compounds. Further, the volume of fermentation broth for the unit production of the desired compound is much smaller in the case of the continuous method using immobilized cells than in the case of the conventional fermentation method. This fact indicates that the former method is very advantageous with respect to water pollution in plant. As this new technique is very efficient and superior to the conventional fermentative and enzymic methods in certain cases, it will be the subject of increased interest in the fermentative industry. Therefore, in future, the studies on the immobilized microbial cells will be developed extensively as well as those on immobilized enzymes. REFERENCES Brownstein, A. M., Vieth, W. R., and Constantinides, A. (1974). Symp., 168th Meet., Am. Chem. Soc., Atlantic City Industrial and Engineering Chemistry Division. Chibata, I., Tosa, T., Sato, T., Mori, T., and Matuo, Y. (1972).In “Fermentation Technology Today” (G. Terui, ed.), pp. 383389. Soc. Ferment. Technol., Osaka, Japan. Chibata, I., Tosa, T., Sato, T., Mori, T., and Yamamoto, K. (1973).Enzyme Eng. 2, 303313. Chibata, I., Kakimoto, T., and Nabe, K. (1974a). Japanese Patent Kokai 81,590/74. Chibata, I., Kakimoto, T., and Nabe, K. (1974b). Japanese Patent Kokai 81,591/74. Chibata, I., Tosa, T., and Mori, T. (1974~).Japanese Patent Kokai 30,582/74. Chibata, I., Tosa, T., and Sato, T. (1974d). Japanese Patent Kokai 132,290/74. Chibata, I., Tosa, T., and Sato, T. (1974e).Appl. Microbiol. 27, 878885. Chibata, I., Kato, J., Watanabe, T., and Uchida, T. (1975a). Japanese Patent Kokai 135,290/75. Chibata, I., Tosa, T., Sato, T., and Yamamoto, K. (1975b). Japanese Patent Kokai 100,289/75. Chibata, I., Tosa, T., and Yamamoto, K. (1975~).Enzyme Eng. 3 (in press). Dinelli, D. (1972). Process Biochem. August 9-12. Franks, N. E. (1971). Biochim. Biophys. Acta 252, 246-254. Fujii, T., Hanamitsu, K., Izumi, R., Yamada, T., and Watanabe, T. (1973). Japanese Patent Kokai 99,393/73. Fujii, T., Matsumoto, K., Shibuya, Y., Hanamitsu, K., Yamaguchi, T., Watanabe, T., and Abe, S. (1974). British Patent 1,347,665. Hackel, U., Klein, J., Megnet, R., and Wagner, F. (1975).Eur. J. Appl. Microbiol. 1,291-293. Hattori, T., and Furusaka, C. (1960).J. Biochem. (Tokyo) 48, 831437. Hattori, T., and Furusaka, C. (1961). J. Biochem. (Tokyo) 50, 312315. Hirano, K., Karube, I., Otani, K., and Suzuki, S. (1975).Annu. Meet. Soc. Ferment. Technol.. Jpn., Osaka p. 249. Ishimatsu, Y., Shigesada, S., and Kimura, S. (1976). Japanese Patent Kokai 86,142/76. Johnson, D. E., and Ciegler, A. (1969). Arch. Biochem. Biophys. 130, 384388. Kakimoto, T., Shibatani, T., Nishimura, N., and Chibata, I. (1971). Appl. Microbiol. 22, 992-999. Kanemitsu, 0. (1975). Japanese Patent Kokai 132,181/75.

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Karube, I., Matsunaga, K., Suzuki, S., Tsuru, S., and Sugama, S . (1975).Annu. Meet. Soc. Ferment. Technol., Jpn., Osaka p. 25. Kinoshita, S., Muranaka, M., and Okada, H. (1975).J . Ferment. Technol. 53, 223-229. Kolarik, M. J., Chen, B. J., Emery, A. H., Jr., and Lim, H. C. (1974). In “Immobilized Enzymes in Food and Microbiol Process” (A. C. Olson and C. L. Cooney, eds.), pp. 71-83. Plenum, New York. Kosugi, Y., and Suzuki, H. (1973).J . Ferment. Technol. 51, 895-903. Kumagaya, H., Sezima, S., Yamada, H., Hino, T., and Okamura, S. (1976).Annu. Meet. Agric. Chem. Soc. Jpn., Kyoto p. 233. Marconi, W., Bartoli, F., Cerere, F., Galli, G., and Morisi, F. (1975).Agric. Biol. Chem. 39, 277-279. Martin, C . K. A., and Perlman, D. (1976).Biotechnol. Bioeng. 18, 217-237. Miyata, N., Kikuchi, T., and Furuichi, E. (1975).Annu. Meet. Agric. Chem. Soc. Jpn., Sapporo p. 58. Mohan, R. R., and Li, N. N. (1975). Biotechnol. Bioeng. 17, 1137-1156. Mosbach, K . , and Larsson, P. 0. (1970). Biotechnol. Bioeng. 12, 19-27. Mosbach, K., and Mosbach, R. (1966).Acta Chern. Scand. 20,2807-2810. Nanba, A., and Matuo, Y. (1970).Annu. Meet. Agric. Chern. Soc. J p n . , Fukuoka p. 251. Nishimaru, H., Izumi, C., Narita, S . , and Yamada, K. (1975). Japanese Patent Kokai 140,680/ 75. Nonomura, S., Watanabe, M., and Inagaki, T. (1976). Japanese Patent Kokai 48,488/76. Novo Industri. (1976a). Japanese Patent Kokai 51,580/76. Novo Industri. (197613). U.S. Patent Appl. 501,292. S d , S. R . , Tani, Y., and Ogata, K. (1975).J . Ferment. Technol. 53, 380385. Sato, T., Mori, T., Tosa, T., Chibata, I., Furui, M., Yamashita, K., and Sumi, A. (1975). Biotechnol. Bioeng. 17, 1797-1804. Sato, T., Tosa, T., and Chibata I. (1976). Eur. J. Appl. Microbiol. 2, 153-160. Shibatani, T., Nishimura, N., Nabe, K., Kakimoto, T., and Chibata, I. (1974).Appl. Microbiol. 27, 688-694. Shimizu, S., Morioka, H., Tani, Y., and Ogata, K. (1975).J . Ferment. Technol. 53, 77-83. Slowinski, W., and Charm, S. E. (1973).Biotechnol. Bioeng. 15, 973-979. Takasaki, Y., and Kanbayashi, A. (1969).Kogyo Gijutsuin Biseibutsu Kogyo Gijutsu Kenkyusho Kenkyu Hokoku 37, 31-37; C.A. 74, 139538 (1971). Toda, K., and Shoda, M. (1975). Biotechnol. Bioeng. 17, 481497. Tosa, T., Mori, T., Fuse, N., and Chibata, I. (1966). Enzymologia 31, 214-224. Tosa, T., Mori, T., Fuse, N., and Chibata, I. (1967).Biotechnol. Bioeng. 9, 603415. Tosa, T., Mori, T., Fuse, N., and Chibata, I. (1969).Agric. Biol. Chem. 33, 1047-1052. Tosa, T., Sato, T., Mori, T., Matuo, Y., and Chibata, I. (1973).Biotechnol. Bioeng. 15, 6 9 4 4 . Tosa, T., Sato, T., Mori, T., and Chibata, I. (1974).Appl. Microbiol. 27, 8864389. Tosa, T., Sato, T., Nishida, Y., and Chibata, I. (1975).Annu. Meet. Agric. Chem. SOC. Jpn., Sapporo p. 323. Tsumura, S . (1969).Annu. Meet. Soc. Ferment. Technol. Jpn., Osaka p. 81. Tsumura, S., Kasumi, T., Ishikawa, M., and Ozawa, 0. (1976).Annu. Meet. Agric. Chem. Soc. J p n . , Kyoto p. 383. Updike, S. J., Harris, D. R., and Shrago, E. (1969).Nature (London) 224, 1122-1123. Venkatasubramanian, K., Saini, R., and Vieth, W. R. (1974).J. Ferment. Technol. 52,268-278. Vieth, W. R . , Wang, S. S., and Saini, R. (1973). Biotechnol. Bioeng. 15, 565-569. Wang, S. S . , Vieth, W. R., and Constantinides, A. (1973).Enzyme Eng. 2, 123-129. Yagi, S, Toda, Y., and Minoda, T. (1976).Annu. Meet. Agric. C h a . SOC. J p n . , Kyoto p. 414. Yamada, H., Yamada, M., Nagazawa, E., Kumagaya, H., Hino, T., and Okamura, S. (1975). Annu. Meet. Agric. Chem. Soc. Jpn., Sapporo p. 336.

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Yamamoto, K . , Sato, T., Tosa, T., and Chibata, I. (1974a).Biotechnol. Bioeng. 16, 1589-1599. Yamamoto, K . , Sato, T., Tosa, T., and Chibata, I. (1974b). Biotechnol. Bioeng. 16, 1601-1610. Yamamoto, K . , Tosa, T., Yamashita, K . , and Chibata, I. (1976). Eur. /. A p p l . Microbiol. 3, 169-183. Yamamoto, K . , Tosa, T., Yamashita, K . , and Chibata, I. (1977). Biotechnol. Bioeng. (in press). Yuta, S., Bhatt, R. R . , Yoshida, T., and Taguchi, K . (1975).Annu. Meet. SOC. Ferment. Technol. / p n . . Osaka p. 250.

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Microbial Cleavage of Sterol Side Chains CHRISTOPH K. A. MARTIN Gesellschaft fur Biotechnologische Forschung mbH, Braunschweig-Stockheim, West G m n y I. Introduction . . . . .

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

11. Complete Microbi adation of Sterols . . . . . . . . . . . . . . . . 111. Mechanism of Microbial Sterol Metabolism . . . .....

A. Degradation Pathway of the Steroid Nucleus . . . . . . . . . . . B. Mechanism of Sterol Side-Chain Degradation . . . . . . . . . . IV. Selective Side-Chain Cleavage of Sterols . . . . . . . . . . . . . . . . . . A. Conversion of Sterols with Modified Structure.. . . . . . . . . B. Conversion of Sterols in the Presence of E Inhibitors ...................... C. Sterol Conversion by Selected Mutants . . . . . . . . . . . . . . . . V. Substrate Addition and Isolation of Fermentation Products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI. Summary and Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

29

30 33 33 35 36 37 44

50 52

54 54

I. Introduction For the synthesis and production of pharmacologically active steroids (e.g., contraceptives, corticosteroids, and geriatric drugs) in principle two methods can be employed: the chemical total synthesis starting with precursors of the cyclopentanoperhydrophenanthrene structure or the partial synthesis from naturally occurring steroids. Since the steroid molecule contains numerous asymmetric centers the total synthesis represents a difficult task.As primary raw material for producing steroid drugs by partial synthesis serves diosgenin from Dioscorea spp., stigmasterol and cholesterol from plant and animal sources, respectively (Petrow, 1969; Wiechert, 1970). As a result of a shortage of diosgenin the conversion of plant sterols (phytosterols), such as soybean sterols (p-sitosterol and congeners admixed with substantial amounts of stigmasterol) or tall oil sterols [about 17 different compounds, of which sitosterol and campesterol comprise about 85%(Conner and Rowe, 1975)]has attracted much attention, and several useful processes have been developed recently. Conventional chemical oxidation of the saturated aliphatic (2-17 side chain results in very low yields (Fieser and Fieser, 1959; Mukhina, 1971). However, using remote oxidation the removal of the cholesterol side chain proceeds rather efficiently (Snider et al., 1975). For the chemical oxidation of unsaturated sterol side chains there exist few methods. The Upjohn Company uses stigmasterol as starting material for the production of progesterone. 4,4,14-Trimethylsteroids containing a C-17 or C-20 keto function can be prepared by the oxidative cleavage of the lanosterol side chain (Habermehl and Volkwein, 1970; Kreiser and Ulrich, 1976). 29

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CHRISTOPH K . A . MARTIN

As alternative to chemical methods, the microbial removal of the aliphatic side chain of phytosterols offers a promising method for use of these sterols and has been studied for some time. More recently, processes for the commercial conversion of sterols to C19-steroids by microorganisms have been developed (Anonymous, 1975). Although numerous reviews on the transformation of steroids by microorganisms covering partly also the microbial side chain cleavage and transformation of sterols have been published (e.g., Charney and Herzog, 1967; Iizuka and Naito, 1967; Sih and Whitlock, 1968; Marsheck, 1971; Vezina et al., 1971; Beukers et al., 1972; Takahashi, 1972; Fonken and Johnson, 1972; Smith, 1974; Heftmann, 1975), a more comprehensive discussion of this topic seems to be necessary. It is the purpose of this review to summarize the data of the microbial conversion of sterols, especially the selective C-17 side-chain cleavage yielding 17-ketosteroids useful for pharmaceutical application.

11. Complete Microbial Degradation of Sterols Since Sohngen’s observation in 1913 that mycobacteria are capable of decomposing cholesterol as sole source of carbon numerous studies on the microbial breakdown of sterols have been published (Haag, 1927; Tak, 1942; Turfitt, 1944, 1947; Schatz et al., 1949; Sobel and Plaut, 1949; Mueller et al., 1951; Curran and Brewster, 1952; Peterson et al., 1962a; Toulmin, 1962; Imshenetskii et al., 1968; Zhukova and Kozlova, 1968, 1970; Kozlova and Fonina, 1971, 1972; Voets and Lamot, 1974; Kozlova et al., 1976). In systematic studies on the cholesterol decomposing activity of 1589 microbial strains Arima et al. (1969) observed complete degradation of cholesterol in the genera Arthrobacter, Corynebacterium, M ycobacterium, Nocardia, Streptomyces, Brevibacterium, Bacillus, Protaminobacter, Serratia, and Microbacterium. A screening method for “anticholesterol substances” which could also be used for the selection of microorganisms capable of transforming sterols has been published by Fukuda and collaborators (1973). This screening method was based on the antagonism between polyene antifungal agents and cholesterol against yeast. The presence of compounds converting the sterols resulted in an inhibition zone in agar medium seeded with Candida sp. Close correlation between respiration rate and cholesterol degradation in Pseudomonas boreopolis (Imshenetskii and Mavrina, 197.213) and Mycobacterium rubrum (Imshenetskii and Mavrina, 1972a) has been reported. In most studies, evidence for the degradation of sterols by microorganisms was based upon the detection of these materials in organic solvent extracts of culture broth. However, since after exposure to the cells the remaining cholesterol concentration was determined usually with color reagents specif-

MICROBIAL CLEAVAGE OF STEROL SIDE CHAINS

31

ically reacting with the 3/3-hydroxy-A5-structure (e.g., LiebermannBurchard reaction), the disappearance of cholesterol did not mean necessarily complete decomposition but could have been the result of conversion of the substrate by the microorganisms into 4-cholesten-3-one or related metabolites. In addition, several investigators studying the uptake of cholesterol by cell suspensions of some microorganisms (Hartman and Holmlund, 1960; Smith and Rothblat, 1960; Gubler et al., 1961; Peterson et aZ., 196213) noted that these strains modified the solubility of cholesterol during incubation, thus preventing its extraction by organic solvents. The enzymes responsible for the primary attack and further breakdown of cholesterol were inducible; only in a few strains constitutive formation has also been reported (Davis et al., 1962; Peterson and Davis, 1964; Kozlova and Fonina, 1971). Although Stadtman and collaborators (1954), utilizing 14C-labeledcholesterol, found that a soil Mycobacterium oxidized C 4 of the steroid ring to C 0 2 about four times more rapidly than C-26, contrary data were obtained for a streptomycete (Peterson and Davis, 1964). These results suggested that there are two points of primary attack on the sterol molecule. Furthermore, these initial reactions occurred either simultaneously or independently. In order to elucidate the mechanism of the microbial sterol metabolism and to isolate the enzymes involved in the oxidative breakdown, several research groups prepared cell-free extracts of cholesterol decomposing organisms. Stadtman et al. (1954) observed that cell-free extracts of their Mycobacterium sp. contained “cholesterol dehydrogenase” that catalyzes the conversion of cholesterol to 4-cholesten-3-one. Cell-free extracts of various different M ycobacteria and of Achromobacter candidans were actively decomposing cholesterol (Imshenetskii et al., 1973, 1975a; Chipley et al., 1975). Again, one of the main metabolites was identified as 4cholesten-3-one. Cell-free extracts from Nocardia erythropolis were capable of decomposing cholesterol in human serum. The authors (Geier and LaPolla, 1974) discussed the possibility of in oivo application of this enzyme preparation for prevention and treatment of atherosclerosis. Although the active enzymes in these cell-free extracts have not been identified and purified, it seems very likely that these preparations contained “cholesterol oxidase,” the enzyme responsible for the oxygendependent conversion of cholesterol to 4-cholesten-3-one. This enzyme is now produced on a commercial scale from various microorganisms capable of using cholesterol as sole source of carbon (Smith and Brooks, 1976). Its main application is the determination of cholesterol in serum. Although the most important aspects of the pathway and mechanism of steroid oxidation will be discussed in Section 111, we will review here some early observations on the metabolism of sterols by microorganisms.

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CHRlSTOPH K. A . MARTIN

The metabolite that had been detected most frequently was found to be 4-cholesten-3-one (Amaudi, 1954; Stadtman et al., 1954; Arima et al., 1969; Zanin, 1968; Efimochkina et al., 1970; Imshenetskii et a l . , 1975a). Thus, oxidation of the 3P-hydroxy function and isomerization of the A5-double bond were thought to be necessary enzymic steps in the ring fission of the steroid nucleus. In addition to this compound, Loomeijer (1958) isolated several cholesterol metabolites containing more than one carbonyl group. Davis and coworkers (1964) and Brown and Peterson (1966) were able to detect trace amounts of 4-cholesten-4-01-3-one in cholesterol-containingcultures of a soil streptomycete and postulated the following degradation pathway: cholesterol + 4-cholesten3-one -+ 4-cholesten-4-01-3-one + * COz. Horvath and Kramli (1947) reported that a species of Axotobacter was capable of oxidizing cholesterol to 4-cholesten-3-one, 5,7-cholestadien3/3-01, and methylheptanone, presumably by the fission of the C-17-C-20 bond. However, the counterpart steroidal component was not isolated. In studies with Proactinomyces sp. they isolated 7-hydroxycholesterol (Kramli and Horvath, 1949), a compound also detected by Loomeijer (1958) but thought to be an autoxidation product of cholesterol. From a prolonged large-scale incubation of 4-cholesten-&one as sole carbon source with Proactinomyces erythropolis Turfitt (1948) isolated in very small yields 3-keto-4-androstene-17P-carboxylicacid, isocaproic acid, and A-nor-3,5-secocholestan-S-on-3-oic acid (Windaus’ keto acid). This latter compound was also isolated from cultures of Mycobacterium sp. in the presence of cholesterol (Stadtman et al., 1954), in addition to 4-cholestene-3, 6-dione and several other unidentified oxygenated metabolites. Recently, Wada and Ishida (1974) reported the biotransformation of cholesterol by Coriolus hirstus. Trace amounts of GP-hydroxy4-cholesten-3one, 6a-hydroxy-4-cholesten-3-one, 3P-hydroxy-5-cholesten-7-one, 5-cholesten-3-/3,7a-diol, and 5-cholesten-3/3,7@-diolwere found. In experiments with a “halo-forming’’streptomycete, Davis et al. (1962) obtained evidence that 4-cholesten3-one, cholic acid, and deoxycholic acid were intermediates of cholesterol degradation. Schubert and co-workers (Schubert and Kaufmann, 1965; Schubert et al., 1968) noted that Mycobacterium smegmatis accumulated cholesterol in the cells when cholesterol was added to a cell suspension. Simultaneously, the oxidative degradation of cholesterol to carbon dioxide took place. The accumulated sterol consisted of Sa-cholestan3P-ol and a mixture of various esters of cholesterol and fatty acids (in a total yield of up to 25%).The fatty acid patterns of the bacterial lipids and of the sterol esters formed were approximately in agreement. With growing cells, two more esters were formed by M . smegmatis which proved to be a succinate of cholesterol with 26-hydroxy-4-cholesten-3-oneor 26-hydroxy-1,4-cholestadien-3-one.

MICROBIAL CLEAVAGE OF STEROL SIDE CHAINS

33

w-Hydroxylated 4-cholesten-3-one was isolated also more recently. Zaretskaya et al. (1968) reported the transformation of cholesterol to 27hydroxy-4-cholesten3-one by Mycobacterium sp. Galli-Kienle et al. (1973) by found a conversion of cholesterol to (25S)-26-hydroxy4-cholesten-3-one M . smegmu tis .

Ill. Mechanism of Microbial Sterol Metabolism

As discussed in Section 11, a variety of microorganisms are capable of utilizing the cyclopentanoperhydrophenanthrene nucleus as well as the aliphatic side chain of sterols as sole source of carbon and are capable of oxidizing it completely to carbon dioxide and water. Since the detailed mechanism of steroid oxidation was reviewed very frequently (e.g., Charney and Herzog, 1967; Sih and Whitlock, 1968; Marsheck, 1971; Smith, 1974), we will discuss this topic very briefly, emphasizing the enzymic steps involved in the conversion of sterols to 17ketosteroids. A. DEGRADATION PATHWAYOF

THE

STEROIDNUCLEUS

The first report of the microbial fission of the steroid ring system was that of T u d t t (1948), who showed the formation of A-nor-3,5-secocholestan5-on-3-oic acid as a product of 4-cholesten-%one metabolism by Proactinomyces erythropolis. Taken together with Stadtman’s observation that carbon atom 4 was rapidly lost when cholesterol was exposed to Mycobacterium sp., these results would seem to indicate that enzymic cleavage of the steroid ring A occurs between either C-3 and C 4 , or C-4 and C-5. However, in subsequent studies such intermediates of steroid ring oxidation have been observed only in few instances, the conversion of eburicoic acid by Glomerella fusarioides (Laskin et al., 1964) and the catabolism of 4-hydroxy-4-cholesten3-oneby Nocardia restrictus or Nocardia corallina (Lefebvre et al., 1974). Through the combined efforts of Dodson and Muir, Sih, Schubert, Tdalay, and their collaborators the general pathway whereby steroid degradation occurs among organisms of the genera Nocardia, Pseudomonus, Mycobacterium, Bacterium, and Arthrobacter has been elucidated completely. As is illustrated in Fig. 1, sterols containing the 3/3-hydroxy-A5 configuration (I) are oxidized first to the corresponding 3-keto-A4 compounds (11). While in some instances the enzymes involved in this step are NAD-dependent dehydrogenases, especially in the oxidation of sterols the enzymes are true oxidases, requiring only molecular oxygen for their action (Smith and Brooks, 1976). A great variety of different sterols are substrates for this enzyme; however, the length of the C-17 side chain is an

34

CHRlSTOPH K . A . MARTIN

FIG. 1. Microbial degradation of the steroid nucleus.

important factor in determining the reaction rate. 4,4-Dimethylsterols are not oxidized; therefore, lanosterol has to be decomposed by a different mechanism. The primary oxidation of the 3P-hydroxy function is followed by the isomerization of the A5-double bond, a reaction which has been shown to be catalyzed by various cholesterol oxidases, too. However, this isomerization can also occur nonenzymically. References to original papers or reviews on the subsequent enzymic steps in the fission of the steroid ring system are cited in the comprehensive report by Smith (1974). Only two more enzymic reactions in the oxidative breakdown will be discussed here. Depending on the organism studied, the further metabolism of 3keto-A4 compounds involves 9a-hydroxylation followed by Cl(2)-dehydrogenation or vice versa. The resulting metabolite (V) undergoes simultaneous aromatization with the cleavage of ring B via a nonenzymic reverse-aldol type reaction to produce a 9,lO-secophenolic derivative (VI). The stereochemistry of the C-l(2)-dehydrogenation process has been studied in Bacillus sphaericus (Ringold et a l . , 1963), Nocardia corallina (Holmlund et a l . , 1962), Septornyxa affinis (Abul-Hajj, 1972), and Mycobac-

MICROBIAL CLEAVAGE OF STEROL SIDE CHAINS

35

terium phlei (Phillips and Ross, 1974). It was shown that the mechanism proceeds via a trans diaxial loss of the la,2@-hydrogens. Studies of the second enzyme acting at the early state of ring degradation, the 9a-hydroxylase, have been carried out by Sih and co-workers (Chang and Sih, 1964) and more recently by Strijewski and Wagner (1976). Although the enzyme, a monoxygenase, has not been purified completely, it has been shown to consist of several proteins forming an electron transport chain. In contrast to other microbial steroid hydroxylases the 9a-hydroxylase of Nocardia sp. apparently does not contain cytochrome P-450. Complexing agents for ferrous ions were shown to inhibit the reaction. Among the substrates tested, 4-androstene-3,17-dione, 4-pregnene-3,2O-dione were good substrates for the 9a-hydroxylase whereas cholesterol and 4-cholesten-3-on were not hydroxylated.

B. MECHANISMOF STEROL SIDE-CHAINDEGRADATION In addition to Turfitt’s (1948) report of the formation of 3keto-4-androstene-17~-carboxylic acid in cultures of Proactinomyces erythropolis first indications on the metabolism of the sterol side chain came from Whitmarsh (1964), who reported that cholesterol was degraded in poor yield by Nocardia sp. into 3-0~0-23,24-dinor-4-cholenic acid, 3-0~0-23,24dinor-1,4-choladienic acid, 4-androstene-3,17-dione, and 1,4-androstadiene3,17-dione. In a series of brilliant experiments Sih and his collaborators (1967a,b, 1968a,b; Lee, 1967) completely elucidated the pathway whereby the C-17 sterol side chain is degraded during the microbial degradation of cholesterol to 17-ketosteroids. In contrast to mammalian systems where 17-ketosteroids are formed via cleavage of the C-20 - C-22 bond followed by cleavage of the C-17 - C-20 bond microorganisms of the genera Nocardia, Arthrobacter, Mycobacterium, and Corynebacterium appear to shorten the side chain by a mechanism similar to the @oxidation of fatty acids (Fig. 2). Following C27-hydroxylation which has been shown to occur in certain Mycobacteriaas was discussed earlier-and presumably oxidation of the resulting alcohol to a C-27 carboyxlic acid subsequently propionic acid, acetic acid, and again propionic acid are removed, thus resulting in the formation of C-24 (11) and C-22 (111)carboxylic acids as intermediates and finally in the formation of the C-17-keto function (IV). Since the conversion of the dinorcholanic acid derivative (111) to 17-keto compounds took place also under anaerobic conditions, it seemed likely that this last step involved dehydrogenation and addition of water followed by aldolytic cleavage to yield the 17-keto function and propionic acid (Sih et a l . , 1968a).

36

CHRISTOPH K. A. MARTIN

+CH,-CH,

-COOH

FIG. 2. Mechanism of microbial side-chain cleavage of sterols.

Experiments supporting the pathway proposed by Slh and co-workers were reported by Hayakawa et al. (I%%), who showed that the C-24 carboxylic acid side chain of cholic acid is degraded by Streptomyces rubescens and Cwynebacterium equi via the C-22 carboxylic acid to the 17-keto compound. Furthermore, Lefebvre et al. (1974) observed the liberation of propionic acid in cultures of Nocardia restrictus or Nocardia cwallina containing 4-hydroxy4-cholesten-3-oneas substrate. At the end of this chapter it should be emphasized that the enzymic reactions involved in the side-chain degradation and ring cleavage do not follow a compulsory order but occur simultaneously and independently. Thus, if the structure of the side chain is modified so that the enzymes normally involved in the degradation are unable to catalyze the fission, the ring system will be attacked resulting in accumulation of metabolites with partly oxidized ring structure. On the contrary, in substrates with modified ring structure blocking the C-1 (2)-dehydrogenationor 9a-hydroxylation the side chain will be degraded resulting in the formation of 17-ketosteroids.

IV. Selective Side-Chain Cleavage of Sterols As discussed in Section I, cholesterol and phytosterols (p-sitosterol, campesterol, stigmasterol, ergosterol) have long been considered to be potential starting materials for the synthesis of steroid hormones as they are available in large quantities at very low cost. Therefore, if an organism could partially decompose the sterol by selectively cleaving the C-17 side chain without degrading the steroid nucleus, such a process might have great commercial

MICROBIAL CLEAVAGE OF STEROL SIDE CHAINS

37

application. Since extensive screening programs in several laboratories were not successful, it appeared that such organisms are very rare. So, in order to cleave the side chain selectively, methods for the inhibition of the ring degradation were developed. C-l(2)-dehydrogenation and 9a-hydroxylation are essential enzymic reactions in the breakdown of the steroidal nucleus. In principle, three different methods have been employed to inhibit one or both of the enzymes involved: (1) structural modification of the substrates, thus preventing the enzymic attack of the ring system; (2)conversion of unmodified substrates in the presence of compounds inhibiting the enzymic mechanism of the C-1 (2)-dehydrogenation or 9a-hydroxylation; (3)mutation of organisms capable of decomposing the sterols completely in order to inactivate the C-1(2)dehydrogenase andlor 9a-hydroxylase. In the following sections, these various methods will be discussed separately. A. CONVERSION OF STEROLSWITH MODIFIED STRUCTURE Dodson and Muir (1961)showed that 19-hydroxy-4-androstene-3,17-dione was converted into estrone readily by Pseudomonas sp. Sih and Rahim (1963) observed the same reaction using Nocardia restrictus ATCC 14,887. Since the estrone formed was not further metabolized by this organism in contrast to 4-androstene-3,17-dioneor cholesterol it became clear to Sih and Wang (1965) that it should be possible to aromatize a 19-nor or 19hydroxysterol with the concomitant degradation of the side chain to a 17ketone. Proving this hypothesis, they prepared 19-hydroxy-4-cholesten-3one (I) and incubated this compound with Nocardia restrictus ATCC 14,887 (Fig. 3). An 8% yield of estrone (111)was obtained (240 hours). Using Nocardia sp. CSD-10 (later named Nocardia sp. ATCC 19,170),a soil isolate using cholesterol as sole carbon source, the same substrate was converted into estrone in 30% yield. Similarly, 19-hydroxy4-stigmasten-3-one (11)was also converted into estrone by Nocardia sp. ATCC 19,170 in 10% yield. No complete degradation of the substrates took place in these fermentations since practically all the steroidal material was recovered, mainly unmetabolized. Taken together with the observation that 4-cholesten-3-one is not only a poor inducer but also a poor substrate for the 9a-hydroxylase when compared to 4-androstene-3,17-dione Sih and Wang concluded that the major pathway of sterol metabolism among microorganisms begins with the side chain degradation. Reasoning that most organisms capable of degrading cholesterol completely contain enzymes which are capable of (a) hydrolyzing acetoxyl func-

38

CHRlSTOPH K. A . MARTIN

/

(n) R=-CH,-CH,

i

%fP& I

I

FIG.3. Conversion of 19-hydroxysterols to estrone by Nocardia spp.

tion at C 3 , (b) converting 3P-OH to 3-keto with concomitant isomerization of the double bond, (c) introducing a C-1 (2)-doublebond, and (d) degrading the aliphatic side chain of sterols, they concluded that an ideal substrate for the microbial conversion to estrone would be 3~-acetoxy-l9-hydroxy-5-cholestene [(IV) in Fig. 31 (Sih et al., 1965; Lee, 1967). Adding this compound to a culture of Nocardiu sp. ATCC 19,170, Sih and co-workers were able to isolate estrone in 72% yield (96 hours). 3~-Acetoxy-19-hydroxy-5-cholestene can be prepared by chemical means in three steps. Apparently the entire process cholesterol to estrone can be carried out in an overall yield of 50% (Charney and Herzog, 1967). Interestingly, 19-norcholesta-1,3,5(lO)-trien-3-ol(V) was not oxidized by Nocardia sp. ATCC 19,170. In the same publication, Sih and collaborators (1965)reported the conversion of 6P, 19-oxido-4-cholesten-3-one [(I) in Fig. 41 by Nocardia sp. ATCC 19,170 into 6P, 19-oxido-4-androstene-3,17-dione (11)in 57% yield (70 hours) (111) in smaller and 6p, 19-oxido-9a-hydroxy-4-androstene-3,17-dione amounts. Since 3&acetoxy-5-a-chloro-6~,19-oxidocholestane (IV)is an intermediate in the chemical preparation of 6P, 19-oxido-4-cholesten-3-one(I) as well as of 3~-acetoxy-~9-hydroxy-5-cholestene and can be prepared from cholesterol acetate in only two steps, it appeared that this compound would be an even better substrate for microbial transformation into (11). Nocurdia sp. ATCC 19,170was capable of converting it into (11)in 36% yield (80 hours). Sih and collaborators concluded from their studies that presence of the 6P, 19-oxidobridge blocked the introduction of the C-l(2)-double bond whereas the C-19-hydroxy function prevented 9a-hydroxylation. Since both enzymic steps are essential in the steroid ring degradation more than side-chain cleavage cannot take place.

MICROBIAL CLEAVAGE OF STEROL SIDE CHAINS

&o -0#

(I)

\

39

(In)

KO&

AC 0

Cl

FIG. 4. Microbial conversion of 6p,19-oxidosterols to 6p,19-oxidoandrostanes.

Although the side chain of C-19-oxygenated sterols was usually degraded by microorganisms completely, resulting in the accumulation of only C17-keto compounds, by shortening the incubation period Sih and co-workers (1968a) were able to isolate the intermediary 23,24-dinorcholanic acid derivatives. The production of these acids as well as of estrone by microorganisms was patented, starting with various C-19-oxygenated substrates (19-OH, 19-formyl, 19-carboxy, 19-nor, 6p, 19-oxido) of the cholestane and stigmastane series (Sih, 1970). As mentioned earlier, Nocardia sp. ATCC 19,170 failed to convert 19norcholesta-1,3,5(10)-trien-3-01. However, working with Nocardia restrictus ATCC 14,887, Afonso et al. (1966) observed that an aromatic A-ring does not prevent the microbial degradation of the C-17 side chain of cholesterol. Prolonged incubation (240 hours) of this compound with N . restrictus ATCC 14,887 yielded 8% estrone. Based on the observations of Sih and his collaborators that introduction of C-19-hydroxy or 6p, 19-oxido functions protect the steroid nucleus from microbial degradation, several other research groups studied the conversion of related compounds by microorganisms. Processes using substrate modification for the selective degradation of the sterol side chain are summarized in Table I. In an attempt to synthesize equilin, 19-hydroxy-4,7-cholestadien-3-one was incubated with Nocardia sp. ATCC 19,170 (Deghenghi et al., 1967). Surprisingly, only estrone was formed. Evidence was shown that reduction of the C-7(8)-double bond must have taken place before the complete removal of the cholestane side chain and aromatization of ring A. Using Mycobacterium sp. (RMTP) the same substrate was converted to 16% equilin, 20% estrone, and traces of equilenin. Similarly, Rakhit and Sin& (cited in Vezina et al., 1971) observed that 19-norcholesta-1,3,5(10),7(8)-

TABLE I SELECTIVE SIDE-CHAIN DEGRADATION OF STEROLS WITH MODIFIEDRINGA/B STRUCTURE ~

Substrate 19-Hydroxysterols, 19-hydroxystenones, other 19-oxygenated sterols, 19-norsterols

19-Hydro~y-A~~~-steroIs 3-Hydroxy-19-norA~23.5(~0)-sterols 3-Hydroxy-19-norA1,3,5(10),?(8)_sterf11s

Product

Microorganism

Estrone

Reference

Nocardia restrictus ATCC 14,887 Sih and Wang (1965) Nocardia sp. ATCC 19,170 Sih et al. (1965), Lee (1967) Mycobacteria Denot et al. (1967), Lebedeva et al. (1970) Arthrobacter simplex IAM 1660 Nagasawa et al. (1970a) Corynebacterium sp. Mallett (1973) Nocardiu spp. Sih et al. (1968a), Sih (1970)

A9""-Estrone Estrone and dinorcholanic acids Equilin, equilenin + estrone Mycobacterium sp. Deghenghi et al. (1967) Estrone Nocardia restrictus ATCC 14,887 Afonso et al. (1966) Equilin, equilenin

+ estrone

Cmynebucterium simplex, Nocardia rubra Nocardiu sp. ATCC 19,170

Rakhit and Singh (1971)

6& 19-oxido-4-andros teneSih et al. (1965), Lee (1971) 3.17-dione 3~-Acetoxy-5a-chloro(fluoro)Mycobacteria SP, 19-oxidosterols Denot et al. (1967) 5a-Bromo-GP-19-oxidoLee (1971) 3P-Acetoxy-5a-bromo-6P,19Nocardia sp. ATCC 19,170 oxidosterols androstane-3,17-dione 3a,5a-Cycloandrostan-17-one Mycobacterium phlei KNGSF 70 Van der Waard et al. (1970) 3a,5a-Cyclos terols Arthrobacter spp., 3a,5a-Cyclo-GP,19-oxidosterols 3a,5a-Cyclo-GP, 19-oxidoShirasaki et al. (1969) androstan-17-one corynebacteria

Sb,19-Oxidostenones,

MICROBIAL CLEAVAGE OF STEROL SIDE CHAINS

41

tetraen-3-ol w a s converted to equilenin and traces of equilin by Corynebacterium simplex, and to equilenin by Nocardia rubra. Denot and collaborators (1967)prepared numerous C-19-hydroxy and 6/3, 19-oxido derivatives of the cholestane and stigmastane series and submitted them to microbial transformation by several mycobacteria. 19Hydroxycholesterol, 19-hydroxysitosterol, and 19-hydroxy-4-stigmasten-3one were converted into estrone by M . phlei, M . acapulcensis, M . runyonii, M . flavescens, and M . peregrinum (19-hydroxycholesterolonly). The same organisms (except M . peregrinum) transformed 6/3,19-oxido-4-cholesten-3one and the corresponding sitosterol derivative to a more polar compound was not (not identified)whereas 3/3-acetoxy-5cr-bromo-6/3,19-oxidocholestane metabolized by M . phlei. On the other hand, Lee (1971) reported that addition of 3P-acetoxy-5abromo-6/3,19-oxidocholestaneto a culture of Nocardia sp. ATCC 19,170 resulted in accumulation of 5cr-bromo-6/3,19-oxidoandrostane-3,17-dione. However, the same organism converted 3/3-acetoxy-5a-chloro-6/3,19-oxidocholestane and the corresponding 5ff-fluoro compound to 6/3,19-oxido-4androstene-3,17-dione. 3/3-Acetoxy-19-hydroxycholesterol was converted into estrone by 31 of 56 proactinomycetes and by 24 of 29 mycobacteria tested in studies by Lebedeva et al. (1970). Maximal output of estrone was obtained after 6-7 hours of cultivation with Nocardia asteroides 438 and Mycobacterium sp. 202 cultures. A""-Estrone was produced from 3/3-acetoxy-19-hydroxycholest-5-ene by Cwynebacterium sp. A 24,014 (Mallett, 1973). Although this observation seems to contradict the findings with 19-hydroxylated substrates mentioned above, it is very unlikely that the C-g(ll)-double bond was introduced by a steroid dehydrogenase. A more probable interpretation would involve either hydroxylation at C-9 or C-11 and subsequent dehydration. The use of C-19 oxygenated sterols as substrates in the selective side-chain degradation apparently does not always result in the aromatization of ring A. Brevibacterium sp. ATCC 19,653 was found to convert 19-hydroxy, 6/3,19oxido, or 19-norderivatives of the cholestane and stigmastane series as well as 19-hemisuccinates thereof to the corresponding 17-keto compounds without concomitant ring A aromatization and fission of the C-10 - C-19 bond (G. D. Searle & Co., 1970). Such fermentation products are useful in the synthesis of 19-norsteroids. In addition to the introduction of C-19 oxygenated functions, a completely different modification of the substrate structure also prevents the breakdown of the steroid ring system when organisms capable of utilizing cholesterol are used. Van der W a r d (1970)reported that exposure of 3a,5a-cyclocholestan6/3-01 [(I)in Fig. 5, i-cholesterol] to cultures of Mycobacterium phlei KNGSF

42

CHRlSTOPH K. A . MARTIN

#-c(P ..._.

......

0

OH

@ OH )

FIG.5. Microbial conversion of 3a,5a-cyclosterols to 17-ketosteroids.

70 in a corn steep medium resulted in the accumulation of two main metabo(11, 22.5% in 96 hours) and 3a,5alites: 3a,5a-cycloandrostane-6,l7-dione cycloandrostan-6/3-ol-17-one (111, 40% in 96 hours). In another experiment i-cholesterol was converted in 47% yield into 3a,5a-cycloandrostan-6/3-01-17one (111) with only small amounts of the corresponding 6-0x0 compound formed. Similarly, conversion of i-sitosterol or i-stigmasterol resulted in the accumulation of the same 6P-hydroxy compound; 6/3-methoxy-i-cholesterol was converted to 6/3-methoxy-3a,5a-cycloandrostan-17-one. A combination of the different methods of substrate structure modification was used by Shirasaki et al. (1969)for the production of 17-ketosteroids. 3a, Fia-Cyclo-GP, 19-oxido-5a-cholestane and the corresponding substrates of the ergostane and stigmastane series were transformed into 3a,5a-cyclo-6P, 19oxid0-5a-androstan-17-one by Corynebacterium equi IAM 1038, C. xerosis, Arthrobacter simplex IAM 1660, and A. ureafaciens IAM 1658. Although this kind of steroid ring A/B structure should protect the steroid nucleus from microbial degradation very efficiently, these investigators reported that addition of compounds which are known to inhibit 9a-hydroxylation increased the yields of the fermentation products. As reported by Barnes et al. (1974), deoxycholic acid could be converted by Pseudomonas sp. NCIB 10,590 to 10% 12&hydroxy-l,4-androstadiene-3, 17-dione. Although not belonging exactly to the microbial conversion of sterols, the selective side-chain cleavage of sapogenins and insect molting hormones appears to be another example of structural modification preventing complete oxidation of the ring system and will be mentioned here briefly. Fermentation of diosgenone [(I) in Fig. 6, 25D-spirost4-en-3-one] and derivatives thereof with cultures of Fusarium solani resulted in the accumulation of 65% 1,4-androstadiene-3,16-dione (11) (Kondo and Mitsugi, 1966). Similar substrates were converted to 20a-hydroxy-4-pregnene-3,16-dione

MICROBIAL CLEAVAGE OF STEROL SIDE CHAINS

43

+

FIG. 6. Microbial side-chain cleavage of sapogenins.

(111) and 3a, 11/3,2Oa-trihydroxy-5a-pregnan-16-one (IV) by Verticillium theobromue and Stachylidium bicolor (Kondo and Mitsugi, 1973, 1975). Canonica et aZ. (1974) observed that crustecdysone [(I) in Fig. 7) and makisterone (11) were degraded by lysed mycelia of Rhizopus arrhizus, R. nigricans, and CurvuZuria lunata to give poststerone (IV) and subsequently rubrosterone (V). Similarly, ponasterone A (111) was converted by Fusurium Zini ATCC 9593 into rubrosterone (V) in 15% yield (Tom and Abul-Hajj,

1975).

FIG. 7. Microbial side-chain cleavage of insect molting hormones

44

CHRISTOPH K. A . MARTIN

B. CONVERSION OF

STEROLS I N THE PRESENCE OF ENZYME INHIBITORS

Several research groups worked out procedures for the selective microbial side-chain cleavage of sterols in the presence of enzyme inhibitors. Inhibition of the steroid 9a-hydroxylase, a key enzyme in steroidal ring fission, yielded the most promising results. Since this enzyme was found to be a monoxygenase consisting of several proteins forming an electron transfer chain the involvement of metal proteins containing, for instance, iron appeared likely. Therefore, the effect of chemical agents capable of removing these metal ions (e.g., chelating agents) or replacing them by similar but inactive species was examined. Processes employing these techniques are summarized in Table II. As mentioned earlier, Whitmarsh (1964) reported that Nocardia sp. degraded cholesterol [(I) in Fig. 81 in low yield into 3-oxo-23,24-dinor-4-cholenic acid (11), 3-oxo-23,24-dinor-1,4-choladienic acid (111), 4-androstene-3,17dione (AD, IV) and 1,4-androstadiene-3,17-dione(ADD, V) in the presence of 8-hydroxyquinoline (0.25 mM). Extending the findings by Whitmarsh and their own earlier experiments indicating that the degradation of ADD to ~7a-oxa-D-homo-l,4-androstadiene-3,17-dione by Fusarium caucasicum could be prevented by certain condensed aromatic bicyclic compounds, preferably p-naphthol and 8hydroxyquinoline, Wix and collaborators (1968) found that chelating agents would inhibit the enzymic steroid ring oxidation of cholesterol. They screened 59 strains of mycobacteria by incubating 4-cholesten-%one (400 mg/liter) and 8-hydroxyquinoline (100 mg/liter) in resting cell suspensions of these strains. Mycobacterium phlei, M . smegmutis, M . fortuitum, M . thermophilus, and M . butyricum were found to be most active and accumulated ADD in yields of up to 55% (48 hours). Since ADD remained unchanged in cultures of M.,phbi in the presence of 8-hydroxyquinoline, but on the other hand 9a-hydroxy-4-androstene-3,17dione was decomposed, they concluded that this iron-chelating agent inhibited the introduction of the 9a-hydroxy group. Screening other chelating agents they discovered that 1,lO-phenanthroline (20 mgliter), 5-nitro-1, 10-phenanthroline (20 mdliter), a,a’-dipyridyl(% mditer), and cupferron (200 mg/liter) were effective in accumulating ADD. Lower concentrations of these compounds did not prevent the cleavage of the ring system whereas higher concentrations inhibited also the side-chain degradation. Numerous other steroids and sterols could also be transformed to ADD by M . phlei. The substrate concentration (cholesterol) could be increased to 1 gmfliter without loss of yield. Later, these investigators reported the conversion of 4P,5/i!-epoxycholestan-3-one to ADD with AD as metabolic intermediate

TABLE I1 SELECTIVE SIDE-CHAIN CLEAVAGE OF CHOLESTEROL AND PHYTOSTEROLS IN T H E PRESENCE OF

Inhibitor Chelating agents

Product

4-Androstene-3,17-dione, 1,4-androstadiene-3,17-dione, and dinorcholanic acids 4-Androstene-3,17-dione, 1,4-androstadiene-3,17-dione, other androstane derivatives

Microorganism

ENZYMEINHIBITORS Reference

Nocardk spp

Whitmarsh (1964), Martin and Wagner (1976a)

Mycobacterium spp. Mycobacterium sp. KNGSF 70 Arthrobacter simplex Bacillus spp., Brevibacterium lipolyticurn, Corynebacterium spp., Microbacterium lacticum, Mycobacterium spp., Norcardia spp., Protaminobacter alboflavus, Serratia mrcescens, Streptomyces tanashiensis Proactinomyces astwoides Arthrobacter simplex and Nocardia corallina (mixed)

Wix et al. (1968) Van der Waard et al. (1968) Arima et al. (1969), Nagasawa et al. (1969, 1970a,b,c)

Lebedeva et al. (1972) Awata et al. (1974), Teramoto and Sano (1975), Okamoto and Teramoto (1974), Makino et a1. (1974)

Inorganic ions

4-Androstene-3,17-dione, Mycobacterium sp. KNGSF 70 1,4-androstadiene-3,17-dione, other androstane derivatives Arthrobacter simplex Proactinomyces asteroides

Redox dyes

1,4-Androstadiene-3,17-dione

Arthrobacter simplex

Nagasawa et al. (1970~)

Rhamnolipids

4-Androstene-3,17-dione and 1,4-androstadiene-3,17-dione

Arthrobacter simplex and Pseudomonas aeruginosa (mixed)

Awata (1975)

Van der Waard et al. (1968), Lilly et al. (1976) Nagasawa et al. (1970~) Lebedeva et al. (1972)

46

CHRlSTOPH K . A . MARTIN

FIG. 8. Selective side-chain cleavage of cholesterol by Nocurdiu sp. (Whitmarsh, 1964).

(Tomorkeny et al., 1969). The 4&5P-epoxy function apparently did not interfere with the side-chain cleavage. Dutch investigators also tried to limit ring degradation by addition of enzyme inhibitors (van der Waard et al., 1968).In a screening program they decided to (1)isolate organisms capable of decomposing cholesterol rapidly and completely, (2) optimize this breakdown process, and (3) add enzyme inhibitors in different concentrations. Best results were obtained with Mycobacterium sp. KNGSF 70 in the presence of nickel sulfate as inhibitor. Under conditions optimal for the formation of ADD (104 mg Ni2+/liter)the conversion yield was generally 65% (800 mg ADD per liter in 72 hours). P-Sitosterol, campesterol, and stigmasterol were transformed under similar conditions, although the conversion rates were low compared with that of cholesterol, Evidence was shown that the conversion of cholesterol to 4-cholesten-3-one was not a rate-limiting step for the whole process. In addition to Ni2+, presence of Co2+, Pb2+, and SeOg- resulted in the accumulation of ADD, too. Trace amounts of Fez+were found to reverse the inhibitory effect of Ni2+. When Mycobacterium sp. KNGSF 70 was cultivated in a medium low in iron, small amounts of ADD accumulated but disappeared after prolonged incubation. Using the same strain, Lilly and co-workers (1976) obtained a 20% yield of ADD from cholesterol. In a series of studies, Japanese investigators of the Noda Institute systematically examined the factors affecting the conversion of sterols to 17ketosteroids. In preliminary experiments they observed that addition of a, a'-dipyridyl or sodium arsenite to cultures of Arthrobacter simplex IAM 1660, Bacillus roseus IAM 1257, B . sphaericus, Brevibacterium lipolyticum IAM 1398, Corynebacterium equi IAM 1038, C. sepedonium, Microbacterium lacticum IAM 1640, Mycobacterium smegmatis I F 0 3083, M . avium,

47

MICROBIAL CLEAVAGE OF STEROL SIDE CHAINS

M . phlei, Nocardia gardneri IAM 0105, N . corallina, N . erythropolis, N . lutea, N . medurae, N . minima, Protaminobacter alboflavus IAM 1040, S e r ratia marcescens IAM 1255, Streptomyces tanashiensis IAM 0016, and S . rubescens in the presence of cholesterol led to the formation of ADD (Arima et al., 1969; Nagasawa et al., 1969). The degradation pathway of cholesterol as well as of campesterol, P-sitosterol, stigmasterol, and 7-dehydrocholesterol was proposed as is shown in Fig. 9. Using Arthrobacter simplex, optimal fermentation conditions were described as follows (Nagasawaet al., 1970a):500-ml shake flasks containing 50 ml of a corn steep-meat extract-molasses medium were inoculated by A. simplex and incubated at 30°C on a reciprocal shaker. After 20 hours, 0.5 gmfliter finely dispersed steroid were added, 6 hours later 124 mg/liter a, a’-dipyridyl dissolved in ethanol. The incubation continued for 48 hours. The ability of A. simplex to degrade the side chain of various sterols decreased in the following sequence: lithocholic acid (63%ADD) > cholesterol (58% ADD) > p-sitosterol (39% ADD) > campesterol (38%ADD)> cholestanol (33%ADD) > stigmasterol (29% ADD) > 7-dehydrocholesterol (16% ADD)> ergosterol (5% ADD). Therefore, the following conclusions were made: (1) cleaving efficienciesof the side chains decreased by increasing the length of the side chain or by introduction of a branch at C-24; (2)presence of a C-22(23)or C-7(8) double bond lowered the reaction rate; (3)esterification or substitution of the C-3-hydroxyfunction led to almost complete resistance of the substrate to microbial breakdown (exception: cholesterol acetate). Although the main product was usually found to be ADD, several other metabolites were formed in minor amounts (Nagasawa et al., 1970b): $3-

HO

Cholesterol

4- Cholesten- 3 -one

1.4-Cholestadien- 3 -one

i

L-Androstene- 3.17dione ( A D )

l.L-Androstadiene-3.17dione ( ADD 1

FIG.9. Degradation pathway of sterols by Arthrobacter simpler (Nagasawa et al. , 1969).

48

CHRISTOPH K. A . MARTIN

androst-l-ene3,17-dione,5P-androst-l-en-17/3-01-3-one, 4-androstene-3, 17-dione,4-androsten-17/3-01-3-one, 1,4-androstadien-17p-ol-3-one, and 5pandrostane3a, 17P-diol. Nagasawa and collaborators concluded that these minor components should be regarded as secondary products from ADD. The effect of 167 compounds on the transformation of cholesterol by A. simplex was also examined (Nagasawa et al., 1970~).Accumulation of ADD was observed with chemicals classified as (1)lipophilic chelating agents, (2) metal ions with similar ion radii as Fez+,(3)inorganic SH reagents, and (4) autoxidizable redox dyes. The most effective inhibitors are listed in Table 111. Only lipophilic chelating agents were found to be effective in the inhibition of 9a-hydroxylation. Presumably hydrophilic agents did not penetrate the cytoplasmic membrane. EDTA became an effective inhibitor by the simultaneous addition of compounds increasing the permeability of this diffusion barrier, e.g., penicillins, cetyltrimethylammonium sulfate. The processes described above for the microbial synthesis of ADD from sterols using chelating agents were patented (Noda Institute for Scientific Research, 1965; Arima et al., 1968). In an additional patent, Nagasawa et al. (1971)reported the stimulation of the conversion of cholesterol into ADD by the addition of various surfactants, e.g., sorbitan monolaurate, in a concentration of 0.1 gmhter. ADD yields were increased by a factor of about 2. TABLE I11 AGENTS EFFECTIVEFOR THE SELECTIVEINHIBITIONOF STEROIDRING DEGRADATION Mechanism of action

Compound

Chelating agents for Fez+

a,a-Dipyridy 1,

Metal ions replacing iron or blocking SH-functions Redox dyes

1,10-phenanthroline, 8-hydroxyquinoline, 5-nitro-l,lO-phenanthroline, cupferron, diphenylthiocarbazone, diethyldithiocarbamate, isonicotinic acid hydrazide, xanthogenic acid, o-phenylenediamine, 4-isopropyltropolone, tetraethylthiuramdisulfide Ni2+, Coz+, Pb2+, S e Q - , As02'Methylene blue, resazurine

MICROBIAL CLEAVAGE OF STEROL SIDE CHAINS

49

Based on the studies of Wix, van der Waard, Nagasawa, and their collaborators, several other research groups described the selective side chain cleavage of sterols in the presence of enzyme inhibitors. As mentioned in the preceding chapter, Shirasaki et al. (1969) reported an improvement in the yields of 3a,5-cyclo-6~,19-oxido-5-a-androstan-17-one by the addition of various inhibitors to the fermentation medium. Lebedeva and co-workers (1972) observed that ADD was obtained in the course of fermentation of a Proactinomyces asteroides culture with cholesterol in the presence of Coz+,a,a‘-dipyridyl or 8-hydroxyquinoline. Induction of the enzymes involved in the transformation enhanced the culture’s activity. In a series of patents investigators of the Japanese Kanebo Co. described the microbiological production of androstane compounds. According to Awata et al. (1974), mixed cultures of Arthrobacter simplex IAM 1660 and Nocardia corallina I F 0 3338 showed enhanced productivity. Cholesterol, 2 gm/liter, was converted to 21% androstane compounds in the presence of 1 mM a,a’-dipyridyl by 1:l mixtures of these strains. A variation of this method consisted in inducing the enzymes by addition of cholesterol acetate 4 hours before addition of the substrate cholesterol (Teramoto and Sano, 1975). Addition of antibiotics such as penicillin G, carbomycin, polymyxin B, cholestin, streptomycin, chloramphenicol, neomycin, and kanamycin apparently increased the permeability of the cell membrane or cell wall. This effect caused %fold stimulation of the cholesterol to ADD conversion by mixed cultures in the presence of 0.5 mM a,a’-dipyridyl (Okamoto and Teramoto, 1974).Rhamnolipids produced from paraffins by Pseudomonas sp. inhibited the steroid ring degradation by several bacterial species (Awata, 1975). Still another variation of the cholesterol to ADD transformation was described by Makino et al. (1974).Using as substrate wastes discharged from wool washings which were hydrolyzed by CaO before addition to a mixed culture ofA. simplex and N . curallina they obtained a total yield of32% AD and ADD after 48 hours of fermentation. Martin and Wagner (1976a) employed similar techniques as Nagasawa et al. (1969) in the conversion of p-sitosterol into 17-ketosteroids. Inhibition of the steroid ring cleaving enzymes by 0.3 mM a,a’-dipyridyl resulted in low yields of AD and ADD (22% from 1.5 gmfliter p-sitosterol), but complete degradation of the substrate occurred to a large extent. They hypothesized that these relatively poor results were caused by incomplete inhibition of the 9a-hydroxylase. However, using higher inhibitor concentrations than 0.3 mM the C-26-hydroxylase (initiating the side-chain oxidation) was also partly inhibited. Therefore, in order to trap the accumulating 17ketosteroids it seemed promising to use hydrophobic organic resins known

50

CHIUSTOPH K. A . MARTIN

to adsorb steroids very efficiently.Addition of Amberlite XAD-2 and similar resins enhanced the ADD yields 2- to 3-fold. In addition to ADD (maximal 50% in 120 hours from 1.5 gm/liter p-sitosterol), 3-0xo-23,24-dinor-l, 4-choladienic acid and 3-0~0-23,24-dinor-1,4-choladienic acid methyl ester were isolated (total yield of 11%).Studies on the mechanism of stimulation by these adsorbents indicated that a,a'-dipyridyl was also adsorbed causing decreased toxicity of this compound although the capability to trap Fez+was not hindered (Martin and Wagner, 1976b). However, the main effect seems to consist in selective adsorption of 17-ketosteroidsbecause of micelle formation of the substrates (Martin and Wagner, 1977). Buckland and co-workers (1976) noted that high dissolved oxygen concentrations caused formation of trace amounts of AD and ADD in cultures of Nocardia sp. NCIB 10554. Tomatidine was dehydrogenated in ring A by Arthrobacter simplex IAM 1660, but no degradation took place (Belieet al., 1975). When the side-chain cleaving enzymes were induced by incubation in the presence of cholesterol and tomatidine added to the incubation broth afterward, ADD in 4.5% yields was formed. This result indicated that the nitrogen atom in the side chain prevented tomatidine from being an inducer for the side chain degrading enzymes. The spiroketal side chain of sapogenins was degraded by Mycobacterium phlei in the presence of 100 mg of 8-hydroxyquinoline per liter (Ambrus and Buki, 1969).ADD was isolated in 3% yields. This result contradicts the findings of Kondo and Mitsugi (1966, 1973), who observed the formation of 16-ketosteroids (with fungi).

C. STEROLCONVERSION BY SELECTEDMUTANTS Recently, mutagenic treatment has been employed for the production of organisms capable of degrading the sterol side chain selectively. Such mutants are biochemically blocked from degrading the nucleus and can be used to efficiently produce steroids from sterols without the necessity of modifying the substrate or of adding chemical inhibitors. The generation and isolation of mutants is a well established process in microbial genetics. Mutagens of choice include ultraviolet light, N methyl-N'-nitro-nitrosoguanidine, etc. Cargile and McChesney (1974) described methodology that allows for the production and selection of mutant organisms capable of specific side-chain cleavage of sterols. This methodology was based upon the mutation of wild-type strains that are capable of completely degrading cholesterol and selection of mutants blocked at the desired conversion.

MICROBIAL CLEAVAGE OF STEROL SIDE CHAINS

51

The production of useful mutants was carried out as follows: cultures of Aerobacter lipolyticus were grown in nutrient broth, then treated with nitrosoguanidine for 15 minutes. After centrifugation the treated cells were resuspended in minimal medium containing cholesterol as sole source of carbon and incubated in the presence of carbenicillin. The enriched cultures were then screened by examining the growth on cholesterol and testosterone agar, respectively. If a clone showed growth on cholesterol but none on testosterone, it was designated a mutant. Although a few stable mutants were produced by this procedure, the investigators were unable to find organisms capable of accumulating steroids in sterol fermentations. A process was described for the microbial degradation of sterols to produce 17-ketosteroids by two newly isolated bacteria designated as Mycobacterium sp. NRRL B-3683 and Mycobacterium sp. NRRL B-3805. No ring degradation inhibitory agents were necessary (Marsheck et al., 1972; Kraychy et al., 1972). Mycobacterium sp. NRRL B-3683 was produced by UV-irradiation of a soil mycobacterium capable of converting 4stigmasten-3-one to ADD. This organism showed increased ability to effect the biotransformation. Further irradiation produced Mycobacterium sp. NRRL B-3805 which lacked the ability to C-I@)-dehydrogenatesteroids and was found to produce AD from sterols. Mycobacterium sp. NRRL B-3683 displayed a preference for substrates possessing the 3/3-OH-A5 system rather than the corresponding 3keto-A4 structure. Decreasing ADD yields were obtained in the sequence: cholesterol (78%, 144 hours) > sitosterols, N.F. (48%, 168 hours) > 4-cholesten-%one (44%, 168 hours) > stigmasterol (37%, 168 hours) > 1, 4-stigmastadien-%one (34%, 168 hours) > 4,22-stigmastadien-3-one (31% 240 hours) > soya sterols residue (20%,240 hours, substrate concentration 4 gmfliter) > 4-stigmasten-3-one (4%, 168 hours); substrate concentration was 1 gmfliter. It should be noted that the calculation of the yields was based upon the substrate consumed (substrate added - substrate recovered). 4-Androstene-3,17-dione (AD) was formed in trace amounts as it was converted to ADD. Various other media gave comparable yields; inclusion of myo-inositol stimulated the growth of the organism. Substrates not con6P, 19-oxidosterols, verted to ADD included 3a,5a-cyclo-6~-hydroxysterols, 3-deoxysterols, cholanic acid derivatives, and progesterone. Interestingly, a new compound in addition to 17-ketosteroids was isolated, characterized as 20ac-hydroxymethyl-l,4-pregnadien-3-one.The production of this compound was believed to represent a side reaction in the side-chain degradation. Although 17-ketosteroids were accumulated in fermentations of this culture containing 3-hydroxy-A5-sterols, this strain apparently still has some 9a-hydroxylase activity depending very much on the medium composition;

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CHRISTOPH K. A . MARTIN

30% degradation of A D D occurred within 144 hours when this compound was added as substrate. Mycobacterium sp. NRRL B-3805 blocked in the C-l(2)-dehydrogenation converted various sterols to 4-androstene-3,17-dione (-4D)in yields of up to 39%. Few other investigators carried out conversions of sterols to 17ketosteroids by Mycobacterium sp. NRRL B-3683. Tall oil sterols (mainly sitosterol and campesterol) are potentially available in huge amounts (20,000 tons per year in the United States). Conner and collaborators (1976) studied their microbial conversion to CISsteroids. Compared to soybean sterols, tall oil sterols were transformed with similar efficiency. Lilly and co-workers (1976)reported that they obtained A D D from cholesterol in very high yield in fermentation with Mycobacterium sp. NRRL B-3683. As conclusion of the discussion of methods for the selective side chain cleavage of sterols we would like to emphasize that almost all the processes described were carried out on a laboratory scale using substrate concentrations of 1 gmAiter or even less. As has been pointed out by Marsheck (1971), to make the conversion competitive with diosgenin degradation or other chemical methods for production of 17-ketosteroids,the substrate concentration would have to be significantly higher than 1 gmfliter. Optimal substrates would have to be soybean sterols or tall oil sterols. V. Substrate Addition and Isolation of Fermentation Products In the processes described in Section IV, usually conventional techniques well known in the microbial transformation of steroids were employed for the substrate addition and isolation of products. These will not be discussed here. However, some problems arose because of the extraordinary hydrophobic nature of the sterols. As determined by Haberland and Reynolds (1973), cholesterol has a and maximum solubility in water or aqueous buffers of 1.8 mg/liter (4.7 undergoes a thermodynamically reversible monomer-micelle equilibrium with a critical micelle concentration (CMC) of 2540 nM at 25°C. These micelles are stabilized by strong intermolecular attractive forces in addition to the hydrophobic repulsion by the solvent. In subsequent publications these investigators determined the hydrophobicity of cholesterol and thermodynamic equilibria of cholesterol-detergent-water systems (Gilbert et al., 1975; Gilbert and Reynolds, 1976). Jones and Baskevitch (1973)hypothesized that one of the factors operating against facile microbiological degradation of sterols would be their aggrega-

a)

MICROBIAL CLEAVAGE OF STEROL SIDE CHAINS

53

tion in aqueous solution. Using kinetic data of the acid-catalyzed or the enzymic (A5-A43-ketosteroid isomerase of Pseudomoms testosteroni) isomerization of 3-keto-A5-stenonesto 3-keto-A4-stenonesas indicator for the degree of aggregation, they observed that these compounds are severely aggregated in aqueous solution and that addition of large proportions of an organic solvent such as methanol was required to effect complete solvation of the sterols. Since this appeared to represent an impractical solution when complex degradative reactions have to be carried out with whole microorganisms, they proposed the introduction of oxygen functions into the side chain which leads to some disaggregation. A logical consequence of these reports was the conversion of sterols in nonaqueous solvents. Buckland et al. (1975) demonstrated that the conversion of cholesterol to 4-cholesten-3-one by Nocardia sp. NCIB 10554 can be done in the presence of very high concentrations of water-immiscible solvents such as carbon tetrachloride. The enzymes involved (cholesterol oxidase and catalase) continued to function under these conditions. Unfortunately, no further breakdown of the substrate did occur indicating that several other enzymes essential for degradative reactions were inactivated. To avoid the problems of high hydrophobicity of the substrates many investigators added nonionic surface-active agents like the Tweens or Spans to their cultures. Water-soluble forms of ergosterol and cholesterol were described by Adams and Parks (1967)and Thompson and co-workers (1973). They discovered that ergosterol added to certain growth media became refractory to solvent extraction. The components capable of solubilizing the sterols were found to be polysaccharides occurring in yeast extract, characterized as yeast cell wall mannan. Sterols bound to the polysaccharide have been shown to be metabolically available to yeast cultures requiring sterols. Since yeast extract and other complex medium constituents have been employed very frequently in the microbial conversion of sterols to 17ketosteroids it might be that similar polysaccharides present in these media, although unknown, facilitated these processes. In order to isolate the fermentation products the broth was extracted usually by organic solvents such as chloroform, dichloromethane, ethyl acetate, etc. This results in extraction of the 17-ketosteroids as well as of the lipophilic substrates. Therefore, laborious separation steps had to follow the extraction (usually preparative column chromatography on alumina or silica gel). However, for the selective recovery of 17-ketosteroids, especially of ADD, from sterol containing fermentation broths, other methods have been described. ADD was found capable of forming molecular complexes with aand @naphthol and certain other aromatic compounds (Chinoin, 1961).According to the examples described in this patent, ADD can be recovered

54

CHfUSTOPH K . A . MARTIN

from its solution in the solvent extracts of the fermentation broth by adding P-naphthol. A precipitate is formed consisting of ADD and @naphthol in a molar proportion of 1:1, which can easily be removed and decomposed. A similar process, using hydroquinone as trapping agent was employed in the patent by Kraychy et d.(1972). As discussed earlier, addition of hydrophobic organic adsorbents resulted in stimulation of the /3-sitosterol to ADD conversion, mainly because of selective trapping of ADD (Martin and Wagner, 1976a). The removal of steroids from biological fluids by adsorption on Amberlite XAD-2 is well known. However, the selective adsorption of ADD compared to the sterols and stenones is probably due to the self-association of the latter. The micelles formed are stabilized by unusual high interaction energies between the monomers. It might be that the interaction energies between monomers are larger than those between monomer and adsorbent thus explaining the weak adsorption on the resins. As result, ADD and other steroids not forming stable self-aggregates could be extracted by adsorption, whereas the unaltered substrates mainly remained in the fermentation broth (Martin and Wagner, 1977). After adsorption, the 17-ketosteroids and traces of substrate were separated easily by washing the resin with aqueous alcohol.

VI. Summary and Conclusion The recent interest in the microbial conversion of sterols to C,, steroids was caused by the increased demand for steroid drugs and the shortage of diosgenin. Based upon studies on the pathway of sterol metabolism by certain bacteria methods for the selective cleavage of the 17-alkyl side chain of cholesterol and phytosterols were developed on a laboratory scale. These methods consist in the inhibition of the enzymes responsible for the primary attack of the steroid ring system. Structural modification of the substrates, fermentation in the presence of enzyme inhibitors, or mutagenic treatment were employed to achieve this goal. Since the microbial process has to compete with chemical methods, high conversion efficiencies in the presence of large substrate concentrations have to be obtained. Some of these processes are used now on an industrial scale for the production of 17-ketosteroids. REFERENCES Abul-Hajj, Y. J. (1972).J. Biol. Chem. 247, 686. Adams, B. G., and Parks, L. W. (1967).Biochem. Biophys. Res. Commun. 28, 490. Afonso, A., Herzog, H. L., Federbush, C., and Charney, W. (1966).Steroids 7, 429.

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Zearalenone and Some Derivatives: Production and Biological Activities P. H. HIDY, R. S. BALDWIN,R. L. GFWASHAM, C. L. KEITH, AND J. R. MCMULLEN I M C Chemical Group, Inc., Terre Haute, Indiana I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Chemistry.. ........................ A. Structure and Termi B. Assay and Recovery. 111. Fermentation . . . . . . . . . . . . . . . . A. Culture Development.. . . . B. Surface Fermentation ............................... C. Submerged Fermentation. ........................... D. Biosynthesis ..........................

59 61 61 62 63 63 64

..................................... A. General Drug Action. . . . . . . . . . . . . . . . ................ B. Endocrine Activities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ................................... D. Safety Evaluation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References ............................. . . . . . . . . . . . . . . .

74 74 75 77 78 81

68 73

I. Introduction During the early 195Os, Commercial Solvents Corporation1 (CSC) examined a number of microorganisms with the hope that they would produce, in controlled forage fermentations, substances having anabolic activity. This approach to a salable product was unsuccessful, but Dr. F. N. Andrews of Purdue University, who has served for many years as a consultant to CSC, called attention to reports (McNutt et al., 1928; Pullar and Lerew, 1937; Koen and Smith, 1945; McErlean, 1952) of estrogenic properties exhibited by moldy corn specimens. During 1957 and 1958, the Purdue University Agricultural Experiment Station had investigated complaints from several widely separated Indiana swine producers. These included vulvular enlargement and mammary gland stimulation in swine herds in Indiana in which the causative agent appeared to be moldy corn. In a joint effort, under an agreement between CSC and the Purdue Research Foundation, workers at Purdue and CSC began to investigate the microflora of these moldy corn specimens to determine whether estrogenicity was related to a particular genus and, if so, whether the active substance had utility in animal nutrition. 'International Minerals and Chemical Corporation acquired Commercial Solvents Corporation May 15, 1975.

60

P.

H. HIDY ET

AL

Among the cultures isolated by the Purdue group and grown on sterile moist ground corn, those of Gibberella produced a substance(s)that could be extracted with ethanol and concentrated by evaporation of the solvent. When this substance was fed to ovariectomized white mice, it induced uterine enlargement. The crude concentrate gave a similar response in mice when it was suspended in sesame oil and injected subcutaneously. Isolation and examination of additional cultures and production of larger quantities of moldy corn were undertaken. As a result of these early efforts, the symptoms observed in swine in the field were duplicated in gilts fed rations prepared from moldy corn. Partially purified extracts were shown to be effective in increasing the rate of gain and feed efficiency in sheep. The production of test material on corn was improved and, with the availability of what was then considered to be large quantities, one of us (R. S. Baldwin) began attempts to prepare the pure substance responsible for the physiologic effects of crude extracts. This work culminated in the isolation of a pure crystalline compound. Some of its properties were described in two publications (Stob et al., 1962; Andrews and Stob, 1965). The intervening years since this first work have seen the accumulation of publications describing the occurrence of the active principle, zearalenone, in stored grains (Eppley et al., 1974; Shotwell et al., 1970; Caldwell and Tuite, 1970); its synthesis by several species of Fusarium (Caldwell et al., 1970; Ishii et al., 1974);the development of techniques for its detection and quantitative estimation (Eppley, 1968; Vandenheuvel, 1968; Mirocha et al., 1968a); and the chemical and physical properties of zearalenone, its derivatives and related compounds. The literature related to the chemistry of zearalenone was reviewed by Shipchandler (1975) and again by Pathre and Mirocha (1976). Interest in the occurrence of zearalenone in feed grains was enhanced by an increasing awareness of the existence of substances produced by a wide variety of fungi that might directly or indirectly affect human health. Much of the work by others has been concerned with this aspect of zearalenone. Some of this information has been reviewed by Mirocha et al. (1971). In our laboratories, however, we were also interested in determining the potential value of this new class of compounds as anabolic substances in animals. This work has covered a period of nearly 15years, during which the production of zearalenone has evolved from the first moldy corn experiments to the present large-scale synthesis by submerged fermentation. The development and testing of a number of chemical derivatives of zearalenone for use in animal nutrition began as soon as adequate quantities of the parent compound became available. Interest in the derivatives extended beyond their potential value in animal nutrition to an examination of their general pharmacological properties. Two derivatives, the pair of dia-

ZEARALENONE A N D DERIVATIVES: PRODUCTION A N D ACTIVITIES

61

stereoisomers obtained by catalytic reduction of the parent compound, exhibited properties suggesting possible value in treatment of the postmenopausal syndrome. One of these isomers is preferred for use as an anabolic agent in sheep and is currently marketed under the trademark RALGRO@. Pharmacological data have been obtained in several animal species in an effort to meet Food and Drug Administration requirements in connection with New Drug applications for these two isomers. The chemistry of zearalenone will be considered here only to the extent necessary to indicate the interrelationships of the parent compound, two derivatives currently being marketed, and the major products of metabolism of the parent compound and one derivative, which will be further considered in Section IV. The gaps in the literature related to zearalenone are in areas of the commercial production by fermentation and of the pharmacological and toxicological data accumulated by CSC. It is the intent of this summary to fill those gaps.

II. Chemistry A. STRUCTURE AND TERMINOLOGY When the structure of the active substance was established, the term zearalenone was selected as a convenient designation for the naturally occurTABLE I RELATED RESORCYLICACID LACTONES Melting point Compound

(“C)

(I)

Zearalenone

164-165

(IV)

Zearalanone

192-193

(V)

Configurationu at C-6’

Trademarks

145-147

RALONE@

182-183

RALONEB, RALGROm

Zearalanol

(Wb

~~

“From W. H. Urry, University of Chicago, Chicago, Illinois, unpublished results. *United States adopted name, zeranol.

62

P. H. HlDY ET AL.

Ni

c

no

OH

\

(X.rn) FIG. 1. Structures and relationships of zearalenone and the derivatives considered in this paper. (See TabIe I for nomenclature.)

ring compound from which at least some of the early derivatives could be named, relating them to the parent compound. This terminology, especially for the parent material, has now found general acceptance. Prior to selection of this name, zearalenone had been referred to as FES or Fermentation Estrogenic Substance (Hodge et al., 1966) and as F-2 by Christensen et al. (1965).A more complete chemical designation applied by Urry et al. (1966)is 6-(10-hydroxy-6-oxo-trans-1-undeceny1)-P-resorcylicacid lactone. Johnson et al. (1970) indicated the size of the macrolide ring as a p-lactone. Chemical Abstracts indexes zearalenone as [S-(E)]-3,4,5,6,9,10-hexahydro-14,16-dihydroxy-3-methyl-lH -2-benzoxacyclotetradecin -1,7(8H)dione. Table I summarizes some information needed to i d e n e the compounds that will be considered in this paper. Figure 1, constructed mainly from Urry et al. (1966), indicates the relationships of the compounds which will be discussed. For the balance of this paper, specific compounds, with the exception of zearalenone, will be indicated by the Roman numeral designation assigned in Fig. 1.

B. ASSAYAND RECOVERY Zearalenone is practically insoluble in water, but is readily soluble in many organic solvents, as shown in Table 11, and in dilute alkali. These properties facilitate both assay and recovery of the compound from fermentations. Whether the zearalenone in fermentation broths is still contained within the mycelial cell or has been released by autolysis, its insol-

ZEARALENONE A N D DERIVATIVES: PRODUCTION A N D ACTIVITIES

63

TABLE I1 SOLUBILITY OF ZEARALENONE Solvent

Gm/100 gma

Water Ethanol Methanol Acetonitrile Acetone Methylene chloride Benzene n-Hexane

0.002 24 18

8.6 58 17.5

1.13 0.05

"25°C

ubility in water ensures that simple filtration with a filter-aid such as Johns Manville Super-Cel will remove more than 99.8% of the total zearalenone in a 10-gm per liter broth. In our high-titer fermentations, the contribution of closely related metabolites to the assay has never been significant. In routine assays of fermentation broths, the zearalenone-containing solids were removed by filtration and the cake was thoroughly extracted with methanol. Ultraviolet absorption spectra were run on the methanol extracts, and titers were calculated by comparing absorbance at 236 or 274 nm with that of pure material. In fermentation samples, corrections may be applied for absorbance not due to zearalenone. Large-scale recovery could be accomplished similarly by extracting fermentation solids with an organic solvent, preferably methanol or acetone, and crystallizing the zearalenone by evaporating the solvent or by adding water to a concentrated solution of the compound. An alternative recovery process based on extraction with aqueous alkaline solutions has been described by Hidy and Young (1971). They slurried the fermentation solids in dilute (0.25%)sodium hydroxide solution (PH 11-12), filtered the slurry, and acidified the filtrate to precipitate the zearalenone. Solutions of crude zearalenone were decolorized with char when necessary and pure product was obtained by the usual methods of recrystallization, generally using isopropyl alcohol as the solvent. @

Ill. Fermentation A. CULTUREDEVELOPMENT As mentioned previously, initial culture isolation studies were conducted at Purdue University. Of the fungal cultures isolated from moldy corn from

64

P. H . HlDY ET A L .

Indiana farms which had reported estrogenic stimulation in swine, only those cultures identified as Gibberella zeae (the perithecial or perfect stage of Fusarium roseum “graminearum”),elicited an estrogenic response in sexually immature female pigs. Culture isolation studies were continued in CSC’s laboratories by examining various moldy corn and soil samples. Additionally, Fusarium cultures were procured from the American Type Culture Collection as well as the Northern Regional Research Laboratories, and their ability to produce zearalenone was tested under surface and submerged fermentation conditions. Three isolates of G. xeae-Dewar (ATCC 20028), Paul S (ATCC 20271), and H331 (ATCC 20272twere selected for further study. A mutation and selection program was initiated concurrently with media and solid support development. Various criteria for selection of improved strains were imposed on the selection process at different times during the program. The first priority was high-producing strains. Using the procedure of Eisenstark et al. (1965) to treat macroconidia of the Dewar strain with N-methyl-N‘-nitro-N-nitrosoguanidine (NTG), selected cultures were again tested under both surface and submerged fermentation conditions. From these studies, a strain (542, ATCC 20273) was selected that not only produced higher titers in surface fermentations, but also produced zearalenone in a suitable medium under aerobic submerged conditions (Keith, 1972). Improvement of this culture (strain 542) was continued using NTG as well as ethyl methane sulfonate as mutagens. This work has resulted in considerably higher zearalenone titers as described in Section II1,C. Other criteria for selection have been applied during the course of developing the submerged process. Because most fusaria produce pigments which can be difficult to remove in the recovery and purification process, some emphasis has been placed on finding colorless mutants with the ability to produce zearalenone. This goal has recently been attained in the laboratory. Strains having high rates of synthesis have been sought, the economic advantage of shortening a 2- to 3-week fermentation being obvious. Here we have been partially successful. Variation in ability of a Fusariurn strain to produce zearalenone has been discussed by Mirocha et al. (1971). Similar variability has not been encountered in our commercial production. Selected strains of G. zeae are preserved both by lyophilization and by submersion in liquid nitrogen.

B. SURFACEFERMENTATION There are few details reported of efforts in other laboratories to produce zearalenone in quantity. In every case, apparently only limited success has been attained utilizing the common grains for support and nutrient supply.

ZEARALENONE AND DERIVATIVES: PRODUCTION AND ACTIVITIES

65

Mirocha et al. (1971) have reviewed the work of the Minnesota group, which has used mainly corn or rice as a substrate either alone or with added glucose. From the data provided, it is difficult to calculate the efficiency of such fermentations, but it does not appear to be high. At the beginning of CSC investigations it was not known whether the active material sought was a metabolic product of the organism or was simply a constituent of grain that was altered by action of the fungus. For this reason our early efforts centered around the use of grains as media, but recovery of a product from the complex mixture including starch, partially digested protein and lipid of the grain base presented problems that were recognized early as barriers to commercial synthesis. Because of these barriers, efforts were directed toward cultivation of the fungus on defined media. Since there was no assurance that the product could be synthesized by Gibberella in submerged culture, a solid support was sought for defined media which could absorb considerable quantities of liquid without becoming waterlogged. Numerous possible supports were tested, but vermiculite, an expanded mica product, was found nearly ideal. Three or four milliliters of medium per gram of vermiculite could be loosely bound by the porous laminar structure so that no liquid separated from the moistened mass even on prolonged standing. This readily available mineral was essentially insoluble in water, dilute alkali, or organic solvents. These properties were valuable not only in the fermentation but also in the recovery process. For the first studies using a vermiculite support, a medium was selected containing a high level of glucose (30%). As later became evident, production of zearalenone was very difficult to initiate at carbohydrate levels less than 20%. It was determined that low initial temperatures were a requirement for a successful synthesis. This requirement has also been reported by others (Mirocha et al., 1971). While growth and production of zearaletione on grain is slow, requiring as much as 6 or 7 weeks to complete, utilization of the glucose on vermiculite was rapid with evolution of heat. Initially, fermentations were carried out in 2-liter Erlenmeyer flasks in which 240 ml of medium was held on 80 gm of vermiculite. Flasks were kept in an incubator room held at 21"-24"C. The temperature in the flasks rose rapidly to above 30"C, and it was obvious that heat transfer was a problem. For this reason, and to increase capacity to produce zearalenone, the flask fermentation was carried out by closely packing the flasks in a tray of running water that could be kept at about 16"-17°C. This provided better heat transfer from the flasks. The production of zearalenone in flasks was encouraging. Yields of 1.52.0 gm per flask (6.25-8.33 gm/liter) were readily obtained. Total production capacity in flasks was still limited and insufficient to provide the quantity of pure material required for structure work and pharmacological study.

66

P. H . HIDY ET AL.

The fermentation was next successfully transferred to 2 0 x 4 0 5-inch ~ aluminum trays with loosely fitted lids. The trays were immersed about 3 inches in a water bath maintained at about 17"-18°C. A single tray held a 3-inch layer of vermiculite weighing about 5 kg. After sterilization by autoclaving in the tray, the vermiculite layer was charged with 13.5 liters of inoculated medium and incubated for 4-5 weeks. Initially, glucose concentrations of 30% were used, but it was shown that 45% glucose could be tolerated and that tray loadings of as much as 17 liters did not greatly affect the efficiency of conversion of carbohydrate to zearalenone. A rack holding 16 trays in four levels was installed in a laboratory to allow increased attention to be given to variables of the process and a pilot plant operation was begun with two similar racks. Later, trays containing integral water baths and designed to be stacked without requirement for a rack were built and operated. Each of these trays had a capacity 2.5 times that of the original trays. Since inoculum preparation and medium composition for flask and tray fermentations were essentially identical, operation of a typical tray process will be described. For inoculum preparation, spores from a Bennett's agar slant or a lyophile culture were suspended in 5 ml of sterile water and added to 100 ml of Bennett's medium in a 500-ml Erlenmeyer flask. After incubation on a rotary shaker at 30°C for 24 hr, 10 ml of this first-stage inoculum was used to inoculate 300 ml of Bennett's medium in a 1-liter Erlenmeyer flask which was also incubated 24 hr at 30°C on a rotary shaker. One flask of second-stage inoculum sufficed to inoculate 13.5-17.0 liters of sterile production medium contained in a 5-gallon stainless steel milk can equipped with tubing to allow siphoning the contents of the can onto the vermiculite in one aluminum tray. Production medium was prepared as shown in Table 111. The concentration of every ingredient of the above medium was varied over a considerable TABLE I11 DEFINEDMEDIUMFOR PRODUCTION OF ZEARALENONE IN SURFACE CULTURE Component Glucose

BYF Yeast KCI

NaNO, N ~ N O S KzHPOi Distilled water to volume

Percent

37 0.1 0.05

0.05 0.2 1.0 0.1

67

ZEARALENONE AND DERIVATIVES: PRODUCTION AND ACTIVITIES

TABLE IV C O U R S E O F A TYPIC&

SURFACE

FERMENTATION^

Zearalenone (gmlliter)’ Tray

3 Weeks

4 Weeks

5 Weeks

1 2 3 4

9.25 9.62 10.36 4.07

13.32 12.95 12.58 10.73

15.91 14.06

17.02

“Strain Dewar. ‘13.5 Liters/tray

range without substantial improvement in yield of the product. No organic nitrogen source was as effective as the combination of ammonium and sodium nitrates shown. Titers of a typical fermentation, consisting of four trays, each containing 13.5 liters of medium, are shown in Table IV. The effect of the culture improvement program on the performance of the surface fermentation is demonstrated in Table V, where strain Dewar was compared to strain 542. A “microfermentation” was developed which proved to be a valuable device for the study of some variables and for evaluation of new cultures. Petri plates (20 x 1OO mm) containing 10 gm of dry vermiculite were autoclaved with lids in place. A total of 25 ml of preinoculated medium was applied evenly over the surface of the vermiculite by pipette. Inoculum volume was 10% of the medium volume. Plates were incubated with lids in place at 19.5”C and at 85% relative humidity. The fermentation was complete in 3 weeks and production of zearalenone on standard medium using strain Dewar was 12 gmfliter. Optimum performance required media containing 30% glucose. This fermentation procedure was easily adapted to the TABLE V COMPARISON OF PERFORMANCE O F STRAIN DEWAR A N D S T R A I N

542

TF114-tray

Strain

Gm/traya.’

Gm/liter

Gmigm CHO

1 2 3 4

Dewar 542 Dewar 542

274 438 247 486

13.5 27.4 15.4 30.37

0.036 0.074 0.042 0.082

“16.0 Literdtray. ’4 Weeks.

68

P . H. HIDY ET A L .

preparation of 14C-labeled zearalenone. Even smaller fermentations in 25-mm Petri plates and requiring only 5 ml of medium were successfully operated but never fully developed. Details of the surface fermentation have been previously described (Hidy, 1971).

C. SUBMERGEDFERMENTATION The discovery of mutant strains of G. zeae capable of producing zearalenone by submerged culture (Keith, 1972) was a major breakthrough in efforts to produce zearalenone on an industrial scale since earlier attempts had been unsuccessful. There have been no reports in the literature of appreciable zearalenone production by submerged fermentation; therefore, the information reported here represents work from our laboratories (Keith, 1972; Woodings, 1972; McMullen, 1972). Even with mutant strains, zearalenone titers from shake flasks were in the range of 1.0 to 1.5 gmhiter using a medium that gave up to 30 gmfliter in surface fermentation. It was obvious, therefore that the development of a successful submerged fermentation required a reinvestigation of the medium components as well as a study of other fermentation parameters. Early work on the submerged process was conducted in shake flasks and small fermentors. The fermentation was subsequently scaled up to 100gallon and 2000-gallon pilot plant fermentors and finally to 20,000-gallon production fermentors. Since the objective of the program was to obtain the highest zearalenone titers in the shortest time with the most economical nutrients, the medium was frequently changed as was the strain employed. Some of the various media and strains used during the course of the study are summarized in Table VI. Inoculum for the submerged fermentation was either a Bennett's broth culture grown for 24 hours at 30°C with aeration or an aliquot of a zearalenone producing fermentation (referred to hereinafter as a whole-beer inoculum). The volume of inoculum was generally 5%although volumes of about 2-20% could be used satisfactorily. Other inoculum media were investigated, but no advantage was seen for them. The optima1 temperature for the zearalenone fermentation was found to be between 21" and HOC-nearer 21°C being preferred for shake flasks and nearer 24°C for fermentors. Zearalenone was generally not produced in fermentations starting at temperatures above 28°C; however, once zearalenone production was established the temperature could be raised slowly to 32°C before production stopped. Temperatures below 20°C were not desirable because of the slower growth rate and lower rate of zearalenone production.

TABLE VI PROGRESSOF MEDIUM AND STRAINDEVELOPMENT PROGRAM Strain Titer (gm/liter) Glucose (grn/liter) NH4N03 NaN03 Urea N-Z-Amine A@ Yeast extract K,HPO, MgS04’ 7HzO KC1 ZnS04.7Hz0 (PP) Inoculum

542 4-5 21 days 300 10.0 2.0

542 7-8 21 days 300 5.0 1.0

542 20-22 21 days 300

542 21-23 21 days 300

4.0

542 16-18 14 days 200

RG-6C 26-28 14 days 200 -

-

4.0 3.0

4.0 3.0

4.0 3.0

-

RG-6C-88- 13 >32 14 days 200

-

-

-

4.0 3.0

1.0 1.0 0.5 0.5

1.0 0.5 0.25 0.25

1.0 0.5 0.25 0.25

0.5 0.25 0.25

0.5 0.25 0.25

0.5 0.25 0.25

0.5 0.25 0.25

Bennett’s broth

Bennett’s broth

0.9 Bennett’s broth

0.9 Whole beer

0.9 Whole beer

0.9 Whole beer

Bennett’s broth

-

-

-

70

P. H . HIDY ET AL

The fermentations were usually run for 14-21 days depending on the amount of sugar in the starting medium and the rate of sugar utilization. Zearalenone production declined shortly after the sugar was depleted but fermentations could be prolonged by feeding additional sugar before zearalenone synthesis completely ceased. Aeration requirement for the fermentation was not great, but maintaining a minimal amount of aeration was essential for production. Shake-flask studies were routinely conducted with 100 ml of medium per 500-ml flask incubated on a rotary shaker at 350 rpm. Reciprocal shakmg or larger volumes of medium per flask reduced titers markedly, as seen in Table VII. Laboratory fermentors were operated with 0.25 to 0.5 volumes of air per volume of medium per minute with a variety of impellor sizes and codigurations. In general, larger impellors at slower speeds (350-500 rpm) were preferred to smaller impellors at higher speeds (750-1000 rpm). Higher speeds also exhibited undesirable sheer on the mycelium. I t was possible to reduce aeration by 40-50% after 6-7 days, but continued zearalenone synthesis required a small amount of air. If fermentations were begun with reduced aeration, zearalenone production was prevented. Higher levels of aeration were not beneficial and caused excessive foaming. The pH of the fermentation was not critical and it was advantageous, from the standpoint of contamination, to allow it to drop naturally to about 3.54.0. Attempts to control pH never resulted in any significant increase in titer and in most cases reduced titers somewhat. The starting pH was about 6.8-7.2. Among the usual carbohydrates tested as carbon sources, glucose, fructose, and galactose were about equal in their ability to support zearalenone production. Xylose, sucrose, maltose, glycerol, and sorbitol performed best in combination with glucose. The more economical forms of glucose, such as CereloseB or Staleydex@,were selected for further development. The less

TABLE VII AERATIONIN SHAKE FLASKFERMENTATIONS Mediuma volume

(4 50

100* 100 200

Shaker

Titer (gm/liter)

Rotary Rotary Reciprocal Rotary

19.6 19.8 0 0

"20%Glucose medium, 14-day fermentations *Standard conditions.

ZEARALENONE A N D DERIVATIVES: PRODUCTION A N D ACTIVITIES

71

refined corn syrups and starch hydrolyzates were satisfactory energy sources, but they required alteration of the other medium components for their variability. Glucose concentration was a very important parameter in the fermentation. Concentrations above 20% were generally required for zearalenone synthesis in a standard medium inoculated with a Bennett’s broth inoculum. It was found, however, that by using a whole-beer inoculum from an established fermentation, zearalenone could be produced at glucose levels of 20% and below. Investigation of this peculiarity led to the observation that extracts of whole beer or a catalytic amount of pure zearalenone could initiate production at lower sugar levels even when a Bennett’s broth inoculum was used. As little as 10 mg of zearalenone per liter of medium could produce the desired fermentation. Table VIII shows the effect of added zearalenone in 20% and 30% glucose media using a Bennett’s broth inoculum. Use of a whole-beer inoculum, containing some zearalenone, eliminated the need to add the pure material and gave titers superior to those in media without added zearalenone. Various nitrogen sources were tested in the submerged fermentation, and in contrast to results from the surface fermentation, organic nitrogen sources gave higher titers than did inorganic sources. Urea was the best and cheapest source tested and, therefore, became a standard ingredient in subsequent fermentations. The optimum concentration was between 0.4and 0.6%. Proteins and protein hydrolyzates were not as satisfactory as urea for zearalenone production, but they did stimulate production when used in combination with urea. Highest titers were obtained with a urea concentration of 0.4% and an N-Z-Amine A* (Humko Sheffield casein hydrolyzate) concentration of 0.3%.Other protein hydrolyzates, casein, steepwater, corn gluten meal, and yeast fractions could also stimulate titers in combination with urea. TABLE VIII EFFECTOF ADDEDZEARALENONE Zearalenone

Percent glucose in medium

Amount added to medium (mgliter)

Amount produced after 14 days (gmiliter)

20 20 30 30

0 20 0 29

0 16.7 9.5 18.0

12

P. H . HIDY ET A L .

Ammonium salts of some organic acids and a number of amino acids, such as asparagine, glutamine, and glycine supported good zearalenone production but were not economically feasible for large-scale fermentations. Inorganic requirements for the fermentation were satisfied by adding dipotassium phosphate, magnesium sulfate, potassium chloride, and zinc sulfate. The optimum concentration of dipotassium phosphate was 0.05% and of magnesium sulfate, 0.025%. Increasing either of these beyond the optimum increased cell yield and sugar utilization but not zearalenone production. Potassium chloride had an optimum of around 0.025%, but varying the concentration beyond the optimum had little effect on the fermentation. Zinc sulfate was found, however, to have a marked effect on the fermentation (McMullen, 1972). Early fermentations contained a small amount of yeast extract, and all attempts to replace yeast extract with vitamins or amino acids were unsuccessful. I t was found that the ash of yeast extract satisfied the requirements. Subsequent experiments demonstrated that the component in yeast extract required by the fermentation was zinc. The amount required was found to be very low, and the optimum appeared to be around 1 ppm (as zinc sulfate heptahydrate). Higher levels of zinc stimulated growth, sugar utilization, and pigment production. The low tolerance for zinc may explain why some crude proteins and carbohydrates failed when tested. Another element for which a requirement was demonstrated was iron, but this held only when reagent grade chemicals were used in the fermentation. Semicontinuous fermentations were run using a variety of withdrawal and replacement levels. A process has been described (Woodings, 1972) wherein 75% of the spent medium from a zearalenone fermentation was withdrawn and an equal volume of fresh sterile medium was added. The replacement was carried out when the glucose was consumed and the zearalenone titer was 10-20 gmAiter. The time interval between replacements was 7-10 days depending on the rate of glucose utilization. The fresh medium added was a complete 20% glucose medium although it was reported that the glucose concentration could be as low as 10%. Other semicontinuous fermentations have been run ranging from a daily 10% replacement to a 75% replacement every 7-10 days. In essence, the use of a 5% whole-beer inoculum could be described as a 95% replacement. A successful semicontinuous fermentation depended on a withdrawal and replacement method that maintained an equilibrium between sugar utilization, growth, and zearalenone production. Once the fermentation had been stabilized, it could be continued for long periods before the production rate decreased significantly. A 2000-gallon pilot plant fermentor has been operated semicontinuously for over 6 months before being terminated for mechanical reasons. Parameters followed during the course of a typical fermentation are presented in Fig. 2. They represent a composite of data obtained from shake

ZEARALENONE A N D DERIVATIVES: PRODUCTION A N D ACTIVITIES

73

Time (days)

FIG.2. Course of a typical fermentation

flasks containing 20% glucose medium inoculated with a 5% whole-beer inoculum and grown at 21°C. The initial level of zearalenone represents carryover from the inoculum. The same parameters followed in a 30% glucose medium would be very similar but would be extended to about 21 days to allow complete utilization of the extra sugar.

D. BIOSYNTHESIS

The zearalenone biosynthetic pathway has been investigated using radioactive potential precursors (Steele et al., 1974). Since the structure of zearalenone suggests a condensation of 9 acetate units, precursors such as acetate, diethyl malonate, senecioate, shikimate, and DL-mevalonic-2lactone have been tested using cultures of G. zeae grown on solid substrates. Only acetate and diethyl malonate were incorporated readily into zearalenone, indicating that zearalenone is synthesized via the polyketide pathway. From 14Cincorporation studies in the CSC laboratories, using cells grown in submerged culture, acetate was also found to be readily incorporated into M sodium acetate, a zearalenone. At an optimum concentration of 8.6 x linear incorporation rate of 8.6 ng/min/mg dry weight of cells for 30 minutes was observed. Acetate concentrations above this optimum proved to be inhibitory to 14C incorporation. This parallels unsuccessful attempts to feed acetate to the submerged fermentation as a technique for increasing zearalenone yields.

74

P. H . HIDY ET A 1

From thin-layer chromatographic examinations of methanol extracts of ['4C]-acetate-exposed cultures, a radioactive fraction other than zearalenone was detected. When this partially purified fraction was used in 14C incorporation studies in place of acetate, its radioactivity was found in zearalenone. At this time, the role, if any, this fraction has in the biosynthesis of zearalenone has not been determined.

IV. Pharmacology Biological studies, begun in 1958, were confined to mouse uterotropic assay of molded grain and fermentation products to establish and confirm that the estrogen syndrome observed in animals was due to a mold metabolite. The assay was selected to follow the course of zearalenone purification and production. Zearalenone and over 150 derivatives have been tested for various pharmacological activities since its initial isolation. A full range of studies to evaluate the potential usefulness of zearalenone and selected derivatives was undertaken following the isolation of the pure material. Data reported herein will be limited to the natural product and those derivatives having either scientific significance or useful application. The biological results reported here deal with the pure materials.

A. GENERALDRUGACTION Single oral doses of the compounds of interest were screened for drug activity in adult male and female mice using oral doses of 4644640 mg/kg. The usual tests for measuring and assessing a wide variety of drug actions were performed. In general, the outward appearance of the animals was unremarkable during the 7-day test period. Specifically, zearalenone and compounds (IV), (V), and (VI) displayed no drug-related effects except compound (VI), which showed a tendency to cause increased uterine weights. These compounds, when tested for central nervous system activity using oral doses of 328 mgkg in the mouse mental health screen test, displayed no significant CNS activity. In another series of tests the compounds were evaluated for their ability to produce behavioral changes in squirrel monkeys (Sidman Avoidance Test) and in food-deprived rats (Food Reinforcement Test). The compounds were without effect when a cumulative oral dose of 150 rng was given to monkeys and 48 mgkg was given to rats. Oral doses of 128 mgkg had no effect on urine output, electrolyte excretion, or propensity to produce gastric hemorrhage when administered to rats. These compounds had no significant effect on gastrointestinal motility when administered orally to mice at a maximum dose of 100 mgkg.

ZEARALENONE A N D DERIVATIVES: PRODUCTION A N D ACTIVITIES

75

TABLE IX BIOLOGICAL ACTIVITIES OF ZEARALENONE AND DERIVATIVES Compounda Activity

I

111

IV

v

VI

Anabolic (mice) Uterine weight (mice) (increase) Oviduct weight (chick) (increase) Vaginal cornification Antiestrogen Antiovulatory Implantation inhibition Gonadotropin inhibition Anti-FSH and HCG Anti-LH Progestational Androgenic Blood cholesterol (lowering) Antiinflammatory Antimicrobial Coccidiostatic Herbicide

+, Active; - , not active. Zearalenone and compounds (V) and (VI) had no direct effect on the cardiovascular system when administered intravenously at doses of 5 m&g to the anesthetized dog. Other parameters monitored, such as blood pressure, electrocardiographic (ECG) and neurophysiologic activities, were not altered significantly by these compounds. Table IX summarizes certain of the biological activities of the parent material and those of the derivatives of current interest.

B. ENDOCRINE ACTIVITIES A total of 46 different classical as well as newly developed endocrine procedures were used to study the range of activities of these compounds. The initial objective of this program, i.e., evaluation for use as an anabolic agent for animals, was expanded to include an appraisal of use in clinical medicine. Zearalenone may be classified as a weak estrogen having anabolic properties when administered to certain animal species. The parent material and those derivatives that possess these properties produce a limited stimulation of the uterus. Thus they are classified as impeded estro-

76

P. H. HlDY ET A L

gens. Perhaps the most remarkable property of these compounds is the difference in relative estrogenic activity displayed among different animal species. Dose-response slopes obtained with zearalenone and its estrogenic derivatives do not parallel those obtained with 17-P-estradiol and estrone. As a result of this divergence, any attempt to assign ratios of biological activities may be misleading. Zearalenone (I) and the derivatives (11), (111), (IV), (V), and (VI) produce estrogenic effects in a variety of assay systems using the mouse, rat, rabbit, chicken, ewe, domestic pig, and monkey. Their relative estrogenic activity listed in decreasing order is (VI), (IV), (I), (V), (111). In classical tests, i.e., uterine weight or vaginal smear, and using (VI) as an example, potency varies from approximately 0.1 to 0.7% of 17-0-estradiol and 1 to 20% of estrone depending upon the particular end point used. The 0ral:parenteral ratio for this compound varies from 0.2 to 3.0. In monkeys, (VI)will produce changes in the vaginal mucosa and the endometrium at a dose of 0.9 mgkg when administered orally. The clinically effective dose of (VI)to treat estrogen deficiency states, projected to be 50-75 mg daily based on animal studies, has been found to be correct. As a result of the advances made in the fermentation process, significant quantities of zearalenone became available in 1961. Evaluation of the anabolic properties of this material was then begun. In two preliminary studies, subcutaneous administration of zearalenone to growing sheep produced a moderate weight gain over untreated animals. Under the procedure of Hershberger et al. (1966),it was found that zearalenone and (VI)produced a positive myotropic effect in castrate male mice. This effect was subsequently confirmed in the Syntex Laboratories and has been reported (Mirocha et al., 196813).Subsequent animal growth trials, in which these two materials were compared as implants administered to steers and heifers, revealed that compound (VI) produced a significant improvement in growth and feed efficiency. The important aspects of the numerous growth trials conducted with (VI) have been reported (Brown, 1970). The same material when administered as an implant was found to produce a significant positive effect on nitrogen retention in sheep. When 6 mg of compound (VI) was compared with an equal weight dosage of diethylstilbestrol, nitrogen retention was improved 23.6%by the former versus 16.4%for the latter. These findings eventually led to the marketing of RALGROm (IMC brand of zeranol) implants for use as a growth promotor in cattle and sheep. Studies concerning metabolic changes produced in growing cattle and sheep have been reported by several investigators (Wilson et al., 1972; Borger et al., 1973a,b). Drug residue studies have shown that edible tissue from animals receiving 36 mg as an implant contain no detectable quantity of (VI) or metabolites 65 days after implantation. The procedure for drug residue

ZEARALENONE A N D DERIVATIVES: PRODUCTION A N D ACTIVITIES

77

analysis, consisting of tissue extraction and gas-liquid chromatography, has a detection limit of 20 ppb. Zearalenone inhibits synthesis and secretion of pituitary gonadotropins, an activity not unexpected for estrogenic substances. This activity was found to be in direct proportion to the uterotropic activity with the possible exception of compound (V). The finding that this compound was more active than anticipated has suggested certain clinical uses. Foreign clinical studies have indicated that effective control of the menopausal syndrome may be achieved with an oral dose of 25-50 mg/day or approximately one-half that of compound (VI). Zearalenone, (V), and (VI)significantly reduce blood lipids when administered to rats receiving a high fat diet. Blood cholesterol lowering appears to be greatest with zearalenone based on relative estrogenicity (5-1O:l). Triglycerides are unaffected by the administration of the above compounds. The oral administration of the parent material, (V), and (VI) to adrenalectomized, diabetic, and fasting rats at doses of 1-10 mg/kg produced no significant alteration of blood glucose levels. Glucose tolerance was likewise unaffected when doses of 1-10 mg/kg of these compounds were given orally to rats. None of the resorcylic acid 'lactones tested had progestational or androgenic activity. Zearalenone and compound (VI)showed antiinflammatory activity using the rat foot edema and the granuloma inhibition tests. However, in view of the thymolytic activity and body weight decreases observed, it was concluded that the activity observed had no practical significance. Compound (VI) showed a highly significant enhancement of cortical bone growth in the rat at an oral dose of 12.5 mg/kg. In summary, zearalenone is a nonsteroidal mold metabolite possessing weak and impeded estrogenic properties in all animals tested. Derivatives prepared from it may possess greater or less activity. The estrogenic nature of (VI) was hrther confirmed with the finding that it will displace tritiated estradiol from the isolated estrogen-binding uterine protein. All compounds tested were well tolerated, even by swine, which is perhaps the most sensitive species studied. It is more active orally than parenterally. The parent material and certain derivatives possess antigonadotropic properties, the effect being exerted at the pituitary level. Certain derivatives, particularly (IV) and (VI), possess significant anabolic properties. C. METABOLISM When biosynthetic 14C-labeled zearalenone was administered orally to rats, 7040% was excreted in the feces and 2030% in the urine. The only significant metabolite found in these studies was identified as (111) (R. S. Baldwin, unpublished results). This compound was found to be one-fourth as

78

P. H . HIDY ET AL

estrogenic as zearalenone. Similar metabolism was found to occur in sheep, where the ratio of metabolite to zearalenone in feces was 2:l. Extensive metabolism studies were carried out with (VI). The major metabolite found in all species studied (rat, sheep, cattle, monkey, dog, rabbit, and man) is (IV). Conjugates of PI)and (IV) have been isolated from the urine and feces of animals. The major route of excretion regardless of the mode of administration, with the exception of the rabbit, is via the feces. The rabbit excretes the major portion of the drug and metabolite by way of the urinary tract. Metabolism of (VI)in cattle, following administration of 72 mg in the ear, has been reported (Sharp and Dyer, 1972).

D. SAFETYEVALUATION Evaluation, in animals, of the effects produced by both single and repeated dose administration of zearalenone and certain derivatives was started by CSC in 1961. These materials have been remarkably well tolerated. The selection of the term “toxin” (Christensen-et d.,1965) used to describe the physiological action of this mold metabolite may have been unfortunate. The acute toxicity data obtained from single-dose experiments for zearalenone and the derivatives (IV), (V), and (VI) are presented in Table X. The oral administration of a single dose of 20,000 mgkg failed to produce deaths in mice or rats. Maximum doses produced slight irritability and hypoactivity during a 14-day observation period. Intraperitoneal doses proTABLE X SUMMARY OF ACUTETOXICITY DATAFOR ZEARALENONE AND DERIVATIVES

Oral Species Mouse

Rat

Compound

Male

Female

Male

Female

(1) (Iv)

-

>20,000

-

>SO0

(v) (VI)

>10,000 240,000

3290 -

2530 4400

(1)

>10,000

>20,OOo >lO,OOO >40,OOO

>500

-

-

>10,000 >10,OOo

5490

(Iv)

-

-

>10,OOO >40,OOO

4200 8900

3480

(VI)

>10,000 >40,000

10,900

(I)

-

>S,OOo

-

2500

0

Guinea pig

Intraperitoneal

ZEARALENONE A N D

DERIVATIVES:

PRODUCTION A N D ACTIVITIES

79

duce irritability, bradypnea, hypoactivity, and hypothermia. The responses were similar for all other compounds. Subacute studies of 2-14 weeks duration have been completed with zearalenone and derivatives (V) and (VI) using several species. Compounds (V) and (VI) have been given orally to rats, dogs, and monkeys for 2 years. Compound (VI) is currently the subject of long-term tests in dogs and monkeys projected to run 7 and 10 years, respectively. Periodic sacrifice and tissue histology are complete through 6 years. Table XI lists the subacute and chronic tests performed. These results have been briefly summarized for each compound. Zearalenone. An oral dose of 1 mgkglday given to rats for 13 weeks was without significant effect. Weight gain suppression but no histological changes were observed at a dosage of 5 mglkglday. A dose of 25 mglkglday produced suppressed weight gain and testicular size and reduced number of corpora lutea. Histological changes observed were similar to those following high dosages of estrogens and included endometrial stimulation and arrested maturation of spermatocytes. In a similar study in dogs, 1 rngkglday orally was without effect while 5 mglkglday produced reduced number of corpora TABLE XI SUBACUTEAND CHRONICTOXICOLOGICAL STUDIESCONDUCTED WITH CERTAINRESORCVLIC ACID LACTONES Dose range Species Rat

Chicken Swine Dog

Monkey

Sex

Drug

Route

(mgikg)

Duration

Subcutaneous Gavage Diet Gavage Diet Diet Diet Diet Intramuscular Diet Gavage Gavage Gavage Gavage Gavage Gavage Gavage

2.5-10.0 25-1600 1-25 1-30 0.25-6.25 1-25 0.1-20 110-220 ppmb 50-200 2.2-4.4 p p d 25-1600 1-30 0.25-12.5 0.025-25 15-37.5 1-30 15-75

2 Weeks 6 Weeks 13 Weeks 13 Weeks 13 Weeks 2 Years 2 Years 4 Weeks 6 Days 61 Days 4 Weeks 13 Weeks 14 Weeks 2 Years 6 Year9 2 Years 6 Years’

“Ongoing studies to continue for 7 years (dogs) and 10 years (monkeys). *In diet as parts per million (ppm).

80

P. H. HlDY ET AL

lutea and arrested spermatogenesisbut no uterine changes. Higher doses of up to 50 mg/kg/day produced similar effects. Growing poultry receiving up to 220 ppm of zearalenone in their diet for 4 weeks showed a slight, nonsignificant, weight improvement. Swine receiving diets containing 4.4 ppm showed signs of estrogenic stimulation in females but not in castrate males. A level of 12.5 ppm of zearalenone can be detected in diets when fed to immature female mice. Compound (V). Experiments of 90-day duration with rats and dogs at a maximum test level of 25 mg/kg/day orally failed to establish a true toxic effect with this compound. Two-year chronic rat and monkey studies with maximum oral doses of 30 mg/kg/day have been completed and data are being analyzed. Preliminary examination of the results have revealed no unexpected findings. Compound (VZ). Based upon the results obtained from the 2-year rat study the highest nontoxic oral dose lies between 6 and 20 mg/kg/day and has been set at the logarithmic mean of 11 mgkglday. Studies of similar duration in dogs have shown that the highest nontoxic oral dose lies between 2.5 and 25 mgkglday and has been set at 8 mgkglday. It is noteworthy that compound (VI)did not increase the incidence of neoplasms in the rat or induce malignant neoplasia in the mammary glands of dogs as has been reported for steroidal substances. Studies completed through 6 years have revealed no gross or histological drug-related evidence of malignant changes in dogs or monkeys. A series of studies was carried out with zearalenone and compounds (V) and (VI) to determine the effects of repeated oral dosage on the mammalian reproductive process. Breeding performance, embryo and teratogenic toxicity, perinatal and postnatal effects were evaluated. Ruddick et al. (1976) reported a no-effect zearalenone dosage of 0.3-1.0 mg/kg/day orally in rats in a teratological study. A CSC study indicated a slightly higher range, 1 . 0 3 . 0 mgkg/day. Higher doses of 5-10 mgkg occasionally produced skeletal anomalies, embryo toxicity, and resorptions. The no-effect dose of compound (VI) in these studies appeared to be between 0.31 and 1.25 mg/kg/ day. A rat three-generation study with compound (VI) showed no alteration of any parameter at a maximum dose of 200 ppb in the diet. This compound had no teratogenic effect in rabbits at a maximum dose of 5 mg/kg/day. Summarizing, safety studies have shown zearalenone and compounds (V) and (VI) are well tolerated upon single and short-term administration. In chronic studies using doses of 20-25 mg/kg/day these compounds may reduce food intake and weight gain in rats, dogs, and monkeys. Morphological changes in endocrine supported tissues, lowered hematocrit, and hemoglobin levels in rats, and elevated sedimentation rates in dogs may occur at oral doses of 20 mgkglday and are directly related to their

ZEARALENONE A N D

DERIVATIVES: PRODUCTION

A N D ACTIVITIES

81

relative estrogenicity. Rats treated with compound (VI) could not be differentiated from control rats on the basis of tumor incidence. Compound (VI) and, to a lesser extent compound (V), have been evaluated in estrogen deficiency states in women and have been found to be effective in a dose range of 0.5-1.0 mg/kg/day. Utian (1973)reported that compound (VI)was superior to natural conjugated estrogen in reducing plasma calcium levels and at least as effective as natural conjugated estrogens in treatment of the postmenopausal syndrome. This compound is currently marketed for this indication in certain foreign countries and is being clinically evaluated in the United States. The pharmacological data discussed were obtained in cooperation with The Endocrine Laboratories, Inc.; Merck, Inc.; and Sandoz, Inc. Safety data were obtained under CSC supervision and in cooperation with Woodard Research Corp.; International Research Corp.; Hill Top Research, Inc.; Institute of Experimental Pathology and Toxicology; and Bio/dynamics, Inc. ACKNOWLEDGMENT The authors are indebted to M. C. Bachman and Jerome Martin for their constructive criticism of our efforts to tell this story, and to Judy Jacobs for preparation of the manuscript. REFERENCES Andrews. F. N., and Stob, M. (1965). U.S. Patent 3,196,019. Borger, M. L., Wilson, L. L., Sink, J, D., Ziegler, J. H., and Davis, S. L. (1973a).J. Anim. Sci. 36, 706-711. Borger, M. L., Sink, J. D., Wilson, L. L., Ziegler, J. H., and Davis, S. L. (1973b).J. Anim. Sci. 36, 712-715. Brown, R. G. (1970).J . Am. Vet. Med. Assoc. 157, 1537-1539. Caldwell, R. W., and Tuite, J. (1970). Phytopathology 60, 1696-1697. Caldwell, R. W., Tuite, I., Stob, M., and Baldwin, R. S. (1970). Appl. Mierobiol. 20, 3 1 3 4 . Christensen, C. M., Nelson, G. H., and Mirocha, C. J. (1965). Appl. Microbiol. 13, 653-659. Eisenstark, A , , Eisenstark, R., and VanSicMe, R. (1965). Mutat. Res. 2, 1-10. Eppley, R. M. (1968).J. Assoc. OH. Anal. Chem. 51, 74-76. Eppley, R. M., Stoloff, L., Trucksess, M. W., and Chung, C. W. (1974)./. Assoc. Off. Anal. Chem. 57, 632-635. Hershberger, L. G., Thompson, C. R., and Clegg, M. T. (1966). Proc. SOC. Erp. Biol. Med. 121, 785-788. Hidy, P. H. (1971). U.S. Patent3,580,811. Hidy, P. H., and Young, V. V. (1971). U.S. Patent 3,580,929. Hodge, E. B., Hidy, P. H., and Wehrmeister, H. L. (1966). U.S. Patent 3,239,345. Ishii, K., Sawano, M., Ueno, Y., and Tsunoda, H. (1974). Appl. Microbiol. 27, 625-628. Johnson, D. B. R., Sawicki, C. A , , Windholz, T. B., and Patchett, A. A. (1970).J . Med. Chem. 13, 941-944. Keith, C. L. (1972). U.S. Patent 3,661,712. Koen, J. S., and Smith, H. C. (1945). Vet. Med. (Kansas City, Mo.) 40, 131-133. McErlean, B. A. (1952). Vet. Rec. 64, 539-540.

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McMullen, J. R. (1972). U.S. Patent 3,661,713. McNutt, S. H., Punvin, P., and Murray, C. (1928).J . Am. Vet. Med. Assoc. 73, 484-492. Mirocha, C. J., Harrison, J . , Nichols, A,, and McClintlock, M. (1968a). Appl. Microbid. 16, 797-798. Mirocha, C. J., Christensen, C. M., and Nelson, G. H. (1968b). Biotechnol. Bioeng. 10, 469482. Mirocha, C. J., Christensen, C. M., and Nelson, G. H. (1971). Microb. Torins 7, 107-138. Pathre, S. V., and Mirocha, C. J. (1976).Ado. Chem. Ser. 149, 178-227. Pullar, E. M., and Lerew, W. M. (1937). Aust. Vet. J . 13, 2 8 3 1 . Ruddick, J., Scott, P. M., and Harwig, J. (1976).Bull. Enuiron. Contam. Toricol. 15,678-681. Sharp, G. D., and Dyer, I. A. (1972).J . Anim. Sci. 34, 176-179. Shipchandler, M. T. (1975).Heterocycles 3, 471-520. Shotwell, D. L., Hesseltine, C. W., Goulden, M. L., and Vandegraft, E. E. (1970). Cereal Chem. 47, 700-707. Steele, J. A., Lieberman, J. R., and Mirocha, C. (1974). Can. J . Microbiol. 20, 531-534. Stob, M., Baldwin, R. S., Tuite, J., Andrews, F. N., and Gillette, K. G. (1962). Nature (London) 196, 1318. Uny,W. H., Wehrmeister, H. L., Hodge, E. B., andHidy, P. H. (1966). TetrahedronLett. No. 27, pp. 31093114. Utian, W. H. (1973). Br. Med. J . 1, 579-581. Vandenheuvel, W. J. A. (1968). Sep. Sci. 3, 151-163. Wilson, L. L., Borger, M. L., Peterson, A. D., and Rugh, M. C. (1972). J . Anim. Sci. 35, 128-132. Woodings, E. T. (1972). U.S. Patent 3,661,714.

Mode of Action of Mycotoxins and Related Compounds’ F.

s. CHU

Food Research Institute and Department of Food Microbiology and Toxicology, University of Wisconsin, Madison, Wisconsin I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Biological Effects of Mycotoxins .......................... A. Acute and Chronic Toxic Effect of Mycotoxins in Animals B. Carcinogenic Effect of Mycotoxins .................... C. Mutagenic and Teratogenic Effects of Mycotoxins . . . . . . . D. Biological Effects of Mycotoxins Other Than in Animal Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . III. Modification of Carbohydrate and Lipid Metabolism by Mycotoxins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Effect of Mycotoxins on Carbohydrate Metabolism . . . . . . B. Effect of Mycotoxins on Mitochondrial Respiration . . . . . . C. Effect of Mycotoxins on Lipid Metabolism . . . . . . . . . . . . . IV. Effect of Mycotoxins on Nucleic Acid and Protein Synthesis . . A. Effect of Mycotoxins on Nucleic Acid Synthesis . . . . . . . . . B. Effect of Mycotoxins on Translational Steps in Protein .. Synthesis . . . . . . . . . . . . . . . . . . . V. Interaction of Mycotoxins with Macromolecules as the Mode of Action of Mycotoxins . . ............................ A. Interaction of Mycotoxins with Nucleic Acids . . . . . . . . . . . B. Interaction of Mycotoxins with Proteins and Organelles . . VI. Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

83 85 85 95 96 98

100 100 102 105 107 108 115 121 122 126 133 135

I. Introduction Mycotoxin is a convenient generic term to describe the toxic substances formed during the growth of fungi. In contrast to the bacterial toxins which are mainly proteins with antigenic properties, the mycotoxins encompass a considerable variety of fungal secondary metabolites with diverse chemical structures and biological activities. In considering the effect of mycotoxins on the animal’s body, it is important to distinguish between “mycotoxicosis” and “mycosis .” Mycotoxicosis is used, in general, to describe the action of mycotoxin(s) and is frequently mediated through a number of organs, notably the liver, kidney, lung, and nervous system. On the other hand, mycosis refers to a generalized invasion of living tissue(s) by growing fungi. Mycotoxins and mycotoxicosis are an especially significant problem for ‘Contribution from the College of Agricultural and Life Sciences, The University of Wisconsin, Madison, Wisconsin. Part of the work described in this paper was supported by Public Health Service Grant No. ES00656 from the National Institute of Environmental Health Sciences and by a grant from the Brown-Hazen Fund of the Research Corporation. 83

84

F. S . CHU

human and animal health, because under certain conditions foodstuffs can provide a favorable medium for fungus growth and toxin production. Because of the relative stability of mycotoxins to heat and other treatment they may remain in foods and feeds for a considerably long period. The moldy andlor toxin-containing food thus become a source of human and animal intoxication. The mycotoxin problem is actually an old one. Ergotism and mushroom poisoning, for example, have been known for centuries. Outbreaks of other types of toxicoses associated with the ingestion of moldy foods and feeds by humans and animals have been also recorded (Forgacs and Carll, 1962). One of the well documented examples is the outbreak of a disease called alimentary toxic aleukia in the Orenberg district of the USSR during World War II. The cause of the outbreak was recognized as toxins produced by fungi growing on grain allowed to stand in the field during winter (Joffe, 1974). Since the discovery in the early 1960s of aflatoxins, a series of highly potent carcinogens produced by Aspergillusflavus, research has focused new attention on this old problem. Developments in the last decade have disclosed not only many new fungal poisons, which are attracting attention because of their diverse effects on animals or other biological systems, but have led also to reinvestigations of a number of fungal secondary metabolites with antibiotic activity which were found to be toxic to test animals (Scott, 1974). The importance of mycotoxin problems in human and animal health resulted in the organizing of several domestic and international symposia (ASM Symposium on Mycotoxins, 1975; I11 IUPAC Symposium on Mycotoxins in Foodstuffs, 1976; Conference on Mycotoxins in Human and Animal Health,

1976). Although mycotoxin research has progressed rapidly in recent years, as is evidence by the large volumes of research papers published each year-more than 300 titles (Austwick, 1 9 7 5 t a n d the books and review articles devoted to this subject, the mode of action for most mycotoxins is not known. As will be seen from this review, information on the mode of action for mycotoxins and related compounds is limited to 7 or 8 types of compounds. The present review is a result of the relatively few comprehensive comparisons on the modes of action of different mycotoxins. It is hoped that this review will stimulate additional research in this area. Owing to the diversity of the chemical structure and biological properties of mycotoxins, it is not possible to generalize on the mode of action for most mycotoxins, but rather the special biochemical lesions caused by certain mycotoxins will be discussed. Because of the volume of research papers on mycotoxins, no attempt will be made to cover all the literature, but rather only the most important recent work relevant to the subject is cited. For additional information on the mycotoxins, reviews and books should be consulted (Ciegler et al., 1972;

MODE OF ACTION OF MYCOTOXINS A N D RELATED COMPOUNDS

85

Goldblatt, 1969; Purchase, 1974; Rodricks, 1977; Steyn, 1977; Wilson and Hayes, 1973).

II. Biological Effects of Mycotoxins Due to the diversity of chemical structures, mycotoxins may exhibit a number of biological effects including both acute and chronic toxic effects as well as carcinogenic, mutagenic, and teratogenic in both prokaryotic and eukaryotic systems. In animal systems, the biological actions of mycotoxins are affected by sex and species of the animal, environmental factors (Campbellet al., 1972),nutritional status (Newberne, 1974a; Wogan, 1975a), and mycotoxin synergism (Lillehoj and Ciegler, 1975). Thus, the biological effects of mycotoxins are as varied as the chemical structures of the toxins. A large volume of information has been accumulated on this subject; consequently, this discussion will be confined to those mycotoxins of which the modes of action have been studied to some extent in recent years. Detailed information related to the biological effect of mycotoxins may be found in recent reviews by Austwick (1975), Ciegler (1975), Ciegler et al. (1971a), Newberne (1974a,b), Purchase (1974), and Wogan (1975b). Review articles on some important specific mycotoxins also are available and will be cited when necessary.

A. ACUTE AND CHRONIC TOXICEFFECTOF MYCOTOXINSI N ANIMALS 1 . Hepatotoxic Effect

Information on the acute and chronic toxic effects of mycotoxins in animals is generally obtained from experimental and naturally occurring mycotoxicosis. Since, in general, mycotoxicosis is mediated through specific organs and tissues with distinct clinical features, mycotoxins can be classified as hepatotoxins, nephrotoxins, etc., as shown in Tables I and II, in which some of the most common mycotoxins and their toxic effects are summarized. The liver is the most common organ attacked by mycotoxins. The hepatotoxic mycotoxins induce both nonspecific liver injuries such as fatty and pale liver, extensive necrosis, and hemorrhage, and specific lesions caused by specific mycotoxicosis (Austwick, 1975). Among mycotoxins causing hepatic damage, aflatoxin B, (afla B,) is the only mycotoxin which has been studied extensively (Butler, 1974; Ciegler et al., 1971a; Goldblatt, 1969; Wogan, 1975b) and is one of the most potent toxins among a series of aflatoxins produced by A. jlavus and A. ,parasiticus. The toxin causes typical proliferation of the bile duct, centrilobular necrosis and fatty infiltration of the liver, and hepatomas in addition to the generalized hepatotoxic effects in

86

F. S. CHU

TABLE I TOXICITYOF SELECTED HEPATOTOXIC MYCOTOXINS

LDm Mycotoxins

Producing fungi

(mdkg)

References

7.2 ( 6 , r, p.0.): 6.0 ( 6 , r, i.p.), 17.9 (9,r, i.p.)

Butler (1974)

Aflatoxin B,

Aspergillus flavus, A. parasiticus

Aflatoxin G,

Aspergillus flavus, A. parasiticus

a-Amanitin

Amanita phalloides and other toxic Amunita sp.

0.1 (h, P.o., lethal)

Haffield and Brady (1975)

Cyclochlorotine

Penicilliuin islandicum

0.33 (m, i.p.)

Enomoto and Ueno (1974)

Luteoskyrin

P. islandicum, P. rugulosum

40.8 (m, i.p.)

Enomoto and Ueno (1974)

-b

Butler (1974)

Ma1toryzin e

Aspergillus oryzae

3 (m)

Iizuka (1974)

Phalloidins

Amanita phalloides

1-2 (h, P.o., lethal)

Haffield and Brady (1975)

Rubratoxin B

P. rubruin, P. purpurogenum

0.36 (r, i.p.), 6.6 (r, p.0.)

Hayes (l977a)

Rugulosin

P. rugulosum

55 (m, i.p.),

Ueno et al. (1971)

44 (r, i.p.)

Sporidesmin

Pithomyces chartaruin

S terigmatocystin

A. versicolor, A. nidulans, and others

-

Atherton et al. (1974)

60-65 (6, r, i.p.) ~

~~

Van der Watt (1974) ~

~

‘Abbreviations used in Tables I and 11. I3 : male, P : female, h: human, m: mouse; r: rat; i.p., i.v., and p.0. represent intraperitoneal injection, intravenous injection, and oral feeding or intubation, respectively. ’Aflatoxin B1 is reported to be approximately twice as toxic as aflatoxin G, in 1-day-old duckling and rainbow trout. ‘Varied with animal species; sheep is most sensitive.

many animal species. The susceptibility of animals to afla B1 varies considerably with the animal species with a decrease in sensitivity (LD5,,:mg/kg) in the following order: ducklings (0.34), rabbits (0.3), mink (0.5-0.6), cats (0.55), pigs (6-7 kg size, 0.6), trout (0.8), dogs (l.O), guinea pigs (1.4-2.0), sheep (2.0), monkeys (2.2), chickens (6.3),rats (5.5 for male, 17.9 female), mice (9.0), and hamsters (10.2) (Butler, 1974; Ciegler, 1975). Outbreaks

MODE OF ACTION OF MYCOTOXINS A N D RELATED COMPOUNDS

87

TABLE I1 TOXICITY OF SELECTED MYCOTOXINS SHOWING LESIONS OTHER THANHEPATOTOXIC EFFECT

Mycotoxiris Nephrotoxins Citrinin Ochratoxin A

Producing fungi

P . citrinuin, P . oiridicatuin Aspergillus ochraceus P . oiridicatum

LD5" (mgikg)

35 (m.

S.C.)~

References

Steyn (1977)

20 (r, i.p.)

Chu (1974a)

7.5 ( 6 , rn, i.p.) 29 (6, m, p.0.) 15-35 (r, in,

Ueno (1974)

and others Neurotoxins Citreovirdin Patulin Penitrein A Roquefbrtine Vomitoxin Genitotoxin Zearalenone

P . citreo-oiride P . patulum and others P . crustosum, P . pulitans P . royueforti

i.v., i.p.) 1.1 (m, i.p.)

15-20 (6,m, i.p.)

Fusuriuiii grarninearuni

F . graminearuiii

Scott et al. (1975) Vesonder et a1 (1973) Mirocha and Christensen (1974)

and others Dermatoxin and others Cytochalasin E

Stott and Bullerman (1975a) Ciegler (1975)

A. claoutus

2.6 (r, i.p.)

Fusarenon X

F . nioale, F . epishuria, and

3.4 ( 6 , m, i.p.)

Nivalenol

F . nioab P. roquefurti

Glinsukon et al. (1975a,b) Saito and Ohtsubo (1974)

others

PR Toxin T 2

F. tricinctuin and others

4.0 ( 6 , m , i.p.) 11 (r, i.p.), 115 (r, p.0.) 3.8 (r, p . ~ . ) 3.04 (m , i.p.)

Saito and Ohtsubo (1974) Wei et al. (1973) Sinalley and Strong (1974)

aAbbreviations are as in Table I; s . c . , subcutaneous injection.

linked to aflatoxicosis in humans have been reported occasionally with the most recent one involving more than 300 people (Krishnamachari et al., 1975). Because some of the clinical features of the Reye-Johnson Syndrome are similar to experimental aflatoxicosis and because afla B1 and/or its derivative has been isolated from the livers of some of these patients (Harwiget a l . , 1975; Chaves-Carballo et al., 1976), the involvement of aflatoxins as one of the possible etiological agents has been suggested (Harwig et al., 1975).

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F. S. CHU

The basic structures of aflatoxins are shown in Fig. 1. As many as 14 dif€erent aflatoxins, produced either by the fungi or by metibolic conversion have been characterized. Because earlier investigations show that the toxicity of 4 basic aflatoxins follows the order of B1 > GI > Bz > G,, it has been suggested that both the dihydrohrofuran moiety and cyclopentenone ring are important for biological activity. This hypothesis was substantiated by subsequent studies on the biological activity of different aflatoxin derivatives. Modification of the C-2-C-3 unsaturated double bond by additional reactions with water (Pohland et al., 1968)or other organic solvents resulted in a substantial loss of toxicity (Wei and Chu, 1973). Likewise, conversion of the carbonyl group in the cyclopentenone ring to an alcohol, i.e. aflatoxicol (Detroy and Hesseltine, 1970), carboxymethyloxime (Chu et al., 1977), and trihydroxyl afla B1 (Ashoor and Chu, 1975b)also resulted in loss of toxicity to different degrees. Other evidence to support the importance of the ring structure is the lesser toxic effect of sterigmatocystin (Fig. 2) and its derivatives in which only the dihydrohrofuran moiety resembles afla B,. With the exception of afla and the mushroom poisons, a-amanitin and phalloidin (Hatfield and Brady, 1975), the significance of other hepatotoxic A

B

s

R1

A H A O H A H A H A H A H AB H AB' H AB OH C H BC H B'C H 0c OH

-

-

FIG. 1. Structures of aflatoxins. The abbreviations S, F, and M indicate structure type, aflatoxin produced by fungi, and metabolite of aflatoxin, respectively. Structure of parasitic01 is not shown.

MODE OF ACTION OF MYCOTOXINS A N D RELATED COMPOUNDS

Ri Rz OH CH, H H H CH,

Aspertoxin Sterigrnatocystin 0-methylsterigrnatocystin

FIG. 2. Sterigmatocystin and its derivatives.

Luteoskyrin Rugulos in

R OH

H

FIG.3. Luteoskynn and rugulosin

C ycbchbrdine FIG.4. Cyclochlorotine.

89

90

F. S . CHU

Rubratoxin A , R=H,OH Rubratoxin 8 ,R=O FIG.5. Rubratoxins.

mycotoxins to human health is not known. Although the yellow rice mycotoxins such as luteoskyrin and rugulosin (Fig. 3), and cyclochlorotine (Fig. 4) (Enomoto and Ueno, 1974), which cause acute liver atrophy, cirrhosis, and tumors, have been considered to be important for the rice-consuming people in Asia, only one natural outbreak in chickens was reported (Enomoto and Ueno, 1974). Other hepatotoxic mycotoxins, such as rubratoxin B (Fig. 5 ) (Hayes, 197713; Newberne, 1974b) and spordesmins (Atherton et al., 1974), all may have economic importance because of their association with some mycotoxicoses in animals. Although different mycotoxins can be classified according to their primary lesion in target organs, damage to other organs and tissues may occur also. For example, both aflatoxin and sterigmatocystin cause liver damage in animals, but injury to kidneys was observed also at higher doses (Butler, 1974; Van der Watt, 1974). Therefore, in characterizing a specific mycotoxicosis, identification of a specific causal mycotoxin(s) or fungi in the feeds and food or animal tissues is essential, as well as the information obtained from clinical, pathological, and biochemical analysis. 2 . Nephrotoxic Effect

Unlike aflatoxins, which are produced only by A . jlavus and A . parasiticus, the nephrotoxic ochratoxins are produced by a number of fungi in the A. ochraceus group, Penicillium viridicatum, and 5 other penicillia. Ochratoxin A (OA), the most toxic member of this group, causes kidney damage, such as degeneration of the proximal tubule, in many animal species, including beagle dogs, chicken, duckling, mice, rats, sheep, swine, and rainbow trout. Liver necrosis and enteritis were also observed in these animals. Hydrolysis of OA to Oa (Fig. 6) resulted in a loss of toxicity. Ochratoxin B (OB), the dechlorinated ochratoxin, was also found to be less toxic. Substitution of phenylalanine with other amino acids in the molecules resulted in different active derivatives with varied toxicity (Steyn et al., 1975; Wei and Chu, 1974). Structure-activity studies revealed that the dis-

MODE OF ACTION OF MYCOTOXINS AND RELATED COMPOUNDS

Ochratoxin A

B C A Methyl ester B Methyl ester B Ethyl ester 4-OH-OA

d

P

;1

?;

a b c c b a OH OH

H CI CI H H CI CI H

91

; 3 H H H H H OH H H

FIG. 6. Ochratoxins.

sociation of the phenolic hydroxyl group in the isocoumarin ring is important for toxicity (Chu, 1974a). Ochratoxin A and possibly other ochratoxins have been found to be associated with the endemic porcine nephropathy in Denmark (Krogh, 1976a) and several other outbreaks in poultry in Denmark (Elling et al., 1975) as well as in the United States. Because of the striking similarities between Balkan endemic nephropathy (a fatal renal disorder in humans occurring in certain areas of Bulgaria, Rumania, and Yugoslavia), and the OA-induced porcine nephropathy, it has been suggested that OA may be involved in the endemic disease. A recent investigation (Krogh, 1976b) which indicated that the contamination of foods with OA in the endemic area was more frequent than in the nonendemic areas reiterates the impact of OA on human health. Citrinin is another nephrotoxic mycotoxin which once was suggested as having possible involvement in +e porcine nephropathy, but it does not cause enteritis in experimental animals. Because citrinin rarely occurs in nature, it was considered less likely to be involved in the endemic nephropathy (Krogh, 1976a,b).

3 . Dermutoxic and Alimentary Toxic Effects All mycotoxins in the trichothecene group contain the basic tetracyclic sesquiterpene structure (Fig. 7) and are produced by various species of Cephalosporium, Fusarium, Myrothecium, Trichothecium, Trichodermu, and Stachybotrys. The 12,13-epoxytrichothecenesare capable of inducing dermal lesions, including several skin irritations, hemorrhaging, and desquamation by external application to the skin of the experimental animal; but because of structural variations, they also have other very diverse biological activities, such as antibiotic, cytotoxic, and neurotoxic (emetic) effects.

92

F.

S. CHU

B

A

SCIRPENE TRICHODERMIN T2 FUSAENON X NIVALENOL TRICHOTKCIN VOMITOXIN

H H OCOCH3 H OCOCH3 OCOCH3 OCOCH3 OH OH OH OH H OCOCH-CHCH3 H OH H OH

H H OH OH

H

H H

OH OH

H OH

H2 H2 CCOCH$H(CH3)2 -4 =O =O =O

R IN STRUCTURE B

FIG. 7 . Trichothecenes.

Neither carcinogenic nor mutagenic effects have been observed. Either oral ingestion or intraperitoneal injection of the toxin to animals causes inflammation of the GI tract, ulceration, hemorrhaging, edema, diarrhea, degeneration of bone marrow, and leukopenia. Specific lesions and biological activity, however, varied with the specific trichothecene. The trichothecenes become inactive upon reduction with LiAIHl or treatment with acid to form the apotrichothecenes. Reduction of the 9-10 double bond by hydrogenation resulted in a marked decreas in biological activity. Thus, both the 12,13epoxide and the 9-10 double ond are essential for all the biological activities. The effect of alterations of the side chain residues on biological responses can be seen from the examples listed in Table 11. Details on the chemistry and biological activity of trichothecenes have been reviewed by Bamburg and Strong (1971),Smalley and Strong (1974),and Ueno (1977). The 12,13-epoxytrichotheceneshave emerged in recent years as an important group of mycotoxins because of their possible association with several mycotoxicoses in humans and farm animals, the latter resulting in considerable economic loss. Although it has been suggested that trichothecenes might be responsible for ATA disease because of the similarity of the toxic effect of these mycotoxins with the clinical symptoms of ATA, it was not proved until Tz toxin was isolated from the suspected outbreak samples by Mirocha and Pathre (1973) and by Yagen (1977).The Tz toxin and other related

\

MODE OF ACTION OF MYCOTOXINS A N D RELATED COMPOUNDS

93

trichothecenes may be involved also in moldy corn toxicosis in farm animals including poultry, swine, and cattle (Smalley and Strong, 1974). The isolation and characterization of several 12,13-epoxytrichothecenes,such as roridin E, stratoxins C, F , G, and H produced by toxic Stachybotrytis atra, a fungus responsible for stachybotryotoxicosis (Eppley and Bailey, 1973; Rodricks and Eppley, 1974; Eppley, 1977) implicated the possible association of the trichothecenes with this mycotoxicosis. Recent investigations on the causative agents of emesis and feed refusal in farm animals show that several trichothecenes, including T2 (Ellison and Kotsonis, 1973), vomitoxin, and two other trichothecenes might be involved also (Y. Ueno et al., 1974a; Morooka et al., 1972; Vesonder et al., 1973, 1976). Other fungal metabolites showing dermatoxic effects include butenolide (Yates et al., 1968),and the photosensitizing furocoumarins produced in celery as the result of infection of pink rot fungus Sclerotinia sclerotiorium. Furocoumarins cause dermatoxic effects only when the skin is exposed to the toxin and sunlight simultaneously. 4 . Neurotoxic Effects

Although manifestation of toxicity of fungal secondary metabolites through

the nervous system were documented for some classical compounds, such as the ergot alkaloids on smooth muscle and the mushroom toxin muscarin, a cholinergic substance acting on the postganglionic parasympathetic synapses, outbreaks of mycotoxicosis associated with the nervous system were identified more recently. Slaframine, which causes excessive salivation in farm animals after either biological or photochemical activation to a quarternary amine, acts physiologically similarly to acetylcholine (Aust, 1974). Penitrem A, a mycotoxin causing several tremors and convulsions, is produced by fungi that were originally involved in outbreaks of mycotoxicosis in sheep and horses (Wilson et al., 1968) and in cattle (Ciegler et al. 1976b). Subsequent work has led to the isolation from penitrem B and C (Hou et al., 1971), fumitremorgin A (Yamazaki et al., 1971), verruculogen (Cole and Kirksey, 1973), and several other active tremorgenic substances (Ciegler et al., 197613). Since the structures of most tremorgens are not known, the modes of their action thus have not been studied in detail. Stern (1971) and Cysewski (1973) suggested that penitrem A acts at the level of the spinal cord in mice and rabbits used as test animals. Wilson et al. (1973) found that penitrem A increased the frequency and mean amplitude of the miniature end plate potential of the isolated rat phrenic nerve diaphragm and suggested that the tremorgen might act at the pre- and postjunctional sites, thus causing the spontaneous release of the transmitter. Details on the tremorgenic substances produced by fungi were covered by Cole (1977) and Ciegler et al. (1976b).

94

F. S. CHU

0-

c=o

CkH PATULIN

FIG. 8. Patulin.

Other mycotoxins known to affect the nervous system are citreoviridin and patulin (Fig. 8). Citreoviridin affects the central nervous system causing paralysis and convulsions with subsequent respiratory and cardiac failure and death. Because the clinical symptoms induced by this toxin are similar to those of cardiac beriberi in humans, the possible association of this mycotoxicosis with cardiac beriberi has been suggested (Ueno, 1974). Data on the natural occurrence of this mycotoxin in foods and feeds are not available, although the fungi producing this mycotoxin has been isolated from moldy foods (Ueno, 1974). Patulin causes pulmonary edema, vomiting, and dermatitis in experimental animals and also has been shown to be carcinogenic to mice. This mycotoxin was originally isolated as an antibiotic, but because it is toxic to both animals and plants, it has not been accepted as a practical antimicrobial agent. With the reports of occurrence of patulin in foods, especially in apple juices, patulin has been considered a potentially hazardous mycotoxin to humans and animals (Scott, 1974; Stott and Bullerman, 1975a; Ciegler, 197713). Another neurotoxic mycotoxin, roquefortine, produces convulsive seizures in mice (Scott et al., 1976).

5 . Other Biological Effects in Animals The naturally occurring zearalenone derivatives produced by F . roseum and other fusaria in cereal products may be of great economic importance even though they have no acute toxic effects on animals. Zearalenone (F-2 toxin, Fig. 9) is not a steroid but produces a true estrus with enlargement of both vulva and uterus and other general responses associated with estrogens. Whereas a number of farm animals such as poultry and cattle may be suscep-

FIG. 9. Zearalenone

MODE OF ACTION OF MYCOTOXINS A N D RELATED COMPOUNDS

95

tible to zearalenone, swine is the most sensitive animal species. More than 10 estrogenic syndromes in swine outbreaks have been documented since 1928. Chemical modification of zearalenone have resulted in a number of compounds with varied activity. For example, reduction of the ketone in the b' position to a hydroxyl group and the double bond at C-1',2' position resulted in two isomers one of which is 4 times more active than the parent compound. This new isomer has been used as an anabolic agent. The hydroxyl group or some equivalent functional group is necessary also, at least in position two of the benzene ring (Mirocha and Christensen, 1974). While some examples regarding the implication of specific mycotoxins in mycotoxicosis in animals have been summarized, the cause of many other types of mycotoxicosis are still unknown. Considerable efforts to identify new mycotoxins and their association with mycotoxicosis have been made. The chemistry and biological effects of mycotoxins other than aflatoxins, trichothecenes, and zearalenone have been reviewed (Steyn, 1977). Low levels of mycotoxins in foods result in an increase in mortality and reduced weight gain and an impairment of resistance and immunity to disease in livestock, thus causing economic loss. Richard et al. (1975, 1977) have reviewed the effects of several mycotoxins on immunity in animals. In general, mycotoxins mostly affect the nonspecific hormonal substances and phagocytic processes. Certain cellular immune mechanisms, including the function of T lymphocytes and delayed hypersensitivity, were found to be affected by aflatoxins (Richard et al., 1977).

B. CARCINOGENICEFFECTOF MYCOTOXINS Perhaps the most significant event in the research of secondary fungal metabolites during the last 15 years was the discovery of afla B, as the most potent liver carcinogen. This toxin primarily causes hepatocellular carcinoma and cholangiocarcinoma in the liver of many animal species, including trout, salmon, duck, rat, mouse, rhesus monkey, and marmoset, after repeated dosing or chronic feedings. Renal adenocarcinomas are observed also in some rat strains (Wogan, 1975b). Other tissues affected by the toxin include the trachea and the skin (Wogan et al., 1971; Lindenfelser et al. 1974). Dietary level of afla B, and G , for the induction of liver tumors in trout and the rat was found to be varied with the animal strains. In the rats, the levels were in the range of 0.5 to 1.5 ppm and 1.0 to 3.0 ppm for afla B1 and GI, respectively (Butler, 1974). The potency of afla M1 to induce liver tumors in trout is about 30% of that of afla B,. Sinnhuber et a2. (1974) observed a 60% incidence of liver tumor in trout when they fed the animal with afla M, at a dietary level of 16 ppb over a period of 1 year. In the rats, the potency of afla M, is much weaker than that of afla B1. M a B2 was found to be as active as G1

96

F. S. CHU

in inducing liver tumors in trout, but it is about 150 times less effective than B, in rats. Atla G, was inactive in trout (Wogan, 197513; Wogan et al., 1971). A number of other mycotoxins have been shown to be carcinogenic, but at a dietary level much higher than that of afla B,. The regular dietary daily dosage (ppm) necessary to induce hepatoma by luteoskyrin, rugulosin, griseohlvin, and sterigmatocystin was reported to be 50-100 (mouse), 200 (mouse), 5OOO-10,OOO (mouse), and 30-100 (rat), respectively (Enomoto and Saito, 1972). Cyclochlorotine induced hepatoma in mice at a daily dose of 60 mgkg (Enomoto and Ueno, 1974). Other mycotoxins reported to be carcinogens are patulin and penicillic acid which cause sarcoma in rat by subcutaneous treatment (Dickens and Jones, 1961, 1965). Citrinin was quoted as a carcinogen (Ueno and Kubota, 1976); details have not been reported. Although the significance of carcinogenic mycotoxins to human health cannot be assessed until more epidemiologicaldata regarding the actual level of exposure to specific mycotoxin and the incidence of special cancer are available, certain circumstantial evidence on the possible association of afla B, with liver cancer in certain parts of the world, such as Thailand (Shank et al., 1972a,b,c) and Kenya (Peers and Linsell, 1973; Peers et al., 1976) has been accumulated;Van Rensburget a2. (1974)showed a significant correlation between the afla B1 content in foods at a level of 7.8 ppb (wet weight, or 20 ppb of dry foods) and the high incidence of primary liver cancer rate of 25-35 per 100,OOO per year. Carcinogenic mycotoxins are potential hazards to human health even though the carcinogenic potential for most mycotoxins has not been studied extensively nor is the level of some known carcinogenic mycotoxins other than afla B, in foods and feeds known.

EFFECTSOF C. MUTAGENICAND TERATOGENIC MYCOTOXINS 1. Mutagenic Effect

While strong evidence for the carcinogenicity of several mycotoxins has been accumulated, some mycotoxins also show mutagenicity. Most of the studies have been centered on aflatoxins (Ong, 1975, 1977). M a B1 causes chromosomal aberrations and inhibition of mitosis in a number of systems. Induction of mutations by afla B1 were reported for Bacillus subtilis in the transforming DNA assay, Neurospora crassa adenine3 (ad-3) sensitive vegetative culture, Saccharomyces cerevisiae growing cell, streptomycinresistant mutation of Chlamydomonusreinhardii, and Chinese hamster cells. In addition, afla B1 is able to induce autosomal recessive-lethal mutations in Drosophila melanogaster (Ong, 1975, 1977). Whereas Epstein and Schafner (1968)reported that a mixture of afla B1 and G, caused the dominant-lethal mutations in male mice at a dose of 68 mgkg, Leonard et al. (1975) found

MODE OF ACTION OF MYCOTOXINS AND RELATED COMPOUNDS

97

that afla Bl at a dose of 5 mgkg did not produce gross structural chromosomal changes in male mouse germ cells in vivo. They attributed this fact to poor penetration or metabolic degradation. In the Neurospora system, the relative mutagenicity of afla B1,GI, B,, and G, seems in line with the relative toxicity (acute) and carcinogenicity of these toxins. A number of other mycotoxins also have been shown to have a mutagenic effect. Patulin causes petite mutations in the exponential growth phase of S. cerevisiae (Mayer and Legator, 1969) and also causes chromosome breakage in salamander eggs during mitosis (Scott, 1974). Furocoumarins are mutagenic in several bacterial and yeast systems when ultraviolet light is applied simultaneously (Musajo and Rodighiero, 1972; Averbecket al., 1975). DNA breakage in HeLa cells was found to be induced by afla B1, patulin, and penicillic acid, but not by luteoskyrin, rubratoxin B, and fusarenon X (Umeda et al., 1972).Although rubratoxin B causes chromosomal aberrations in HeLa cells (Umeda et al., 1970) and dominant-lethals in mice (Evans et al., 1975), Kuczuk et al. (1977)were unable to demonstrate the mutagenicity either in the Ames Salmonella system (Ames et al., 1973) or the S . cerevisiae system. Both luteoskyrin and rugulosin were able to produce respiratory deficient mutants of S. cerevisiae (Ueno and Nakajima, 1974). Ueno and Kubota (1976) surveyed the mutagenic and carcinogenic potentials of 35 mycotoxins or mycotoxin derivatives by testing the DNA-attacking ability in the recombination-deficient mutant of B. subtilis M 45 (rec-) and the parent strain H 17 (rec+). Among the toxins tested, atla Bl and G1, citrinin, luteoskyrin, patulin, penicillic acid, PR toxin, rugulosin, sterigmatocystin, 0acetylsterigmatocystin, 0-acetyldihydrosterigmatocystin, zearalenone, and zearalenol b gave positive results. Although 8 of the 13 mycotoxins which gave positive results have been reported as carcinogens, the significance of these results is not known, especially since some of these mycotoxins affect the DNA polymerase enzymes and some of them including griseofulvin and cyclohlorotine are reported to be carcinogens but give negative results in the recombination assay. The increasing evidence of the close relationship between mutagens and carcinogens and the theory that metabolic activation is a prerequisite for most carcinogens, led Ames et al. (1973) to devise a mutagenic testing system including an activation step by liver homogenate (S9) or microsomal preparation. Afla B1 as activated by rat, human, and hamster liver homogenate, was shown to be both toxic and mutagenic to Salmonella typhimurium strains TA 1530, 1531 (60 p M ) , 1537, 1538 (1 pg/ml) (Ames et al., 1973; McCann et al., 1975) and to N. crassa (0.67 ,uM Ong, 1975). The activated afla B1acted as an inducer for Escherichia coli K12 (A) and had a mutagenic affect on A phage (Goze et al., 1975). Subsequent studies with S. typhimurium strain TA 98 (Wong and Hsieh, 1976) revealed that afla Bl was the most potent mutagen among the aflatoxin series. The relative mutagenic-

98

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S . CHU

ity of afla are listed in decreasing order as follows: Afla B1 (potency = 1)> aflatoxicol(O.228)> G, (0.033) 5 M, (0.032)> H1 (0.020) > Q1 (0.012) > Bz (0.002) > PI (0.001) 3 Gz (0.001). Afla Bz actually exhibited an activity lower than the controls, thus afla Bz, P1 and Gz were considered to be nonmutagenic. In general, the mutagenic potential of different mycotoxins in this system is in accordance with the in uivo carcinogenicity. The microsomal enzymes which activated afla B1, G , , aflatoxicol, and sterigmatocystin were found also to induce chromosome breakage, DNA damage and lethality in cultured human fibroblasts (Stich and Laishes, 1975). Induction of such mutagenic defects by activated afla B1 and sterigmatocystin was found to be much more pronounced in the DNA-repair-deficient cells of xeroderma pigmentosum than in the control human fibroblasts. Neither these compounds without activation nor afla Bz and G, with activation show the mutagenic defects in human cells. Stich and Laishes (1975) did not find significant differences in the capacity for activation of d a B1 and sterigmatocystin by S9 &actions of liver, kidney, and lung obtained from various animal species in the human cell system.

2 . Teratogenic Effect of Mycotorins Although embryotoxicity of some mycotoxins has been known for some time, the teratogenic effect of most mycotoxins was not known until the last few years. Teratogenicity of afla B1 to hamster, rats, and mice was demonstrated as early as in the mid 1960s (Di Paolo et al., 1967). Evidence of teratogenicity to mammals of other mycotoxins including griseofulvin (Klein and Beall, 1972; Scott et al., 1975), OA (Brown et aZ., 1976; Hayes et al., 1974a; More and Galtier, 1974), rubratoxin B (Hood et al., 1973; Koshakji et al., 1973) and Tz (Haynes, 1976) have been reported. In addition, a teratogenic effect of cytochalasin B, (Linville and Shepard, 1972), patulin and patulin-cysteine adducts (Ciegler et al., 1976a) in the chicken embryo was demonstrated. Hayes (197713)using data obtained from his laboratory as well as others, concluded that afla B1, OA, rubratoxin B and Tz are teratogens, and that citrinin, viriditoxin, and alternariol are embryocides and/or growth retardants. No adverse effects have been observed for penitrem A, penicillic acid, maniliformin, and sterigmatocystin (Hayes, 1977b). While the data on the mycotoxin teratogenicity in mice and hamsters are diacult to extrapolate to humans, the accumulated data indicate the potential hazard of mycotoxins as environmental teratogens. D. BIOLOGICALEFFECTSOF MYCOTOXINSOTHERTHAN I N ANIMAL SYSTEMS In addition to the various toxic effects of mycotoxins in animal systems, there are other biological effects including antimicrobial, antiprotozoal, an-

MODE OF ACTION OF MYCOTOXINS A N D RELATED COMPOUNDS

99

titumor, as well as cytotoxic and phytotoxic effects. Interest in the investigation ofthese specific biological effects developed for the following reasons: (1) to search for a simple and sensitive method for confirmation and analysis of mycotoxins; (2) to provide a simple system for investigation of mode of action; and (3)to investigate the possibility of some fungal metabolites such as patulin and some trichothecenes as therapeutic agents. Because the review of this subject could be treated as a separate entity, only the sensitivity of some lower organisms or tissue cultures for selected mycotoxins will be summarized. In a survey on the sensitivity of329 microorganisms to afla B1, Burmeister and Hesseltine (1966)found that only one strain of Bacillus brevis and two of B . megaterium were most sensitive. Subsequently, B . megaterium NRRL 1368 was used as a strain for the microbiological assay of afla B1, 1 pg/ml (Clements, 1968a), OA, 4 pg/ml (Clements, 1968b), and patulin, 2 pg/ml (Stott and Bullerman, 1975b). B . cereus var. mycoides LSU was also sensitive to ochratoxins A and B, 1.53 p g (Broce et al., 1970). Among the number of gram-positive and gram-negative bacteria and fungi that are sensitive to patulin (Scott, 1974), Stott and Bullerman (1975b) considered B . megaterium to be the most sensitive one. Of 4 strains of B . thuringieus which are sensitive to patulin (200 pg/ml) and other mycotoxins to a certain degree, Boutibonnes (1975) found that only two are sensitive to afla B, and G,. Boutibonnes and Auffray (1975) found that coumarin suppressed the inhibitory effect of afla B1 on the sensitive bacillus and suggested that they may have similar modes of action. Heller and Roschenthaler (1976) and Heller et al. (1975) found that OA inhibits gram-positive bacteria but not gramnegative ones. Inhibition of the growth of both B . subtilis and Streptococcus fuecalis by OA was found to be pH dependent and could be reversed by addition of Mg2+, and cyclic AMP and GMP. Ochratoxin A only exhibited a bacteriostatic property but no killing effect. Reduction of protein and RNA synthesis by OA was observed in both bacteria but not in DNA synthesis. The antagonistic effect of cyclic nucleotides could be as signifcant as the mode of action of OA in bacteria. Whether similar application can be made to animals is not known, especially since OA does not inhibit bacteria at a pH above 6.8, but the dissociation of the phenolic group does appear necessary for OA action in animals. Inhibition of E . coli strain F-11, B . subtilis, Staphylococus aureus (50% inhibition all at 0.2-1.5 pg/ml), Alcaligenes fuecalis and Smatia murcescens (at 0.354.5 pg/ml) by luteoskyrin and rugulosin was reported (Enomoto and Ueno, 1974). Antiprotozoal activity was observed for several mycotoxins. Luteoskyrin and rugulosin (R) were most effective in inhibition of Tetrahymena pyrifbrmis with LDS0at 1-2.5 pg/ml and 5-10 pg/ml, respectively (Enomoto and Ueno, 1974). This organism is the only lower organism sensitive to rubratoxin B (Hayes, 1973; Hayes and Wyatt, 1970). It is less sensitive to

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trichothecenes (Saito and Ohtsubo, 1974), insensitive to OA (Hayes et al., 1974b) and degraded afla B, and G, (Teunisson and Robertson, 1967). Mycotoxins also have toxic effects to brine shrimp Artemia salina, zebra fish larvae, and tracheal organ cultures from chicken or Japanese quail. Arternia salinu is most sensitive to afla B1 with LD50 of 0.5 pg/ml (Brown, 1969; Harwig and Scott, 1971). The sensitivity of sterigmatocystin, gliotoxin, and afla B1 to larvae of zebra fish Brachydanw uerio was less than 1 pg/ml. Afla Bz, G1, and GZ, stemphone, diacetoxyscirpenol, OA, aspertoxin, and patulin, are toxic to the zebra fish but in higher concentrations (Abedi and Scott, 1969). In tracheal organ cultures, the minimum detection levels were in nanogram quantities, sterigmatocystin being the most sensitive (12 ng), followed by afla B1 (B)gliotoxin , (85), afla G1 (89), sporidesmin (139), sporidesmin B (la), ochratoxin A (175), patulin (750), and afla B2 (1367) (Cardeilhacet al., 1972). The trichothecenes have shown a very wide range of biological responses. Some have a good antibiotic effect and others are devoid of antimicrobial activity (Smalley and Strong, 1974; Saito and Ohtsubo, 1974). The cytotoxic effect of some trichothecenes was found to be most pronounced (Perlman et al., 1969). Ueno and his colleagues (Ueno and Shimada, 1974) have employed a bioassay for the trichothecene mycotoxin using criteria of their inhibition on the incorporation of [ 'Tlleucine into rabbit reticulocytes. In this system, roidin A was most sensitive (LD5o at 0.006 pglml), followed by verrucarin A (0.015 pg), Tz(0.03pg), 7-OH-deacetyls cirpenol (0.3 pg), and 7,8-dihydrodiacetylcalonectin(20 pg). Because both roidin A and verrucarin A have a monocylic ring, they concluded that the inhibitory potency of trichothecenes in this system depends upon the hydrophobic nature of the side chain in the molecules. Ill. Modification of Carbohydrate and Lipid Metabolism by Mycotoxins

A. EFFECTOF MYCOTOXINS ON CARBOHYDRATE METABOLISM The effect of mycotoxins on carbohydrate metabolism has been generally shown from the alteration of hepatic glycogen and serum glucose levels after the animal received the toxin. For most mycotoxins, including atla B1 (Shank and Wogan, 1966),cyclochlorotine (or islanditoxin), (Uenoet al., 1963;Hara, 1964), OA (Suzuki and Satoh, 1973; Chang, 1975), and rubratoxin B (Hayes and Wilson, 1970), a reduction of hepatic glycogen level was observed. Shank and Wogan (1966)found that the liver glycogen and glycogenesis in

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ducklings decreased 30-70% after the birds received a sublethal dose of afla B1 (60 pgkglday) for 5 consecutive days, but they did not detect any change in liver glycogen level in weanling rats treated under similar conditions except at a higher afla B1 level (600 pgkglday; rats are less susceptible than ducklings). Because the feeding efficiency was found to be decreased appreciably in the duckling experiment, they attributed the observed glycogen reduction in duckling liver to the diminished food consumption. The mechanism by which afla B1 caused the reduction in hepatic glycogen level in 1-day-old chicks was studied by Shankaran et al. (1970). These investigators found a significant decrease in UDP-glucose-glycogen transglucosylase, glycogenphosphorylase, phosphoglucomutase, and glucose-6phosphatase activity accompanied by an increase in the combined hexosemonophosphate dehydrogenase activities of chicken livers 48 hours after having received a single i.p. injection of 2.7 mg afla BJkg. In addition, the incorporation of [14C]-glucoseinto glycogen and into total livers was found to be decreased substantially. These results suggested that the alfa B1 induced glycogen depletion in chicken livers was due to an impairment of the synthetic process. Marked reduction of hepatic glycogen with an accompanying increase of serum glucose in mice was observed after the animal received a small amount of cyclochlorotine (Ueno et al., 1963; Hara, 1964). From studies on the mode of action Ueno et al. (1963) found that cyclochlorotine exerted its effect on glycogen metabolism both in decrease in synthetic ability and in an increase in metabolism of the precursors. Incorporation of ['4C]-glucose or [14C]-acetateinto liver glycogen was found to be suppressed after the mice received the toxin. Using liver slices, they found a decrease in the incorporation of [14C]-glucosein the presence of the toxin. While these investigators did not find significant change in the enzyme activity of phosphorylase, D-glucose-1-phophohydrolase, ~-fructose-l,6-diphosphate-l-phosphohydrolase and anaerobic glycolysis, they found a decrease in UDP-glucoseglycogen-transglucosylase activity and an increase in glucose-6-phosphate dehydrogenase activity in the cell-free system treated with the toxin. Because only a small amount of cyclochlorotine caused the reduction of glycogen in liver, this effect was considered to be one of the major biochemical lesions of the toxin. Although it has been considered that interference of carbohydrate metabolism by OA may play a role in its toxic effect, there were some contradictory reports regarding the mode of its action. In an earlier report, Purchase and Theron (1968)found that OA caused an accumulation of glycogen in the cytoplasm of rat liver cells. This observation led to the speculation that possibly there was impairment of phosphorylase enzymes by OA. In an in vitro study, Pitout (1968) demonstrated the inhibition of the phos-

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phorylase enzyme system by OA, but he did not find any inhibitory effect on the individual enzyme involved. Thus, the exact site of enzyme inhibition remains unknown. Investigations in a number of other laboratories revealed that liver glycogen decreased appreciably after rats received the toxin (Chang, 1975; Suzuki and Satoh, 1973; Suzuki et al., 1975). A single dose of OA (15 mgkg) to rats caused a significant decrease in liver glycogen in both intact and adrenalectomized rats 4 hours after administration of the toxin, but with only slight increase in cardiac glycogen level. The nontoxic ochratoxin a, however, did not cause any alteration of glycogen level in both tissues. Because OA had no effect on the glycogen level of the liver or heart when the adrenalectomized rats were pretreated with hydrocortisone, Suzuki and Satoh (1973) postulated that OA may interfere with endocrine balance. Subsequent work by Suzukiet al. (1975)revealed that the marked depletion of liver glycogen was accompanied by an increase in serum glucose, lactate, and liver lactate in the OA-treated rats. N o alteration of hepatic lipids and proteins was found. Glycogen synthetase activities (type I and total) were reduced, but the phosphorylase activity was increased. From the observation of the reduction of incorporation of ['4C]glucose into hepatic glycogen in OA-treated liver, these investigators suggested that the OA-induced depletion of hepatic glycogen in rats could be attributed to the inhibition of glycogenesis in rat liver, suppression of transport of glucose into liver, and the acceleration of glycogenolysis. B. EFFECT OF MYCOTOXINS ON MITOCHONDRIAL

RESPIRATION Because cellular necrosis in the tissues or organs induced by some foreign compounds could be due to the lack of energy in the cells, there were a number of investigations regarding the possible inhibition of mitochondrial respiration by mycotoxins. Earlier information on such possibility were obtained from the observation of inhibition of some enzymes in the Kreb's cycle by mycotoxins (Clifford and Rees, 1967a; Brown, 1965; Ueno et al., 1964). Direct evidence was obtained mostly from in vitro studies in which mycotoxins, including afla B1, GI, and MI (Doherty and Campbell, 1972, 1973; Pai et al., 1975; Svoboda et al., 1966) luteoskyrin (Ueno, 1!366), OA (Moore and Truelove, 1970; Meisner and Chan, 1974), and rubratoxin B (Hayes and Hannan, 1973) inhibited oxygen consumption in various tissue homogenates of different species at toxin concentration in the range of lop4 M or lower when succinate or other substrate was used. Inhibition of mitochondrial respiration by aflatoxins was demonstrated both in vitro and in vivo. Whereas Svoboda et al. (1966)found a depression of oxygen consumption using the liver mitochondria prepared from rats fed

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with 0.45 mg afla B1 per kilogram of body weight, Clifford and Rees (1967a) failed to show any alteration of respiration capacity or P/O ratio using liver mitochondria prepared from rats dosed orally with 7 mg of toxin per kilogram. Inhibition of oxygen uptake using liver homogenates of male mice which received 7 mg afla B1 per kilogram was observed by Hayes and Hannan (1973). Except in weanling rats, these investigators did not find any inhibition in oxygen consumption by afla B1 at a concentration of 3.3 x lop5 M with mouse, rat, pigeon, and squirrel liver homogenates in vitro. The mechanism by which afla Bl inhibits rat liver mitochondrial electron transport was studied in detail by Doherty and Campbell (1972, 1973). Afla B1 at a concentration of 2.5 x M depressed oxygen consumption to a minimum of 40% in ADP-coupled mitochondria, and 60% in DNPuncoupled mitochondria using either pyridine nucleotide-linked substrate or succinate. Since afla B1 had no effect on the ascorbate-N,N,N',N'-tetra methyl-p-phenylenediamine (TMPN) shunt, and since the stage I11 respiration inhibition caused by afla B1 can be reversed by TMPD, it was concluded that the site of inhibition is between cytochrome b and cytochrome c 1 or c. This conclusion is consistent with the observation of Strufaldi et al. (1970), who found that afla B1has no inhibitory effect on NAD- and FAD-dependent substrates other than succinate. The site of afla B1 inhibition on the respiratory chain was confirmed by Pai et al. (1975), and these investigators further demonstrated that afla M l and GI also act on the same site. Afla B, was found to be most effective for such inhibition, while G I was least effective. At a concentration of 1 x M , afla M I uncouples oxidative phosphorylation and reduces ADP/O ratio whereas afla B1 at this concentration only uncouples the oxidative phosphorylation. Afla B1 at a concentration of 1 x lop5M effects both processes, but afla GI only acts as an uncoupler at 1 x M . Preincubation of mitochondria with all the aflatoxins resulted in inhibition of both respiration and uncoupling. These results thus show that afla concentration and structures greatly affect the two processes, either as an uncoupler or as an inhibitor (Pai et al., 1975). Because Doherty and Campbell (1973) found that the inhibition of respiration was more pronounced when submitochondrial particles were used and that aflatoxins bind with mitochondria (Pai et al., 1975), afla may therefore have a direct effect on the specific sites for oxidative phosphorylation rather than affecting the transport systems in the mitochondrial membrane. The effect of luteoskyrin on the mitochondrial respiration was studied by Ueno et al. (1964). Luteoskyrin inhibited a number of Krebs cycle enzymes at a concentration of 1 x 10V M , but the level required for the decrease of the ADP/O and an increase in the ATPase activity was found to be much lower than that required for the respiratory chain. The toxin was found also to interfere with the ATP-Pi, ATP-ADP exchange and inhibited both swel-

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ling and contracting of rat liver mitochondria in uitro (Ueno, 1966; Ueno et al., 1964). Ueno (1966) suggested that this mycotoxin may act similarly to oligomycin and DNP as an oxidative phosphorylation inhibitor and as an uncoupler. In view of the high &nity of this mycotoxin to mitochondria (I. Ueno et al., 1974) and the distortion of mitochondrial swollen structure in viuo as observed in electron microscopy (Saito, 1959, cited by Enomoto and Ueno, 1974), luteoskynn, therefore, may affect the transport system of mitochondria. Although it has been shown that the liver obtained from animals treated with cyclochlorotine showed an inhibition of respiratory enzyme and coupled phosphorylation, this effect was considered to be secondary when compared to cyclochlorotine’s effect on glycogen metabolism (Enomoto and Ueno, 1974). Ochratoxin A (OA) inhibited state I11 respiration completely at a concentration of 4 x M, but slightly stimulated state IV respiration. The state I11 respiratory inhibition caused by OA was released by DNP (Moore and Truelove, 1970). Ochratoxin a,the hydrolyzed product of OA, was found to be more potent than OA itself as a respiratory chain inhibitor. The inhibitory effect of OA on the respiratory chain was considered to be due to the interference of the toxin on the mitochondrial transport systems in a later study by Meisner and Chan (1974). These investigators confirmed the previous observations of Moore and Truelove (1970) but also found that the ADP/O ratio decreased progressively in the presence of OA up to a concentration of 4 x lop4M. The inhibition of respiration control was more pronounced than the decrease of ADP/O ratio. Ochratoxin A also inhibited the m-chlorocarbonylcyanide phenylhydrazone (uncoupler) stimulated ATPase activity; however, this effect was overcome by the addition of succinate. Studies on the effect of OA on metabolic anion uptake by mitochondria was carried out in the presence of blocking agents such as atractyloside and oligomycin (Meisner and Chan, 1974), and the result showed that OA competitively inhibited the binding of substrates succinate and malonate, ADP, and Pi. The role of OA on the transport system was further demonstrated from the observation that OA had no effect on respiration when submitochondrial particles were used, whereas 64% of inhibition was found at a OA concentration of 10 times less than when whole mitochondria were used. From these results, Meisner and Chan (1974) concluded that OA acts as a competitive type of inhibitor of mitochondrial transport carrier proteins. Ochratoxin A also selectively induced swelling of nonenergized mitochondria by Na+, NH4+C1,but not by K+, Li+, Rb+, or Cs+Cl. When lowering the osmolarity to 0.08 M, OA induced swelling with all monovalent cations except Li+. Inhibition of mitochondrial respiration by rubratoxin B was demonstrated by Hayes and Hannan (1973). This mycotoxin at a concentration of 3.28 X

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lop4M inhibited in vitro oxygen consumption in mice but had no effect on the ADP/O ratio or glycolysis. The inhibition was greater in males, and greater when brain and ileum homogenates were used versus the liver homogenates. Succinate was the only Krebs cycle substrate inhibited by rubratoxin B to a greater extent in vitro than in uivo. Studies on the effect of a mixture of rubratoxin B and afla B1 on the mitochondrial respiration in vitro revealed that rubratoxin B was the only causing agent. Because rubratoxin B affected Krebs cycle activity both in vivo and in vitro, Hayes and Hannan (1973) considered that such an inhibitory effect in mouse liver appears to be primary. Testing with liver homogenates obtained from different animal species (rats, mice, pigeons, and squirrels), these investigators found that the female squirrel appeared to be more susceptible than the other animals. No inhibitory effect on the oxygen consumption was observed when pigeon liver was used. Details on the mechanism of the inhibition of mitochondrial respiration by rubratoxin B were reported by Hayes (1976). Rubratoxin B at M depressed oxygen consumption for both the a concentration of 2.8 x ADP-coupled and 2,4-dinitrophenol uncoupled mitochondria prepared from male mice. Using different inhibitors, Hayes (1976)revealed that the site of action was between cytochrome c1 or c and the termination step of electron flow. Thus, the site of action is different from other mycotoxins. Because investigation in Hayes’s laboratory also showed that rubratoxin B inhibited different ATPase activities in many tissues (Desaiah et al., 1977), the inhibitory effect by this toxin in mitochondria may also be related to membrane transport. The effect of patulin on the inhibition of aerobic respiration was summarized by Singh (1967). Although a number of enzyme systems associated with respiration were found to be inhibited to a certain degree by patulin, the exact site(s) of action was not revealed. Nevertheless, because of the relative insensitivity of the terminal electron transport enzymes, Singh (1967) suggested that patulin may act prior to the terminal stages of respiration. Another neurotoxic mycotoxin citreovirdin was recently discovered as a potent mitochondrial inhibitor with both the toxin and its diacetate considered to have a direct effect on the enzyme ATPase (Beechey et al., 1974). C. EFFECTOF MYCOTOXINSON LIPID METABOLISM

Alteration of lipid metabolism by mycotoxins appears to be more frequent than the effect of mycotoxins on the carbohydrate metabolism. Knowledge of this subject was accumulated mostly from studies with afla B1, and the subject of lipid and vitamin metabolism during mycotoxicoses has been well covered in a recent review (Hamilton, 1975). Although the effect of mycotoxins on lipid metabolism in the overall mode of action is not known, the

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impact of their effect on public health cannot be neglected. Hamilton (1975) pointed out the problem of the relationship between the interactions of mycotoxins with lipid and vitamin metabolism and the overall mycotoxicoses. One of the pronounced effects of the mycotoxins on lipid metabolism is the accumulation of lipids in livers of animals receiving mycotoxins. This effect has been observed for a number of mycotoxins including afla B1, OA, luteoskyrin, rubratoxin B, T,, citrinin (Hamilton, 1975)and penitrem A (Cysewski et al., 1975). In experiments with chickens under essentially steady conditions, Tung et al. (1972) found that, even at the smallest dose (0.625 ppm) tested, afla B1 affects the transport triglycerides, phospholipids, and cholesterol. Because dietary afla B1 concentration required for inhibition of lipid transport (0.625 ppm) was considerably lower than those required for the inhibition of growth rate and RNA synthesis (2.5 ppm) and for the induction of fatty liver (1.5 ppm), Tung et al. (1972) suggested that the inhibition of lipid transport is a primary lesion during aflatoxicosis in the chicken and is not secondary to the general effect on nucleic acid metabolism. Concomitant with an accumulation of hepatic lipids, especially triglycerides, was a decrease in serum lipids in monkeys 3.5 hours after an oral administration of 2.0 mg afla B1 per kilogram of body weight (Rao, 1971). Nevertheless, a 50% inhibition in protein synthesis was observed under the same conditions; therefore it is not known whether the inhibitory effect of aflatoxin on lipid transport is a result of the defect in protein synthesis or not (Rao, 1971). Inhibition of triglyceride transport in mice treated with luteoskynn was observed (Enomoto and Ueno, 1974). While afla B1 and other mycotoxins in general induce fatty liver, the incorporation of radioactive precursors, such as acetate or glucose, into hepatic lipid fractions was found to be inhibited after animals received afla B1 (Shank and Wogan, 1966; Kato et al., 1969; Donaldson et al., 1972). In the chicken system, Donaldson et al. (1972) demonstrated that the decrease in incorporation of acetate into long-chain fatty acids of chicken livers was dose related and that the specific activities of the fatty acid-synthesizing enzyme system obtained from the afla Bl-treated chicken livers was lower. Because afla Bl had no effect on the activity of fatty acid synthetase obtained from the control chicken liver in vitro, they suggested that afla B1 exerts its effect on lipid synthesis by affecting the formation of the enzymes. Because recent investigations indicate that aflatoxin metabolism plays a key role in aflatoxicosis, one cannot rule out the possibility of the involvement of the aflatoxin metabolite(s) in such inhibition. Lo and Block (1972), however, reported an inhibition of lipid synthesis in an in vitro system using human skin. Because the incorporation of [‘“C]acetate into all lipid fractions was greatly inhibited (72-100%) by d a B1 at a concentration of 20 pglml in this

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system, but the incorporation of [3H]acetyl-CoAinto lipids was not affected,

Lo and Block (1972)postulated that the interaction of afla B1 with thiokinase is the possible site of action. In addition, the limitation of available cofactors such as ATP and CoA might be involved also. The in vitro inhibition of incorporation of ['%]acetate into different lipid fractions and fatty acids by afla B1 also has been demonstrated by Chou and Marth (1975) using liver slices or intestinal preparation obtained &om mink. Marked increase in incorporation ['%]acetate into lipids of mink was observed 20, 28, and 48 hours after the animal received a single dose of afla B1 (0.6 mgkg). Chou and Marth (1975) attributed the variation of these results from earlier reports to the difference in the route of afla B1 administration and also the difference in animal species. Inhibition by citrinin of cholesterol synthesis in liver and yeast has been noted recently (Endo and Kuroda, 1976). Contrary to the effect of afla B1, liver slices obtained from mice treated with cyclochlorotine stimulated the incorporation of 2-[ 14C]acetate into lipids (Ueno et al., 1963).This effect was attributed to the increased level of NADPH in the livers of the intoxicated animals. Aflatoxin B1 not only affects lipid synthesis and transportation, but also may interfere with lipid absorption and degradation. A decrease in bile salts and pancreatic lipase were observed in chickens with experimental aflatoxicosis. These observation may be the cause of the increase in excretion of lipids in those chickens (Smith and Hamilton, 1970).

IV. Effect of Mycotoxins on Nucleic Acid and Protein Synthesis There are a number of mycotoxins that affect the overall protein synthesis system which generally can be classified into four groups. The first group of mycotoxins, which includes afla B1 and G1, a-amanitin toxin, luteoskyrin, PR toxin, and furocoumarins, inhibits the nucleic acid synthesis, thus directly affecting the transcription processes. The second group of mycotoxins, such as 12,13-epoxytrichothecenes,impairs protein synthesis directly through the inhibition of one or more steps in the translation processes. The third group, which includes afla B1 and G1, inhibits both transcriptional and translational processes. Finally, the fourth group of mycotoxin, consisting of only zearalenone, alters cellular permeability, thus accelerating protein and nucleic acid synthesis (Ueno and Yagasaki, 1975). For most mycotoxins, the inhibition of protein and nucleic acid synthesis is considered to be the major biochemical lesion causing the acute and carcinogenic or other effects in animals; in others, this inhibitory effect may be secondary. For example, inhibition of nucleic acid synthesis by afla B, has been considered to be related to its mutagenic and carcinogenic effect,

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whereas the inhibitory effect of afla B1 and 12,13-epoxytrichothecene mycotoxins on protein synthesis is considered to be important in the acute toxic effect in animals. The effect of mycotoxins on nucleic acid and protein synthesis has been discussed by MoulC (1977) and McLaughlin (1977) in two recent symposia. A. EFFECTOF MYCOTOXINSON NUCLEIC ACID

SYNTHESIS The effect of mycotoxins on the nucleic acid synthesis and the possible site of action are summarized in Table 111. Afla B1 and a-amanitin toxin have been studied most extensively among those mycotoxins listed. Inhibition of RNA and DNA synthesis by afla B1 has been observed in vivo in animal liver and kidney systems as well as in a number of other systems (Clifford et al., 1967; Mouli: and Frayssinet, 1968; Sporn et al., 1966;Aka0 et al., 1971; Pong and Wogan, 1971; Wogan and Pong, 1970). One of the most prominent effects after an animal receives afla B1 is the decrease in RNA content and in RNA polymerase activity in the nuclei of the liver (Clifford and Rees, 1966; Sporn et al., 1966) in the treated animals. Incorporation of radioactive nucleotides into rat liver nuclear RNA was almost completely inhibited after the rat received a single sublethal dose (i.e., 5.0 mg/kg) (Sporn et al., 1966; Clifford and Rees, 1966)of afla Bl. In an in vitro study, Clifford et al. (1967) showed that afla B1 produced a much greater inhibiting effect than G1 and G,. The ability of afla B1,Bz,and Gl to inhibit RNA polymerase activity and decrease the RNA content in rat liver hepatocytes was studied by Edwards et al. (1971). These investigators found that the inhibitory effect of different

TABLE 111 EFFECTOF MYCOTOXINS ON NUCLEIC ACIDSYNTHESIS Mycotoxins

Level of inhibition

Metabolic activation

Aflatoxins B,, G,

Affecting the template function by binding with DNA covalently Nucleoplasmic RNA polymerase

Yes

Aflatoxin B, metabolite(s) a-Amanitin Furocoumarins Luteoskyrin Patulin PR toxin

(RNA polymerase 11) RNA polymerase I1 Template function Both template function and RNA polymerase activity RNA polymerase activity RNA polymerase I, I1

“Activated by ultraviolet light. bNot determined.

-

No a

No b

No

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aflatoxin derivatives on the RNA polymerase was similar to the carcinogenic and acute and subacute toxic effects, and that afla B, was approximately three times more active than G , in reducing the nuclear RNA synthesis while B2 had no effect even at a very high dose (200 mgkg). The structure activity investigation thus led to the concept of the importance of the 2,3 unsaturated double bond in the dihydrofurofuran moiety in afla B1. Inhibition of liver nuclear RNA synthesis was observed also for the synthetic, racemic afla M1 and B1 when these toxins were given to Fisher male rats (Pong and Wogan, 1971). Afla M1 and B1 caused a similar acute effect and inhibition of RNA synthesis in rats and seemed to be approximately equal in potency. Because the dose for producing a similar level of inhibition of RNA synthesis and the fine structural changes in liver parenchymal cells for the synthetic, racemic aflatoxins was about twice that required for natural B1, Pong and Wogan (1971) suggested that only one of the isomers in the racemic mixture of aflatoxins may be active. In addition to the inhibition of RNA synthesis by afla B1, DNA synthesis has also been found to be inhibited by this mycotoxin in a number of systems including human embryonic lung cells (Legator et al., 1965), E . coli (Wragg et al., 1967), Flavobacterium aurantiacum (Lillehoj and Ciegler, 1967), and B. cereus (Lillehoj and Ciegler, 1970). Other mycotoxins, including luteoskyrin (Ueno et al., 1967), patulin (Schaeffer et al., 1975), PR toxin (Mouli: et al., 1975; Wei and Chang, 1976), rubratoxin B (Hayes, 1977a), and sterigmatocystin (Engelbrecht and Altenkirk, 1972) also have been shown to inhibit nucleic acid synthesis. Inhibition of DNA synthesis by trichothecene mycotoxin, such as nivalenol, has been observed. This effort was considered to be secondary as compared with the inhibitory effect on protein synthesis (Ueno and Fukushima, 1968).

1. Inhibition of Nucleic Acid Synthesis through Modification of Template Function Inhibition of RNA synthesis by mycotoxins may be due either to their direct effect on the template function, such as those observed for the actinomycin D, or their inhibitory effect on RNA polymerase. There is overwhelming evidence indicating that afla B1, and perhaps its metabolite(s), directly modifies the template rather than having a direct effect on the enzyme. Such evidence is summarized as follows: (a) afla B1 does not inhibit the transcription in vitro using calf thymus DNA as the template and RNA polymerase obtained from either bacterial (Mouli: and Frayssinet, 1968; King and Nicholson, 1969) or afla B1 untreated rat liver (Edwards and Wogan, 1970) or rat testicles (Roy, 1968); (b) when altered chromatin, i.e., chromatin obtained from afla B,-treated rats, was incubated with RNA polymerase obtained from normal rats, a reduction of RNA synthesis was observed (Edwards and Wogan, 1970);(c) when thymus DNA was incubated

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with RNA polymerase obtained from either the toxin-treated rats or normal rats, again the transcription was not impaired (Edwards and Wogan, 1970). Portman and Campbell (1970), however, demonstrated that transcription was significantly inhibited when normal rat chromatin was incubated with E . coli polymerase in the presence of different amounts of afla B1 in uitro. Inhibition of DNA template activity in uitro by afla B, was observed also by Maher and Summers (1970) in the E . coli RNA polymerase system. These results indicated other mechanisms including inhibition of the enzymes or the direct effect of afla B1 on DNA might be involved as well. The mode of the inhibition of template function by afla B1 has been studied extensively in recent years, and it is believed that the toxin metabolized to an active form and subsequently interacts with nucleic acid covalently. Details of such interactions have been reviewed by the author (Chu, 1975, 1977)and will be treated later (Section V). Inhibition of nucleic acid synthesis by sterigmatocystin may also involve the same mechanism as afla B1. Luteoskyrin is another mycotoxin possibly affecting the template function in RNA synthesis. The mycotoxin inhibits both E . coli RNA polymerase activity and the synthesis of nuclear RNA in Ehrlich ascites tumor cells and also binds strongly to DNA in uitro (Ohba and Fromageot, 1967; Uneo et al., 1968).The mode of action, however, is different from that of afla B1 and GI, because metabolic activation of the toxin by liver microsomes appears not to be necessary for its action (Ueno et al., 1968). Furthermore, evidence presented by Ruet et al. (1973) indicates that the toxin exerts its effect by interacting with the transcription complex. The toxin reduced the number of chains initiated but the chain growth rate was unaltered at the initial phase of transcription; however, during transcription, the transcription complex remained undissociated but chain elongation was blocked. Inhibition was independent of DNA concentration and the kind of template used and was overcome by an increase in RNA polymerase concentration. These dual properties, i.e., inhibition either on the level of template or enzyme alone, thus led to the postulation by Ruet et al. (1973) that “luteoskyrin acts by first binding loosely to the DNA-bound enzymes followed after initiation of RNA synthesis by irreversible binding of the toxin to exposed single-stranded segments of DNA immediately in front of the enzyme, thus preventing chain elongation.” The mode of action resembles that observed for the antibiotic kankanomycin.

2 . Inhibition of RNA Synthesis by Mycotorins through Their Direct Effect on RNA Polymerase and Other Mechanisms It has been known for some time that a-amanitin (Hatfield and Brady, 1975), the most toxic mushroom poison, affects the liver primarily by damag-

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111

ing nuclei (Fiume and Laschi, 1965). Biochemical investigations revealed that nuclear RNA synthesis was significantly inhibited by a-amanitin in the early stages of intoxication (Fiume and Stirpe, 1966; Stirpe and Fiume, 1966). Unlike for afla B,, the site of action is believed to be on the RNA polymerase itself rather than on the template function, because the inhibitory effect can be reduced by increased enzyme concentration in vitro but cannot be reversed by alteration of DNA concentration. Investigation of the inhibitory effect of a-amanitin on RNA synthesis originated from its specific effect on the type I1 (or B) polymerase (Kedinger et al., 1970, 1971; Lindell et al., 1970) which is situated in the nucleoplasm and is thought to be primarily responsible for the transcription of chromosomal genes and messenger RNA synthesis. The toxin is also specific only to eukaryotic polymerases including sea urchin, rat liver, and calf thymus polymerases, but not to the bacterial or prokaryotic enzyme (Chambon, 1974). This property thus has been used in probing the functions of different nuclear polymerase. For example, Mahy et al. (1972) used a-amanitin as a probing agent for the elucidation of the mode of influenza virus replication. The strong inhibitory effect of a-amanitin on the replication of influenza virus in the chicken ovary duct led to the conclusion that DNA transcription for influenza virus replication is essentially mediated by type I1 polymerase. The specific inhibition of RNA polymerase I1 by amanitin has led also to the suggestion that different types of RNA polymerases are structurally daerent, and that the inhibitory effect is a result of specific interaction of the toxin with the enzyme or the DNA-enzyme complex which leads to the inhibition of chain elongation (Kedinger et al., 1971). a-Amanitin is not the only mycotoxin shown to inhibit the RNA polymerase enzyme. PR toxin (Fig. lo), a mycotoxin produced by P . roquefbrti (Wei et al., 1975), was found to inhibit RNA synthesis both in vitro in E . coli and Ehrlich ascites tumor cells and in vivo rat liver systems (Mouk et al., 1976; Wei and Chang, 1976). Because the in vitro inhibitory effect in E . coli polymerase system was reversed upon addition of enzyme, the inhibition was considered to be due to its effect on the enzyme ( M o d 6 et al., 1976). The inhibitory effect of PR toxin on RNA synthesis was altered neither by the change of ionic strength in the assay system nor by a-amanitin, which indicated that both types of polymerases (types I and 11) were

AcO

FIG. 10. PR toxin.

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affected (MoulC et al., 1976). The toxin also does not require an enzyme activation before interfering with the in vitro RNA synthesis. Kinetic analysis on the pyrophosphate exchange and the polynucleotide synthesis by MoulC et al. (1976) revealed that both processes were inhibited by the toxin to the same extent, thus the toxin affects both the initiation and elongation steps. Furthermore, the inhibitory effect can be reversed upon addition of high concentration of (NH4),S04. PR toxin has a very reactive aldehyde function group and two epoxy rings (Wei et al., 1975). Wei and Chang (1976) have shown that the inhibitory effect on the RNA synthesis was significantly decreased after the aldehyde and ketone group were reduced. Therefore, it is possible the observed inhibitory effect is due to the interaction of the reactive groups with nucleophiles such as -NH2 and -SH groups in the enzyme either specifically with the critical residues in the enzyme active center or nonspecifically with these groups in the enzyme. If the aldehyde indeed played a sole role in the inhibitory effect, such interaction would be nonspecific in nature, and the significance of such inhibition for the overall toxicity of the toxin should be reexamined. Because the evidence for the inhibition of template function by afla B1 metabolite(s)is overwhelming, the metabolite(s)is also considered to have a direct effect on RNA polymerase itself. As early as 1968, MoulC and Frayssinet had demonstrated that aflatoxin crude extracts obtained from A. flaous were effective in inhibiting RNA synthesis in vitro using RNA polymerase from rat liver and M . lysodeikticus (Mouli. and Frayssinet, 1968), but not when the purified toxin was used. Subsequent studies (MoulC and Frayssinet, 1972)showed that a new metabolite (called compound X) produced by incubation of afla B, with rat liver microsomal preparation was able to inhibit E . coli RNA polymerase activity in vitro. The level of inhibition was found to be dependent on the inhibitor concentration, independent of DNA concentration, and was relieved by addition of more enzymes. Because addition of the metabolite 2 minutes after the reaction did not affect the chain elongation, MoulC and Frayssinet (1972) suggested that the inhibition was primary at the initiation step. The new metabolite can be extracted from the incubation mixture with chloroform, and is more polar than afla B1. The structure for this metabolite has not been determined. Inhibition of rat liver nucleoplasmic RNA polymerase I1 (4040%) and cytoplasmic RNA polymerase I11 (2540%)in vivo by afla B1 was observed by Akinrimisi et al. (1974)when they analyzed the enzyme activity in vitro using the enzyme preparation obtained from rat livers 2 hours after afla Bl treatment. Nuclear RNA polymerase I was not affected. These investigators also confirmed previous observations that the toxin had no effect on all three types of enzymes in vitro. However, upon preincubation of afla B, with rat liver microsomal preparation, inhibition of purified RNA polymerase I1

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(2040%), polymerase I11 (10-20%), and total incorporation of [3H]-UTPinto isolated nuclei (38%)were observed in vitro; and again, type I enzyme was not affected. Studies on the effect of different subcellular &actions on the conversion of afla B1 to this active form to inhibit RNA polymerases revealed that only microsomal fractions were responsible for such activation. Since the afla Bl metabolite(s) primarily affects type I1 polymerases, the mode of action appears to be similar to those of a-amanitin. Nevertheless, it must be pointed out that a-amanitin does not affect on the bacterial enzyme; thus, it brings up the question whether the metabolite that inhibited E . coli polymerase in vitro as described by MoulC and Frayssinet (1972) is the same metabolite(s)as that which inhibited the liver RNA polymerase. Or it might be possible that the same metabolite is involved in both systems, but has a different mode of action than those described for a-amanitin. A marked, but reversible, disaggregation of polyribosomes along with a generalized decrease in mouse hepatic RNA was observed after the mice were treated with rubratoxin B (Hayes, 1977a). Because a pronounced decrease in the nucleoplasmic DNA-dependent RNA polymerase (type 11)was observed within 60 minutes after the mice received a single dose of rubratoxin B (0.5 mg/kg), Hayes (1977a) suggested that the observed polyribosome disaggregation was probably due to the suppression of the rate in the initiation of translation step. Although patulin and penicillic acid caused DNA breakage in HeLa cell cultures 1hour after incubation with the toxins at a concentration of 32 pg/ml and 320 pg/ml, other mycotoxins, including afla B1, fusarenon X, luteoskyrin, and rubratoxin did not (Umeda et al., 1972);the role of these mycotoxins on nucleic acid and protein synthesis is still not clear. Inhibition of RNA synthesis was observed 20 minutes after treating Chang's liver cell with 2.5 pg patulin per milliliter, and suppression of protein synthesis occurred 60 minutes after introducing the toxin to the medium (Schaeffer et al., 1975). However, neither protein nor RNA synthesis was completely shut down by the mycotoxin. Similar results were obtained in HeLa cell cultures (Kawasakiet al., 1972). In addition, Schaeffer et al. (1975) observed that the inhibition of incorporation of [ 14C]methylgroup was more pronounced than the inhibition of incorporation of nucleotide, thus leading to the suggestion that the inhibition in RNA synthesis more likely occurred at the level of transcription from DNA. Mode and Hatey (1977)found that patulin primarily inhibits the initiation step in the transcription process. 3 . Effect of Mycotoxins on Enzymes of Posttranscriptional Process

Investigation is being carried on in several laboratories on the problem of whether the observed decrease in RNA or DNA synthesis caused by some

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mycotoxins in vivo is due, at least in part, to an increase in the rate of breakdown. Ribonuclease activity in the liver of afla B1-treated rats did not change significantly as compared with the control animals, thus stimulated RNase activity does not appear to be an important factor in reducing the nuclear RNA by afla B1 (Edwards and Wogan, 1972).A single dose of 2.5 mg of rubratoxin B per kilogram to mice resulted in an increase in RNase activity of 10.8% and 26.5% in the supernatant mouse liver homogenates at 0.5 and 1.0 hour after dosing, respectively. No change in the RNase in the pellet fraction was observed (Hayes et al., 1975a). In the in vitro studies, Schabort and Pitout (1971) found that aflatoxins B1, B2, and M2 stimulated DNase I activity; afla B2 was more effective than B1, with Mz the weakest activator. M a B,, and GZa,however, inhibited DNase I activity, and the inhibition was found to be related to the binding of these two toxins with the enzyme. In the in vivo investigations, the acid DNase I1 of rat liver nuclei was found to be markedly increased after afla B1 treatment, but was not affected by afla B2 and sterigmatocystin (Pitout et al., 1971). Because afla B1 did not interact with DNase in vitro, these investigators suggested that aflatoxin metabolite(s) was responsible for this effect. Subsequent in vivo and in vitro studies (Pitout and Schabort, 1973)revealed that afla B1 altered the lysosomal membrane, thus releasing the acidic DNase from liver lysosomes. Since increased level of tRNA methylase activity or capacity has been observed in a wide range of tumors and transformed cells, Busby et al. (1975, 1976) investigated the possible correlation of toxic effects of afla B1 with tRNA methylase function in rat liver. Dose-independent methylase activity, which was measured by the rate function with excess of tRNA and limited enzyme, increased approximately 40% within 3 days after dosing with afla B1 and decreased thereafter. Transfer RNA methylase capacity as determined by maximum incorporation of the methyl group with excess enzyme and limited tRNA, however, exhibited a linear dose-response relationship with levels increased as high as 100% over the controls (Busby et al., 1975). The methylase activity and capacity in livers of rats dosed with afla B1 at a level to induce carcinoma but not toxic effect for 8 weeks (25 pg per day and 5 days a week) were also examined (Busby et al., 1976). Whereas no significant change of methylase activity was observed during the 55 weeks of the experiment, a significant increase in methylase capacity was detected in two distinct periods, one between 6 and 8 weeks (20%)and the other between 24 and 29 weeks (40%). An increase in both methylase activity and capacity (30%)was observed at least as early as one week after dosing with a high level of afla B, (37.5 pg per day, and 5 days a week), producing acute liver damage. An increase in both methylase activity and capacity was also detected in hyperplastic nodules, with and without histological evidence of carcinoma, excised from livers of rats fed continuously with 2 ppm of afla Bl.

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From these data, it is clear that measurements of methylase activity or capacity in the aflatoxin model were not reliable indicators of neoplastic transformation. The elevation of methylase activity may possibly be used as an indicator for acute toxicity rather than for carcinogenicity. Stimulation of hepatic tRNA methylase activity by the noncarcinogenic mycotoxin rubratoxin B has also been noted (Hayes, 1977a).

B. EFFECTOF MYCOTOXINS ON TRANSLATIONAL IN PROTEINSYNTHESIS

STEPS

Although many mycotoxins have been shown to inhibit protein synthesis in the translational steps, the mechanism and exact role of such inhibition on the overall toxic effect in animals for most mycotoxins is not clearly defined. The main reason for lack of such understanding is due to the complexity of inhibitory processes in certain cases, for example, the difficulty in discriminating between the translational and transcriptional steps. Afla B1 is one of the best examples of this. It has been known for some time that the inhibition of protein synthesis by afla B1 was due to its inhibitory effect on the RNA synthesis, and that the alteration or loss of RNA subsequently results in decreased protein synthesis. Nevertheless, recent investigations of some mycotoxins in the 12,13-epoxytrichothecenegroup which primarily inhibit the protein synthesis but not RNA synthesis (Ueno and Fukushima, 1968) have led to an understanding of the mode of action of some of these mycotoxins. Even for afla B, the direct role of the translational steps is emerging. 1. Inhibition of Protein Synthesis by

12,13-Epoxytrichothecene Type Mycotoxins or Antiobiotics through Possible Blocking of the Initiation Step Inhibition of protein synthesis by 12,13-epoxytrichothecenes has been demonstrated in a number of eukaryotic systems including Ehrlich ascites tumor cells (Ueno and Fukushima, 1968), HeLa cells (Cundliffe et a l . , 1974; Wei et al . , 1974b) rat liver, mice tissue, protozoa (Ueno and Matsumoto, 1975), rabbit reticulocytes (Ueno and Matsumoto, 1975; Wei and McLaughlin, 1974), human tonsils (Carrasco et al., 1973), and yeast (Barbacid et a l . , 1975; Carrasco et al. , 1973; Cundliffe et a l . , 1974; Stafford and McLaughlin, 1973). The potency of the inhibition varied considerably with the chemical structures of the side chain; thus, the mode of their action on the translational steps also varied considerably. The mode of action on protein synthesis has been most recently summarized by McLaughlin (1977). In an early investigation, Wei and McLaughlin (1974) compared a number of trichothecenes for their effect on the overall protein synthesis, on the termi-

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nation reaction, on the peptidyltransferase activity, on poly (U)-directed synthesis of polyphenylalanine, and on polyribosome degradation. These investigators found that trichodermol and some esters or other derivatives of trichodermol, verrucarol, and 15-deacetylcalonectrin did not affect the polyribosome pattern at a level of 90% of inhibition of protein synthesis; however, at this level of inhibition of protein synthesis, scirpentriol, 15acetoxyscirpendiol, diacetoxyscirpenol, verrucarin A, and calonectrin induced polyribosome breakdown. All the mycotoxins (or antibiotics) studied by Wei and Mclaughlin (1974) except calonectrin and 15-deacetylcalonectrin inhibited peptidyltransferase activity to some degree. Although from these data alone one cannot clearly identify the stage of inhibition for most mycotoxins, it is clear that calonectrin inhibits the initiation and that trichodermol, verrucarol, and 15-deacetylcalonectrinaffect protein synthesis beyond the initiation step. Wei and Mclaughlin (1974) also suggested that those mycotoxins inducing polyribosome breakdown probably inhibit at the initiation step. Extensive polyribosome breakdown was also observed when rabbit reticulocytes were incubated with T2,fusarenon X, diacetoxyscirpenol, and neosolaniol (Ueno and Matsumoto, 1975). Nivalenol, T2-toxin, and verrucarin A caused rapid and quantitative breakdown of polyribosomes in H-HeLa cells and in yeast spheroplasts (Cundliffe et al., 1974). Because the concentration required for induction of polyribosome degradation was found to be 10 times lower for T2and diacetylscirpenol(O.01pglml) than for fusarenon X and neosolaniol, and because both T2and fusarenon X inhibited the peptidyltransferase activity at approximately the same concentration (Ueno and Matsumoto, 1975), T2toxin and diacetylscirpenol were considered to have a different site in inhibiting protein synthesis as compared with fusarenon X. In order to find the mode of action of these mycotoxins, CundliEe et al. (1974) examined the polyribosome pattern of H-HeLa cells andlor yeast spheroplasts by preincubation with protein inhibitors of known functions such as anisomycin, cycloheximide, and trichodermin followed by the addition of mycotoxins. By this technique, these investigators were able to identlfy nivalenol, T2 toxin, H-T,, diacetylscirpenol, and verrucarin A as the highly selective and potent inhibitors of polypeptide chain initiation in eukaryotes, because the polyribosome breakdown in H-HeLa and yeast spheroplasts caused by these mycotoxins was inhibited by preincubation with anisomycin, cycloheximide, and trichodermin. Trichodermin, on the other hand, was considered to be an inhibitor in the elongation andlor termination because: (a) this antibiotic does not induce polyribosome disaggregation; (b) it prevents the polyribosome disaggregation caused by other agents; and (c) it prevents the release of nascent peptides from ribosomes by puromycin. Likewise, trichothecin and trichodermol resemble trichodermin

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in their mode of inhibition of protein synthesis in eukaryotic systems. A similar conclusion was reached for trichodermin, trichodermol, trichothecin, fusarenon X by Barbacid et al. (1975) using the yeast system. Rubratoxin B caused a marked decrease in liver protein with a slight decrease in liver RNA in mice and rats. A single dose of rubratoxin B (0.25 mg/kg) caused a marked disaggregation of polyribosomes within 0.5 hour after dosing. However, the mechanism of inhibition of protein synthesis is not known (Hayes et al., 1975a).

2 . Inhibition of Protein Synthesis by 12,13-Epoxytrichothecene Type Mycotoxins through Their Direct Effect on Peptidyltransferase Center in Ribosomes Studies on the effect of 12,13-epoxytrichothecene-type mycotoxins on protein synthesis were done primarily with antibiotics in the sesquiterpenoid group produced by fungi. Only limited information on Tz and other related mycotoxins is available. In the previous section, inhibition of protein synthesis in eukaryotic cells with different modes of action by mycotoxins and/or antibiotics in this group was discussed. Some of these affect the peptidyltransferase center on the 60 S ribosomes of eukaryotic, thus inhibiting either the elongation or termination step. Additional evidence strengthening this conclusion is summarized as follows: (a)Trichodermin (Wei et al., 1974b) and fusarenon X had no effect on the binding of aminoacyl-tRNA to ribosomes of yeast, human tonsil (Carrasco et al., 1973), and rat liver (Ueno and Matsumoto, 1975), (b) Trichodermin and its derivatives, fusarenon X, neosolaniol, and Tz, are potent inhibitors of peptidyltransferase as measured by the formation of aminoacyl-puromycin complex in yeast, human tonsils (Carrasco et al., 1973), rabbit reticulocytes (Wei et al., 1974b) and rat liver (Ueno and Matsumoto, 1975). The potency of such inhibition for different derivatives in yeast and human tonsil systems (Carrasco et al., 1973) is as follows: verrucarin A > hsarenon X > anisomycin > trichodermol; and in rabbit reticulocytes system it is: scirpentriol 3 15-acetoxyscirpenol 3 diacetoxyscirpenol > verrucarin A > trichodermol > verrucarol (Wei and McLaughlin, 1974). (c) Whereas concentration required for overall protein synthesis by neosolaniol, nivalenol, and hsarenon X varied slightly and T2 required 4-10 times less than the others, all four mycotoxins inhibit the release of peptidyl-puromycin complex from ribosomes at similar levels (Ueno and Matsumoto, 1975). (d) Trichodermin, trichodermol, and fusarenon X did not affect the elongation factors (EF1 and EF2) in human tonsil cells (Carrasco et a l . , 1973), rabbit reticulocytes and rat liver (Ueno and Matsumoto, 1975; Wei et a l . , 1974b); thus, the translocation step was not affected. (e)Binding of substrates such as CACCA-Leu-Ac and UACCA-Leu to donor and acceptor sites of the peptidyltransferase center was inhibited by

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trichodermin, venucarin A, fusarenon X, and trichodermol, but the inhibition was found to be slightly less effective than anisomycin (Carrasco et al., 1973). In vitro and in vivo studies by Wei et al. (1974b)indicated that trichodermin inhibits the termination steps in protein synthesis. In these studies, Wei et al. (197413)found that although trichodermin inhibits both the elongation step, assayed with the poly (U)-directed synthesis of polyphenylalanine, and the termination step, probed by the poly (AGU) as the source of release codons, the effect on the termination was more pronounced. Anisomycin and sparsomycin were found to act as inhibitors for elongation and termination, respectively, when analyzed under the same conditions. In the in vivo experiments, trichodermin was found to have no effect on the rate of polyribosome reformation in HeLa cells which had been pretreated with a translational initiation inhibitor, 0-methylthreonine, or heat-treated cells, whereas cycloheximide, an elongation inhibitor, slowed down the rate of reformation. Another piece of evidence supporting the involvement of trichodermin in the termination step came from a study on the binding of the antibiotic to the 60 S subunits of yeast 80 S ribosomes obtained from an antibiotic-sensitive yeast and a resistant mutant (Jimenez et al., 1975). The mutant also shows cross resistance in both in vitro protein syntiiesis and in vivo growth of the yeast with fusarenon X, trichothecin, verrucarin A, and anisomycin. Whereas anisomycin binds to the ribosome subunit equally well in both mutant and wild type, trichodermin binds to the mutant yeast much less efficiently than the wild type. Jimenez et al.(1975) suggested that anisomycin and trichodermin have different sites on the 60 S subunit of eukaryotic ribosomes. T2 and trichodermin also have different sites in ribosomes. Cannon et al. (1976) demonstrated that although both T2and trichodermin can inhibit peptide bond formation on ribosome at the level of the peptidyltransferase catalytic center, but T2 is excluded from the interaction with ribosomal receptor site by the presence of nascent polypeptide chains. Thus, T2 toxin may not be considered as an unique inhibitor in the initiation step.

3. Inhibition of Protein Synthesis by Aflatoxins and Possible Site(s) of Action It has been observed that protein synthesis was depressed after animals received afla B1 (Clifford and Rees, 196713; Shank and Wogan, 1966; VillaTrevino and Leaver, 1968).The incorporation of amino acids into rat liver proteins decreased immediately after administration of afla B1, which was followed by a phase of recovery or increase in synthesis in the first few hours (Sarasin and MoulC, 1973a; Shank and Wogan, 1966);prolonged inhibition of incorporation of amino acid into protein after this initial biphasic phenomena lasting for 24-48 hours was observed both in vivo and in vitro. Inhibition of protein synthesis was observed also both in vitro and in vivo in a number of

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other systems including the induction of several enzymes, such as hydrocortisone-induced tryptophan pyrolase, tyrosine transaminase (Wogan and Friedman, 1968;Kato et al., 1970),benzopyrene-induced zoxazolamine hydroxylase (Pong and Wogan, 1970), and mitochondrial proteins (Belt and Campbell, 1975). The impact of aflatoxin inhibition on protein synthesis, however, has been overlooked because of the overwhelming evidence of the effect of aflatoxin on the transcription process. Nevertheless, recent investigations (Sarasin and Mouk, 1973a, 1975) have shown that, at least in the early phase immediately after the animal receives the toxin, inhibition of protein synthesis is the direct effect of afla B,, or perhaps its metabolite, on certain translational step(s). The inhibition of translational steps in protein synthesis by afla B1 was divided into two steps by Moult. and her colleagues (Mouk, 1977). In the first 5-7 hours after the animal received the toxin, the inhibitory effect on protein synthesis is direct and specifically at the polyribosome level, but beyond 7 hours the inhibitory effect is secondary and is primarily a consequence of impairment of transcription, which leads to an inhibition of the initiation of protein synthesis. A number of investigators, in comparing the extent of protein synthesis inhibition by afla B, and the level of polyribosomes disaggregation in rat livers, found that these two processes are closely related to each other. Disaggregation of liver polyribosomes occurred as early as 3 hours after the animal received afla B1, and lasted as long as 36 hours (Moulk, 1977; Pong and Wogan, 1969; Roy, 1968).Villa-Trevino and Leaver (1968)found that afla B, caused an increase in monomers and dimers of ribosomes and suggested that ribosomes are the site of inhibition of protein synthesis. Whereas the 18 S RNA continued to be excised from a normal 45 S RNA in the presence of afla B, (Moult5 and Sarasin, 1974), it emerged in the newly synthesized 40 S subunits, which still possess active particles capable of initiating protein synthesis. While analyzing monosomes from the disaggregated polyribosomes of the livers of rats treated with afla B1,Hayes et al. (1975b)found that these monosomes lack tRNA and mRNA, because the 80 S monosomes were readily dissociated into 40 S ribosomal subunits in a linear sucrose gradient containing 0.3 M KC1. The 4 S to 5 S molar RNA ratio of the monosome was 0.6, indicating that only 0.6 mole of tRNA and/or aminoacyl tRNA was present per ribosome molecule with no peptidyl tRNA present. Kinetics analysis of the inhibition of protein synthesis and polyribosome disaggregation induced by afla B1 (Sarasin and Moult., 1973a) revealed that whereas the profiles of both processes were nearly the same beyond 7 hours after the rats received the toxin, the rate of polyribosome disaggregation was slower than the rate of inhibition of protein synthesis in the initial phase. These data suggest that afla B, (perhaps its metabolite) inhibits protein synthesis at the initiation step beyond a certain period after dosing but do not explain the early phase of inhibition.

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In order to elucidate the mode of inhibition of protein synthesis by afla B1, Sarasin and Moulk (1975)further analyzed the kinetics of both the incorporation of nascent peptides into polyribosomes and the overall protein synthesis in the supernatant. The following observations have led them to conclude that afla B,(metabolite) has a direct and specific role in the inhibition of

elongation andlor termination step in the early period after receiving the toxin. (a) The kinetics of labeling of nascent peptide in polyribosomes of rat livers treated with afla B1 for 1hour and pulsing with [“C] leucine at various intervals was ddferent from the controls, and the profile resembled those reported for elongation and termination inhibitors. (b) The overall protein synthesis decreased in the first 1-2 hours after afla B1 treatment, but the nascent peptide synthesis either increased or was unaltered during this phase, indicating that the toxin affects the peptidyltransferase center in the ribosomes. (c) The polyribosome profile obtained from livers of rats treated with afla B1 for 1hour was found to be different from those of the controls, as they appear to be longer (or heavier) and contain more ribosomes per unit of mRNA than the control; (d) The extent of inhibition of poly (I)-directed synthesis of polyleucine by postmitochondrial supernatant of rat liver treated with afla B1for 1hour was found to be the same as the inhibition of overall protein synthesis in the absence of poly (I), indicating that the initiation step was not affected. The dose and time study in this investigation revealed that direct inhibition in protein synthesis, in the elongation and/or termination step occurred at a low toxin dose and at an early stage. At high doses and longer exposure time where inhibition of transcriptional process occurred, all the steps in the protein synthesis were affected. Additional evidence for the direct involvement of afla B1 in inhibiting protein synthesis is the observation that extensive helical polyribosome structures occurred in the cytoplasm of rat livers and kidney cells from rat and mouse within a short period (1 hour or less) after the animal received afla B1and G1 (Monneron, 1969; Sarasin and Moul6, 1976). After comparing the data with a number of other chemicals and/or carcinogens which were shown to induce the helical structures and to inhibit protein synthesis, Sarasin and Mouli! (1976)postulated that induction of helical structures of polyribosomes by some of these chemicals and/or carcinogens, including d a B1 and Gl, is due to the inhibition of release of ribosomes from mRNA, thus generally implicating the impairment of protein synthesis in the termination process. Because the enzyme converted afla B1 metabolite(s) does inhibit protein synthesis in vitro system whereas afla Bl does not (Sarasin and Mouli!, 1973a,b), it is believed that the in vivo early phase of inhibition of protein synthesis is due to the aflatoxin metabolite. Sarasin and Mouli! (1975) using reconstituted postmitochondrial supernatants of afla Bl-treated rat livers for incorporation of amino acid into protein, found that the damage due to the afla B1 derivative(s)is observed mostly on a microsomal level. Because inhi-

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3'

5

Ribosome subunits

Ribosome

FIG. 11. Simplified ribosomal cycle in protein synthesis. A typical polyribosome is shown in the center of the cycle. Ribosomes move along the mRNA in the 5' -+ 3' direction and are independent of each other in their function. Mycotoxins may block one or more of the three sites. M a B, metabolite(s) (7 hours after dosing), 15-acetoxyscirpenol, calonectrin, diacetoxyscirpenol, nivalenol, rubratoxin B, T2, H-T,, and verrucarrin A block at site A. Afla B, (GI) metabolite(s) (early phase after dosing), fusarenon X, neosolaniol, verrucarol, T2trichodermol, and trichothecin block site B and (or)C. Trichodermin and possibly also afla B, (GI)metabolites block specifically at site C.

bition of protein synthesis was not observed when polyribosomes obtained from the afla B,-treated rat livers were used, these investigators suggested that an afla B1 derivative(s) possibly bound to the ribosomal site noncovalently was removed during isolation of the polyribosomes. Afla B1 metabolite(s) also was believed to be responsible for the inhibition of the mitochondrial protein synthesis (Belt and Campbell, 1975). Whereas 0.01 mM and 0.1 mM of afla B1 did not affect the mitochondrial protein synthesis and 0.4 mM produced only 14% inhibition, a 25% inhibition was observed with mitochondria isolated from rat livers 2 4 hours after a single i.p. injection of 5.0 mg afla B, per kilogram of body weight. A 23% inhibition of protein synthesis in mitochondria was also observed when the mitochondria was isolated from a 600g supernatant fraction which had been incubated with 0.1 mM afla B1 in an NADPH regenerating system.

V. Interaction of Mycotoxins with Macromolecules as the Mode of Action of Mycotoxins It is apparent from the previous discussions that most mycotoxins exert their toxic effect by altering certain vital biochemical processes either di-

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rectly or after metabolic activation. The basis of such alteration lies in the ability of the toxins or their metabolites to interact with cellular macromolecules and functional organelles, including such as nucleic acids, proteins, mitochondria, and ribosomes. Modification of protein (enzyme) and substrates results in an alteration of secondary and tertiary structures of such macromolecules and organelles and subsequently leads to a change in their functions. In addition, binding of mycotoxins with protein involved in the transport process and membrane function may induce certain adverse biological consequences. The significance and details of the interaction of mycotoxins with macromolecules have been summarized in two recent symposia by the reviewer (Chu, 1975, 1977). In the present paper, only the most significant works and the correlation of the binding phenomena with the biological and biochemical events discussed in the previous sections will be covered. Mycotoxins may either react with macromolecules to form a covalent bond(s) or may react with macromolecules noncovalently. Both bindings may play a role in the mode of their action. Some mycotoxins which have reactive group(s) readily form covalent bonds with macromolecules, but for most mycotoxins metabolic activation is essential in the formation of covalent bonds. Therefore, metabolism plays a significant role in the interaction of such mycotoxins with macromolecules. This is especially true for afla B1 and GI. In addition to forming an active intermediate, metabolism may lead to the formation of either more toxic or less toxic compounds, thus possibly altering the nature of noncovalent bindings. Unfortunately, except for &a B1, the metabolism of most mycotoxins is not known. The role of metabolism on the toxicity of afla B1 was emphasized in a review by Patterson (1973), who suggested that once the toxin has entered the liver cells, it is the rate of metabolism that determines whether the agent will cause tissue injury in a particular animal species. It must be pointed out, however, that because of the complexity of metabolism, its rate is affected by many factors; thus, the bindings and biological activity could be manifested in a number of ways (Campbell and Hayes, 1975). The relationship of various afla Bl metabolites to their biological activities or toxic lesions was reviewed by Campbell and Hayes (1975, 1976), Hsieh (1977) and by Wogan (1976). The reader should refer to these reviews for details. A.

INTERACTION OF

MYCOTOXINSWITH NUCLEICACIDS

1. lnteraction of Mycotoxins with Nucleic Acid without Metabolic Activation

A great deal of attention has been focused on the interaction of mycotoxins with nucleic acids, enzymes, and organelles involved in the protein synthe-

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I23

sis, because the major biochemical lesion for several mycotoxins is their inhibition of the translational and transcriptional processes in protein synthesis. Early studies (Chu, 1975, 1977) indicated that aflatoxins have a high affinity for nucleic acids and polynucleotides, and it was assumed that this type of interaction might result in the alteration of template functions. However, subsequent studies on the structure-function relationship of different aflatoxin analogs (Edwards et al., 1971) and the observation that aflatoxinDNA complex failed to inhibit RNA polymerase activity (Edwards and Wogan, 1970) indicate that this type of binding may not be related to the biological activity, although the high affinity of the toxin for nucleic acids might facilitate subsequent reactions. Because of a better understanding of the role of metabolism in the toxicity of aflatoxins, it is now felt that metabolic activation is a prerequisite for aflatoxin action. The other two types of mycotoxins shown to interact with nucleic acids without biological activation are furocoumarins and the anthraquinone-like mycotoxins, i. e., luteoskyrin and rugulosin. Furocoumarins interact with nucleic acid either through a photocatalyzed (365 mm) addition reaction between the furocoumarins and thymine base (Musajo and Rodighiero, 1972) or through the photocatalyzed interaction of the toxin between two base pairs in DNA (Dall'Acqua et al., 1974). The formation of the cross-linked adducts has been demonstrated both in vitro and in vivo and was found to be correlated to the biological activity of the furocoumarins (Hanson et al., 1976). Although the interaction of the anthraquinone type mycotoxin luteoskyrin was considered possibly to be through intercalation, the interactions appeared to be more complicated because Mg2+played an important role in such interactions (Ohba and Fromageot, 1967; Ueno et al., 1968). A threecomponent complex, i. e., DNA-M2+-luteoskyrin, has been isolated. More recently, complexing of the rugulosin and luteoskyrin with DNA and M$+ or Mn2+ was demonstrated. Bouhet and Chuong (1976) reported that these mycotoxins not only form complexes with either single- or double-strand DNA in the presence of Mg2+or Mn2+,but also complex with M$+ or Mn2+ to form helical polychelates having a 27 A pitch and 4 monomeric units per turn. Dissociation of the double-strand DNA-M$+-toxin complex resulted in a complex having spectral properties resembling those of polychelates. These authors suggested that Mg2+ would be chelated between the middle cage of the ligand molecule and the phosphate group of DNA first, and then stabilized by hydrophobic interaction of one of the two plane halves in the ligand structure with purine base in nucleic acid. The involvement of the Mg2+ ion in the complexation also was suggested by Ruet et al. (1973), but these investigators emphasized the involvement of the interaction of the toxin with RNA polymerase for the overall inhibition of RNA polymerase activity.

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s. cnu

The possibility of binding of rubratoxin B to DNA was suggested by Neely et al. (1977) from the observation that the toxin shifted the ultraviolet spectrum of DNA and RNA. Details on the mode of their interaction are not known.

2. Metabolic Activation as a Prerequisite for AfZatoxins B I and GI-Macromolecule Interaction Lijinsky et al. (1970), using radioactive afla B1 and GI, demonstrated that both toxins were found in DNA, RNA, and protein fractions in rat livers and other organs. Because the bound radioactivity cannot be extracted with organic solvents, these investigators suggested that the bindings were covalent in nature; however, afla Bl and GI as such do not react with macromolecules covalently in vitro. Because the noncovalent bindings of aflatoxins with nucleic acid were found to neither affect the transcriptional process nor correlate with the acute toxicity and carcinogenicity, the observed covalent binding of the toxins with macromolecules must therefore arise from aflatoxin derivative(s)formed in vivo. The possibility of conversion of afla B, to active metabolites was investigated in a number of laboratories, because considerable evidence was available to show that most chemical carcinogens require in vivo metabolic activation to electrophilic compounds before they can react with cellular nucleophiles. Much of the work originated from Professors E. C. and J. A. Miller’s laboratory at the University of Wisconsin. In a series of reports during the past few years, the Millers and their colleagues (Gamer et al., 1972; Garner, 1973a; Swenson et al., 1973, 1974, 1975, 1977) have presented evidence showing that covalent binding of afla B1 to macromolecules requires metabolic activation of afla B1 to a putative “metabolic active aflatoxin B1 2,3-oxide,” which acts as the electrophilic precursor, by liver microsomes in the presence of NADPH and its regeneration system. Covalent bindings of afla to RNA (Swenson et al., 1973), DNA, and a number of other polynucleotides (Garner, 1973b) were demonstrated after incubation of the nucleic acid with rat or hamster liver microsomes in vitro. After purification of the tRNA-afla B1 adducts and mild acid hydrolysis, Swenson et al. (1973) isolated an aflatoxin derivative, i.e., 2,3-dihydro-2,3dihydroxy afla B1 (afla B1-dihydrodiol).A similar observation was made when human liver microsomal preparations were used (Swenson et al., 1974). In addition, Swenson et al. (1974) also showed that afla B1 bound covalently to rat hepatic DNA, RNA, and proteins in vivo. The specific covalent binding of afla B, to nucleic acid (aflatoxin bound per milligram nucleic or protein) was considerably higher than to proteins. Again, afla B1 dihydrodiol was identified after mild acid hydrolysis. In the investigation of binding of afla B1 with different nucleic acids and polynucleotides, Garner (1973b)and Gurtoo and Dave (1975) found that that poly (G) and high contents of guanine nucleic

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acids were most efficient in serving as a nucleophilic acceptor for such bindings, thus suggesting the possibility of involvement of guanine residues in such interactions. Lin et al. (1977) isolated two additional derivatives, both containing guaninyl residues, after hydrolysis of the atla B1-RNA adducts prepared in vitro using hamster liver microsomes. Hydrolysis of the new derivatives with hot acid yielded datoxin dihydrodiol. Further evidence to support the postulation that the putative, 2,3-aflatoxin B, oxide is the common electrophilic precursor for dihydrodiol B1 and the nucleic acid (and protein)-aflatoxin adducts (Lin et al., 1977) is: (a) formation of free dihydrodiol was decreased upon addition of DNA and RNA in the incubation mixture, and (b)the formation of both dihydrodiol and nucleic acid adducts were inhibited by an epoxide hydrase inhibitor, 2,3-epoxy-3,3,3trichloropropane.

PI

FIG. 12. Postulated mechanism for binding of afla B, to macromolecules. M a B, was metabolized to an intermediate, possibly 2,3-oxide, by microsomal enzymes (1) and subsequently subjected to nucleophilic attack by nucleic acid (2), protein (3),and -SH group compounds (5) to form afla B,-macromolecule adducts. 2,3-Dihydro-2,3-dihydroxy-afla B1 was formed after mild acid hydrolysis of the adducts (4),or by hydrolysis of the chemically synthesized (7) osrnic ester (S), or p-chlorobenzyl ester (6) of afla B,. 2,3-Dichloroafla B, was synthesized from afla B, (9), and has been shown readily to react with nucleic acid (10)and proteins (11).Hydrolysis of RNA-afla B, (14) resulted two quaninyl derivatives in addition to the dihydrcdiol.

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The structural requirement for the interaction of aflatoxin with nucleic acid was found to be consistent with the structural requirement for carcinogenicity and toxicity. Swenson et al. (1973) found that afla Bz was not activated by the microsomal fraction, hence it did not react with the nucleic acid. Small amounts of afla B2bound to the nucleic acid (1%)were considered to be due the conversion of this toxin to afla B1 (Swenson et al., 1977). Gurtoo and Dave (1975) confirmed that the microsomal mixed-function oxygenase-catalyzed oxidation of the C2-C3 double bond of aflatoxin is a prerequisite for the formation of aflatoxin-nucleic acid adducts, because afla Gl also formed nucleic acid adducts after activation while a number of other aflatoxin derivatives, including afla B2, G2, and B,, and 5,7dimethoxycyclopentenone 2,3-C coumarin which lack a C-2-C-3 double bond, did not form nucleic acid adduct upon incubation with microsomes. Although the putative metabolic active afla B1 2,3-oxide cannot be experimentally isolated, Swenson et al. (1975) have synthesized a model compound afla B1 2,3-dichloride, and studied its chemical and biological properties. This compound is very unstable (the half-life of hydrolysis in 10% dimethyl sulfoxide is about 30 seconds) and is highly reactive with protein, DNA, and RNA. Biologically, it is also highly carcinogenic to rats and mutagenic to S . typhimurium TA 98 and TA 100. Interaction of this compound with nucleic acids was considered to be between C-2 of afla B, and the nitrogen or oxygen atoms in the nucleic acid to form glycoside type bonds. Like the putative afla B, 2, 3-oxide, the dichloride also reacted most effectively with poly (G) among many homopolymers tested. The overall relationship between the postulated intermediate of & B, and its interaction with macromolecules is summarized in Fig. 12.

B.

h’I”TRACTION OF

MYCOTOXINS WITH

PROTEINS AND

ORGANELLES

A number of mycotoxins have been shown to interact with enzymes and functional proteins through either noncovalent bindings or covalent interactions. Both metabolic activation or nonactivation and the covalent or noncovalent interaction with proteins and organelles all appear to be important for some biological and biochemical events discussed in various previous sections. The overall role of the binding of mycotoxins with macromolecules is shown in Fig. 13.

1. Interaction of Mycotoxins with Enzymes Several mycotoxins have a reactive group(s) and thus readily react with proteins and enzymes, either specifically or nonspecifically. Patulin and penicillic acid, for example, have been shown to react covalently with -SH

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0

127

00

+ooo 00

FIG. 13. Mode of action of mycotoxin. Mycotoxins may interact with carrier proteins noncovalently, (1) transport to target organs, tissues or subcellular fractions, and followed by (2) dissociation into free toxin. The free toxin may (3)directly react with functional macromolecules or organelles noncovalently to induce conformational change or (4) may occupy the active substrate binding site. Likewise, mycotoxin metabolite (5) may react with the functional macromolecules (6) and (7). The free mycotoxin may convert to an active species (8)and then react with functional macromolecules covalently (10) inducing secondary and tertiary structural changes. The activated mycotoxin may convert to mycotoxin metabolite(s)(9). Some mycotoxins may form covalent bond with macromolecules without activation. Conjugation of mycotoxins or metabolite(s)with small molecular weight substances may involve in the detoxification process.

and also possibly -NHz groups of proteins through either addition or substitution reactions with their a , &unsaturated double bonds conjugated to the lactone ring (Ciegler et al., 1971b, 1972; Hofmann et al., 1971; Ashoor and Chu, 1973a,b). The inhibitory effect of patulin and penicillic acid on certain -SH enzymes, such as lactate and alcohol dehydrogenase, aldolase and several bacterial enzymes, was considered to be due to the blocking of -SH and -NH2 groups in the enzymes. As much as 12-16 moles and 8-11 moles of either patulin or penicillic acid were found to be bound with the -NHz and -SH groups in aldolase when 70430% of the activity was lost owing to their interaction (Ashoor and Chu, 197313).Because blocking of the double bond with -SH reagents resulted in loss of biological activity, it was considered that in most cases such interactions may be important for biological activity; however, there were some exceptions. Ashoor and Chu (1973a) found that inhibition of lactate dehydrogenase by both patulin and penicillic acid were reversible and Ciegler et al. (1976a) demonstrated that the patulin and cysteine adduct(s) still had a teratogenic effect. These results suggest that there may be some other mechanisms in addition to these already proposed. Patulin and penicillic acid are not the only type of mycotoxins which bind with the -SH group of proteins or enzymes, as the 12,13

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epoxytrichothecenes may also react with the -SH groups. Ueno and Matsumoto, (1975)found that T, toxin and fusarenon X interacted witha number of “-SH enzymes,” thus causing a reduction in enzyme activity. Although it has been shown that afla B1 is converted to an active intermediate and subsequently reacts with the nucleophiles, there has been no report regarding a specific interaction with enzymes. However, several aflatoxin metabolites have been shown to interact with enzymes and proteins. M a Bza and G,, which were shown to be very reactive with proteins through the interaction of the dialdehyde groups in afla B,, and the -NH2 groups in proteins to form Schiffbases (Patterson and Roberts, 1970;Ashoor and Chu, 1975a; Gurtoo and Campbell, 1974), have been shown to be very effective inhibitors of DNase in vitro (Schabort and Pitout, 1971). Another unidentified afla B, metabolite(s), which was reported to inhibit RNA polymerase activity, may also bind to the enzyme, possibly through noncovalent interaction. Noncovalent interactions of aflatoxins and other mycotoxins with enzymes have been demonstrated in several instances, and the enzyme activities in certain cases have been modified as the result of interaction. Binding of afla B,, B2, and G, in vitro caused an increase in DNase activity (Schabort and Pitout, 1971). The specific inhibition of eukaryotic RNA polymerase I1 by a-amanitin toxin was considered to be due to its interaction with the enzyme. The structural integrity was considered to be the major factor for such interaction (Kedinger et al., 1970). Although OA has been shown to have high affinity for a number of enzymes including aldolase, arginase, thrombin, catalase (Chang, 1975), and carboxypeptidase A (Pitout and Nel, 1969), the relationship between the binding of the toxin with the enzymes and their effect on enzyme activity are not known. Binding of OA with arginase in vitro did not affect the enzyme activity, but the OA and carboxypeptidase A interaction resulted in enzyme inhibition (Pitout and Nel, 1969).

2. Binding of Mycotoxins with Serum Proteins Binding of mycotoxins with serum proteins may greatly affect the disposition of the mycotoxins to different tissues and to different enzyme systems, thus playing a significant role in regulating the toxin concentrations in organs and tissues. In addition, their binding to the serum may also affect the binding of other biological active substrates andlor cofactors to the serum, resulting in a secondary effect. Although most mycotoxins may interact with serum proteins, only the binding of aflatoxins and ochratoxin with serum protein have been studied in detail. These interactions were found to be associated exclusively with the albumin fraction. Interaction of afla B1 with serum albumin was demonstrated by electrophoresis (Rao et al., 1968; Wei and Lee, 1971), column chromatography (Scoppa and Borle, 1971), spectrophotometric analysis (Scoppa and

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Marafante, 1971), and equilibrium dialysis (Bassir and Bababunmi, 1973). Scoppa and Marafante (1971), using spectrophotometric methods, revealed that bovine serum albumin (BSA) and afla B1 formed a 1:l complex with an apparent association constant of 1.6 x 10" M. I n comparing the binding of afla B, and 4-hydroxycoumarin with human (HSA) and bovine serum albumin, Bassir and Bababunmi (1973)found that HSA has two available sites for the ligands, but BSA has only one. The affinity of afla B, for both types of albumin was found to be higher than for 4-hydroxycoumarin. The interaction of different ochratoxins with BSA was studied in detail by Chu (1971, 1974b). Bovine serum albumin bound with 2.47, 1.93, and 3.24 moles of ochratoxins A, B, and C at neutral pH with binding constants of 3.17 x lo6 M-', 7.1 x 105 M-', and 8.9 x 105 M-', respectively. Interaction of the hydrolyzed products, ochratoxins a and p, with BSA was found to be much weaker; i.e., only one binding site for each with a binding constant of 1.5 x 106M-' and 5.0 x 105 M-', respectively. These data indicate that the binding of OA with albumin was considerably stronger than the afla B, or other coumarins. Both ionic and hydropholic interactions are important for such bindings. The structural integrity of BSA was found to be very important for BSA-OA interaction. Chang (1975) found that OA induced a Cotton effect in BSA after the protein bound with 2 moles of the toxin, when analyzed by the circular dichroism method, but it was not observed when 8 M urea was present. Interaction of albumin with OA also was demonstrated in in vivo studies. Both Galtier (1975) and Chang and Chu (1977) observed the association of OA with the albumin fraction after rats received the OA by either oral administration or i.p. injection. The amount of OA bound with albumin in vivo was found to be dependent on the dose (total OA in the serum) and also possibly on the age of the animals (Chang and Chu, 1977). Because the affinities of different ochratoxins with BSA correlated with the acute toxicity of these toxins (Chu, 1975), it was suggested that albumin-OA interaction might be important in bringing the toxin to the target organs (such as the kidney) for its action. The possibility of inducing antibodies against OA by the albumin-OA complex was suggested (Galtier, 1975), but there is no evidence to substantiate this postulation. 3. Znteraction of Mycotoxins with Proteins Involving Regulatory Functions

Although it has been shown that some mycotoxins bind with proteins involving regulatory function, the role of such interaction in the toxic effect for most cases is speculative. The interaction of afla B1 with histone in vitro was demonstrated by Black and Jirgensons (1967).These investigators found that afla B1 interacts with lysine-rich histone, having an apparent equilibrium constant of 700-1000 M-' with 6 5 3 5 moles of afla B, bound to 1 mole

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of histone. Binding of afla B1 to histone resulted in an increase in viscosity of the histone. In a study of the interaction of DNA with afla B1, Fujimoto and Ohba (1975) noted that the binding sites of DNA for alfa B1 doubled in the presence of histone as analyzed spectrophotometrically.They suggested that this may be due to the interaction of the toxin with histone. Edwards and Allfi-ey (1973) noted that administration of afla B1 to rats caused a sudden increase in the rate of release of the acetyl group from histone. This effect was not observed for afla B2 and actinomycin D. They found no difference in the histone deacetylase activities of rat liver from normal and afla B1-treated animals. These authors suggested that the observed effects of afla B1 on the histone deacetylation process reflects a sudden modification of the system which controls the activity of the deacetylation enzyme or the availability of the histone substrates. Whether the binding of afla B, or its metabolites with histone is involved in such a reaction remains to be answered. Because modification of histone structures may alter the interaction between histone and DNA, thus affecting the structure of chromatin, interaction of aflatoxin metabolites with histone warrants further investigation. The in vitro binding of afla B1 with chromatin was studied by Prasanna et al. (1976) using fluorescence quenching and fluorescence polarization techniques. Interaction of afla B1 was found to be strongest with intact chromatin and decreased in affinity for the chromatin components in the following order: histone-free chromatin, histone, DNA, and weakest with nonhistone residue. These results indicate that the structure integrity of chromatin is very important for such interaction. Interaction of afla G1 with chromatin was found to be less than B1. Since it has been shown that in vitro incubation with isolated chromatin did not affect the RNA polymerase activity, the role of these interactions on the biological activity of the toxin remains to be revealed. The interaction between the hormonal action and mycotoxin action has been considered to be very important because: (a) some reports show that the toxicity of mycotoxins can be modified by sex difference in animals and the hormonal status of the animals under study; and (b)mycotoxins also can affect the hormone-induced biochemical reactions as well as possibly affect the hormonal imbalance. Alteration of the hormonal status in animals might possibly affect the metabolism of the mycotoxins (Schwartz and Perantoni, 1975; Swenson et al., 1977), thus affecting the toxicity and binding with macromolecules. The possibility of interaction of mycotoxins with hormone receptors has been considered also. Williams and Rabin (1969)and Williams et al. (1973), while studying the effect of afla B1 on the dissociation of ribosomes from the rat liver endoplasmic reticulum membrane, found that afla B, competed with the steroid-dependent ribosomal binding sites, which suggested the possibility of interaction with the steroid hormone receptors.

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Kyrein (1974) found that afla B1, GI and G2 did not bind to calf uterus estrogen receptors in vitro but afla M1 did when the analysis was made by a competitive binding technique. The affinity of afla MI for the receptors was found to be 200 times smaller than that of the 17B-estrogen. Thus, it was suggested that the -OH group at the 4 positions in afla is important for such bindings. The effect of afla B, on the binding capacity of rat liver cytoplasmic glucocorticoid receptors and nuclear binding of toxin-receptor complex was investigated by Kensler et al. (1976). Although no change in kinetics of the binding of dexamethasone with the cytosol receptors was observed after the rat had received 1 mg of afla B1 per kilogram, the concentration of the nuclear receptor sites for the steroid and the binding constant in the treated rat liver decreased 33% and %%, respectively. Such inhibition of binding capacity was found to be dose related. Similar results were obtained when [3H]-dexamethasonewas administered to the afla Bl-treated rats. Actinomycin D resulted in a decrease in the nuclear receptors concentration but did not affect the binding constant. Therefore, the inhibitory effect of afla B1 on some glucocorticoid-stimulated processes is more likely due to the binding of the toxin (or metabolite) to the nuclear hormone receptors rather than interference with the cytosol hormone receptor-hormone interaction. The possibility of binding of aflatoxicol with hormone receptors has been suggested also (Patterson and Roberts, 1972), because this afla B, metabolite is formed possibly by the catalysis of the 17-ketosteroidreductase in the liver cytosol fraction (Patterson and Roberts, 1972; Salhab and Hsieh, 1975; Schoenhard et al . , 1976)and microorganism (Detroy and Hesseltine, 1970). Although no experimental evidence has been shown for such interaction, owing to the structural similarity with the steroid and the nature of its formation, aflatoxicol would be the most likely candidate of the aflatoxin groups to bind with the receptors. There has been evidence in recent years regarding the importance of this metabolite to the toxic effect of afla B,. Aflatoxicol may convert to afla B, in vivo. Whether a flatoxicol will bind with receptor sites and thus play a significant role in altering the hormoneregulated gene expression remains to be seen. Other mycotoxins may also be capable of binding with the hormone receptors. For example, the estrogenic mycotoxin F-2 (zearalenone)which has recently been shown to bind with a protein-type hormone receptor in fungal mycelium and subsequently affect the perithecium formation (Mirocha, 1977)may also have high affinity for the estrogenic receptors in animals. 4 . Binding of Mycotorins with Functional Subcellular Organelles and Membrane Structure Although information on the distribution of mycotoxins in subcellular organelles has accumulated (Chu, 1975, 1977), details on the mode of the

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binding with such organelles are generally not available. In the previous discussion it was suggested that mycotoxins may affect the transport process through binding with the mitochondrial membrane, thus causing inhibition in mitchondorial respiration, yet only luteoskyrin was shown to have a high affinity for mitochondria. In a distribution study, I. Ueno et al. (1974) found that almost 50% of the total radioactivity in mouse liver homogenate was in the mitochondrial fraction after the animal received [ 3H]luteoskyrin. Of the total radioactivity in the mitochondria, as much as 80%was found to be intact luteoskyrin. For aflatoxin B1, it ranges from 29 to 36% in the mitochondria. Meisner and Chan (1974) found that OA bound with the rat mitochondrial membrane, but Uenoet al. (I. Ueno, P. S. Sun, and F. S. Chu, unpublished observation) found that the toxin in rat liver mitochondria was not significantly high. Inhibition of protein synthesis by the 12,13-epoxytrichothecenetype of mycotoxins was considered to be due directly to their interaction with ribosomes. Binding of T2, fusarenon X, and a number of other 12,13epoxytrichothecenes with eukaryotic ribosomes and with yeast 60 S ribosome subunits has been reported. The binding constants range from 0.6 x 106 to 1.5 x lo6M+' (0.44 mole of toxin per ribosome; Wei et al., 1974a; Jimenez and Vazquez, 1975). Because both the epoxy group and some of the side chains are important in determining the potency for inhibition of protein synthesis and the ribosome bindings, Cundlif€e et al. (1974) and McLaughlin (1977) suggested that the conformation of the toxin molecule, which is determined by the sizes and types of the side chain at C-15 position, plays a significant role by exposing the epoxy group (possibly through opening up the ring) for the interaction of the toxin with ribosomes. Because it has been found that the epoxy group can react with -SH enzymes, Ueno (1977) pointed out the role of epoxy hydrase and epoxy GSH-transferase in determining the overall in vivo toxicity of T2 and other 12,13-epoxytrichothecenes. In addition, both T2 and fusarenon X have been found to bind with rabbit reticulocyte membrane. Because the binding of T2 with membrane was stronger than that for fusarenon X, such bindings appear to be correlated to the toxicity of the toxin (Ueno and Matsumoto, 1975). Interaction of afla B1 or its metabolite(s) with ribosomes was suggested from the observation that the toxin-induced polysome degradation and that the radioactivity was associated with both polysomes and monosomes after rats received [3H]aflaB, (C. Y. T. Shih and F. S. Chu, unpublished observation). Sarasin and Moult5 (1975) suggested that the early phase of in vivo inhibition of protein synthesis in rat liver by afla B1 was due to the noncovalent binding of afla B1 metabolite(s) with polyribosomes. Noncovalent binding of phalloidin was also reported (Wieland and Wieland, 1972), and the binding site appeared to decrease when the CCl,-treated rat liver was used (Kroker and Frimmer, 1974).

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Since microsomal mixed-function oxygenases are the sites for the afla B, metabolism, Gurtoo (1973) and Gurtoo and Campbell (1974)investigated the possible binding of afla B1 with microsomes in vivo. These investigators, using mixed function oxygenase inhibitor such as SKF-525 A, found that metabolic activation is necessary, and they also demonstrated that it was due to the interaction of the metabolite B,, with the -NH2 group of protein to form a Schif€ base-type bond. In the in vitro spectrophotometric study, Norpoth et al. (1974) also reported the interaction of afla B1 with microsomes. Oxidative metatolism of afla B1 was found to be necessary for the formation of afla Bl-glutathione conjugate (Raj et al., 1975).

VI. Concluding Remarks From the foregoing, it is apparent that research on the mode of action of mycotoxins and related compounds has progressed rapidly during the last 2 or 3 years. Whereas major biochemical effects of mycotoxins involve the alteration of normal metabolic and other vital processes, the mode of their action appears to be primarily based on their ability to interact with macromolecules, subcellular organelles and organs. Such interactions, however, are complicated by the nature of toxin involved, requirement of metabolic activation of some mycotoxins and factors affecting such metabolism, biological individuality, especially species, sex, and age, and a number of other factors, such as dietary effects, hormonal status, and interactions with other toxins including mycotoxin synergism. Thus, there is no generalized mechanism that can apply to all mycotoxins. The mechanism for certain mycotoxins in mammalian systems may not necessarily be applicable to other systems, such as plants and microorganisms, especially if metabolic activation is involved. Several model and putative mechanisms have been established for some mycotoxins and have been shown to be applicable to structurally related compounds with or without some modification. For instance, the putative “2,3-epoxide afla B1 intermediate” mechanism is applicable also to sterigmatocystin and related compounds. The question of whether epoxidation and other types of metabolic activiation for other mycotoxins are involved remains to be answered. Trichothecenes have a different biochemical effect as compared to afla B1, as the primary interaction with proteins possibly is through opening the 12,ELepoxide. Structure-function studies also show the importance of the 9-10 unsaturated double bond. Information regarding the metabolism of trichothecenes and a comparative study on the binding capacity of trichothecenes and their derivatives (i.e., the 9-10 double bond saturated) with macromolecules will certainly help in determining whether the 9-10 unsaturated double bond, through epoxidation or other mechanisms, is involved also.

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Examples of structurally related mycotoxins with different modes of action have also been noted. Patulin and penicillic acid interact with nucleophiles through Michael addition, but owing to steric effects, the Michael addition of nucleophiles was not demonstrated for the a-b unsaturated double bond in the cyclopentenone ring in d a B1 (J. V. Rodricks, private communication). Instead, this part of the afla B1 moiety plays a significant role in the noncovalent binding with macromolecules and electron donating compounds perhaps through charge transfer interactions (Noh and Chu, 1972). Because the biological activity of mycotoxins can be affected by a number of factors, investigation of the binding of mycotoxins with macromolecules under different conditions has become a very attractive approach in determining the mode of action of some mycotoxins. Garner and Wright (1975) compared to the binding of [l"C]-aflaB, to liver and kidney macromolecules of rats and hamsters. They found that the radioactivity in the rat liver macromolecules(predomiBately in the nucleic acid)was considerablyhigher than those in the hamster (afla Bl-resistant species) liver. Likewise, mouse liver slices exhibited a lower degree of inhibition of RNA synthesis and lower binding of afla B1 to the nuclear fraction than rat slices after the animals received the toxin (Goday and Neal, 1976). Phenobarbital, which has been shown to inhibit the afla Bl-induced hepatocarcinogenesis in rats, also was shown to inhibit the covalent binding of afla B1 to liver macromolecules (Garner, 1975; Swenson et al., 1977). However, phenobarbital has a different effect in mice. Whereas phenobarbital treatment before dosing with afla B1 resulted in a decrease in RNA synthesis in rat liver, significant enhancement transcription inhibition was observed in mouse liver (Mouk et al., 1975). Alexandrov and Frayssinet (1974) found that phenobarbital and 3-methylcholanthrene stimulated the binding of afla B1 to rat liver DNA, apparently they also included the noncovalent bindings. Effect of sex and hormonal status on the mycotoxin-macromolecule interactions continues to be an interesting research area. Male rats were found to have a higher capacity to form the DNA-alkylating adduct than females, which was considered to be due to their higher rate of metabolism (Gurtoo, 1976; Gurtoo and Motycka, 1976). Likewise, accumulation of luteoskyrin in the male mouse liver mitochondria was found also to be much higher than in females (I. Ueno et al., 1974), although metabolism did not involve this mycotoxin. The levels of hepatic afla B1-DNAadducts were found to be only one-half of the control level in the hypophysectomized rats while the rRNA-, protein-afla B1 adduct were similar to those of controls (Swenson et al.. 1977). Dietary conditions also have a great effect on the afla B1 macromolecule bindings (T. C. Campbell and J. K. Hayes, private communications). Rats (male) fed with a choline-deficient diet were shown to bind less afla B1 to the liver macromolecules either covalently or noncovalently than

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the controls did (I. Ueno, P. S. Sun, and F. S. Chu, unpublished observations). When male weanling rats were fed with a protein-deficient diet, Preston et al. (1976)found that there was a consistent decrease in binding of afla B, to chromatin, DNA, and chromatin protein. While progress on the mode of action of some mycotoxins (especially afla B,) has been made, the mechanism for most other mycotoxins is still unknown. Present knowledge and research experience on the mode of some of those mycotoxins will undoubtedly be useful in the elucidation of other mycotoxins. It is hoped that, through the understanding of the mechanism of action of these toxic compounds, methods for control and prevention of mycotoxin problems can ultimately be developed. ACKNOWLEDGMENTS The author wishes to express his appreciation to Professors M . S. Bergdoll, M. W. Pariza, and D. Perlman, all of University of Wisconsin, for their critical suggestions in the preparation of this manuscript; to Drs. A. Ciegler, A. W. Hayes, C. S. Mc Laughlin, Y. Moulb, J. L. Richards, and P. S. Styen for their prepublication manuscripts on related subjects.

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Schoenhard, G. L., Lee, D. J., Howell, S. E., Pawlowski, N. E., Libbey, L., and Sinnhuber, R. 0. (1976). Cancer Res. 36, 2040. Schwartz, A. G . , and Perantoni, A. (1975). Cancer Res. 35, 2482. Scoppa, P., and Borle, W. 0. (1971). Boll. SOC. Ztal. B i d . Sper. 47, 201. Scoppa, P., and Marafante, E . (1971). Boll. SOC. Ztal. Biol. Sper. 47, 198. Scott, F. W., de LaHunt, A,, Schutz, R. D., Bistner, S . I., and Riis, R . C. (1975). Teratology 11, 79. Scott, P. M. (1974). In “Mycotoxins” (I.F.H. Purchase, ed.), p. 369. Elsevier, Amsterdam. Scott, P. M., Merrien, M. A., and Polonsky, J. (1976). Erpa’entia 32, 140. Shank, R. C., and Wogan, G . N. (1966). Toricol. Appl. Pharmacol. 9, 468. Shank, R. C., Gibson, J. B., Nondasuta, A,, and Wogan, G. N. (1972a). Food Cosmef. Toxicol. 10, 61. Shank, R. C . , Gordon, J. E., Nondasuta, A , , Suhhamani, B., and Wogan, 6. N. (1972b). Food Cosmet. Toxicol. 10, 71. Shank, R. C., Bhamarapravati, N., Gordon, J. E., and Wogan, G. N. (1972~).Food Cosmet. Toricol. 10, 171. Shankaran, R . , Raj, H. G . , and Venkitasubramanian, T. A. (1970). Enzymologia 39, 370. Singh, J. (1967). In “Antibiotics” (D. Gottlieb and P. D. Shaw, eds.), p. 621. Springer-Verlag, Berlin and New York. Sinnhuber, R. O., Lee, D. J., Wales, J. H., Landers, M. K., and Keyl, A. C . (1974).J . Natl. CancerInst. 53, 1285. Smalley, E. B., and Strong, F. M. (1974). In “Mycotoxins” (I.F.H. Purchase, ed.), p. 199. Elsevier, Amsterdam. Smith, J. W., and Hamilton, P. B. (1970). Poult. Sci. 49, 207. Sporn, M. B., Dingman, C. W., Phelps, H. L., and Wogan, G . N. (1966). Science 151, 1539. Statford, M. E., and McLaughlin, C. S. (1973).J . Cell. Physiol. 82, 121. Stern, P. (1971).Jugos. Physiol. Pharmacol. Acta 7, 187. V. Steyn, P. S. (1977). In “Problems in Human Health and Disease in Modern Society” Rodricks, ed.), NIH, HEW (in press). Steyn, P. S., Vleggar, R . , Du Preez, N. P. , Blyth, A. A,, and Sieger, J. C. (1975).Toxicol. Appl. Pharmucol. 32, 198. Stich, H . F . , and Laishes, B. A. (1975). Znt. J . Cancer 16, 266. Stirpe, F., and Fiume, L. (1966).Biochem. J. 105, 779. Stott, W. T., and Bullerman, L. B. (1975a).J . Milk Food Technol. 38, 695. Stott, W. T., and Bullerman, L. B. (1975b).J . Assoc. Oj$ Anal. Chem. 58, 497. Strufaldi, B., Noyueria, D. M., and Pedroso, F. I. (1970). Rev. Farm. Bioyuim. 8, 1. Suzuki, S., and Satoh, T. (1973).Jpn. J. Pharmacol. 23, 415. Suzuki, S . , Satoh, T., and Yamazaki, M. (1975). Toricol. A p p l . Pharmucol. 32, 116. Svoboda, D., Grady, H . , and Higgiuson, J. (1966). Am. J. Pathol. 49, 1023. Swenson, D. H., Miller, J. A., and Miller, E. C . (1973). Biochem. Biophys. Res. Commun. 53, 1260. Swenson, D. H., Miller, E. C., and Miller, J. A. (1974).Biochem. Biophys. Res. Commun. 60, 1036. Swenson, D. H . , Miller, J. A,, and Miller, E. C . (1975). Cancer Res. 35, 3811. Swenson, D. H . , Lin, J. K . , Miller, E. C . , and Miller, J. A. (1977). Cancer Res. 37, 172. Teunisson, D. J.. and Robertson, J. A. (1967). A p p l . Microbiol. 15, 1099. Tung, H . T., Donaldson, W. E., and Hamilton, P. B. (1972). Toricol. Appl. Pharmucol. 22, 97. Ueno, I . (1966). Seikagaku 38, 741. Ueno, I . , Ueno, Y., Tatsuno, T., and Uraguchi, K. (1964).Jpn, J. E r p . Med. 34, 135. Ueno, I . , Hayashi, T., and Ueno, Y. (1974).Jpn. J. Pharmacol. 24, 535.

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Some Aspects of the Microbial Production of Biotin YOSHIKAZU IZUMIAND KOICHI OGATA* Department of Agricultural Chemistry, Kyoto Unizjwsity, Kyoto, lapan

..

..

..

11. Biosynthetic Pathway of Biotin . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Pimelyl-CoA Synthetase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. KAPA Synthetase., . . . . . . . . . C. DAPA Aminotransferase . . . . . D. DTB Synthetase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Biotin Synthesizing Reaction . . . . . . . . . . . . . . . . . . . . . . . . . 111. Regulation of Biotin Biosynthesis . . . . . . . . . . . . . . . . . IV. Preparation of Biotin and Its Vitamers. .................... A. Chemical Processes for Preparation of Biotin . . . . . . . . . . . B. Microbial Synthesis of Biotin and Its Vitamers . . V. Biotin Antimetabolites and Their Actions on Biotin Biosynthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Actithiazic Acid . . . . . .... .. B. a-Dehydrobiotin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. a-Methylbiotin and a-Methyldethiobiotin . . . . . . . . . . . . . . D. Amiclenornycin and Stravidin . . . . . . . . . . . . . . . . . . . . . . . . . E. Adenine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI. Biodegradation of Biotin and Its Vitamers VII. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

145 146 148 149 149 151 153 155 158 158 160 163 164 166 167 168 169 170 172 173

I. Introduction It is well known that biotin, vitamin H and coenzyme of carboxylase, plays a vital role in the process of microbial production of amino acids, as a control factor for the regulation of the amount of product. The action mechanism of biotin in glutamic acid production, which is closely related to the membrane permeability of glutamic acid-producing organisms toward glutamic acid, has been elucidated by extensive studies in a variety of ways. This information has led to the recent establishment of a unique new method for glutamic acid production from n-paraffin by a glycerol-requiring mutant of an nparsn-utilizing bacterium. Thus, in the field of industrial fermentation, biotin is closely linked with the microbial production of amino acids (Kinoshita and Tanaka, 1972). This is also indicated by the fact that, as described later, studies on the microbial production of glutamic acid have greatly contributed to the clarification of the biosynthetic pathway of biotin in microorganisms. *Professor Ogata is deceased

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YOSHIKAZU IZUMl A N D KOICHI OGATA

One of the most common practical uses of biotin is as a supplement of culture media for amino acid fermentation. In addition, biotin has been attracting increasing interest recently as a food and fodder supplement. At present, the industrial preparation of biotin is carried out through a chemical process; a fermentative process remains to be established. Because of intricacy of the chemical process, an efficient and easy biotin fermentation is highly desirable for the supply of large quantities. Here we will review the biosynthesis and biodegradation of biotin from the viewpoint of industrial fermentation.

II. Biosynthetic Pathway of Biotin Studies on the biosynthesis of biotin in microorganisms started directly after the determination of its chemical structure (Melville et al., 1942) with stuhes on the nutritional requirements of microorganisms (Mueller, 1937a,b; du Vigneaud et al., 1942; Eakin and Eakin, 1942; Dittmer and d u Vigneaud, 1944; Dittmer et al., 1944; Lilly and Leonian, 1944; Stokes and Gunness, 1945)and contemporaneous studies on its total synthesis (Harris et al., 1943, 1944a,b, 1945). Subsequently, through radio-chemical and genetic biochemical studies (Tatum, 1945; Pontecorvo, 1953; Ryan, 1956), part of the biosynthetic pathway was proposed. Thereafter a hypothetical pathway was established by Okumura et al. (1962a-e) through their investigation of the microbial production of glutamic acid. In glutamic acid production using biotin-requiring organisms, Brevibacterium lactofmentum and B . flavum, they found that, besides dethiobiotin (DTB)and pimelic acid, a variety of chemically synthesized pelargonic acid derivatives including, 7,8-diaminopelargonic acid (DAPA), 7-keto-8-aminopelargonic acid (KAPA), 7-amino-8-ketopelargonic acid, and 7&diketopelargonic acid, and also oleic acid, satisfied the biotin requirement. They strongly suggested that these compounds might be precursors in the biotin biosynthesis, from the relation in the chemical structure. They also investigated the amount of each biotin vitamer’ which gives a maximum yield of glutamic acid, and the amount of intracellular biotin formed on incubation in the presence of each of these biotin vitamers. Subsequently, Ogata et al. and Iwahara et al. made extensive investigations of biotin biosynthesis and verified the main pathway of Okumura et al. using growing and resting cells. Their evidence was as follows: (1)Various microorganisms synthesized large amounts of biotin vitamers, mainly DTB, in the presence of pimelic acid (Ogataet al., 1965a,b; Iwahara et al., 1966a);(2)various bacteria ‘The term “biotin vitamer” is used in this review to indicate a compound that can satisfy the biotin requirement in the growth of biotin auxotrophs.

147

MICROBIAL PRODUCTION OF BIOTIN

synthesized KAPA from pimelic acid (Ogata et al., 1965b; Iwahara et al., 1967); (3) resting cells of Bacillus sphaericus synthesized DTB both from pimelic acid (Iwahara et al., 1965, 1966e)and from the KAPA obtained from the culture filtrate of Bacillus cereus (Iwahara et al., 1966b); (4) various microorganisms had the ability to synthesize biotin from DTB (Iwahara et al., 1966~). Using biotin auxotrophic mutants of Escherichia coli, Rolfe and Eisenberg (1968)and Pai (1969a) also verified the proposed pathway of Okumura et al. by means of cross-feeding tests, the identification of accumulated biotin vitamers and growth experiments with each vitamer. They confirmed the pathway of biotin biosynthesis to be pimelic acid 4 KAPA + DAPA + DTB + biotin. In this manner, studies of the biosynthetic pathway of biotin progressed using whole cells. The first demonstration of the enzymic system involved in biotin biosynthesis was made by Eisenberg and Star (1968).Since that time,

COAT

L-Alan,ine

COOH CHz(CHd4COOH

Pimelic mid

ATP, Mg"

&-SCoA CHz(CH&COOH

Pimelyl -CoA

PLP

v+ ,

9

7-Keto-8-amino-

H V - 7 pelargonic acfd H3C CHz(CHz)4CWI-I (KAPA) tjHz NHz

H~-+H H3C , CHz(CH?)LCOOH HCO3ATP. Mg2'

7,B-Diaminopelargonic a (DAPA)

d

0

F,YH HY

Dethiobiotin

(DTBI

J

B Hy/'\yH

Biotin

I?&&3-l&COOH FIG. 1. Biosynthetic pathway of biotin in microorganisms.

148

YOSHIKAZU IZUMI AND KOlCHI OGATA

studies have used cell-free systems to determine the enzymic steps in biotin biosynthesis, and so far revealed all the steps from pimelic acid to DTB, as shown in Fig. 1. The final step from DTB to biotin has not been enzymically resolved yet. The following sections describe the four enzymes involved in the synthesis of DTB from pimelic acid, and the final step of biotin biosynthesis. A. PIMELYL-COASYNTHETASE

I t was assumed that pimelyl-CoA, the activated form of pimelic acid which condenses with L-alanine to form KAPA, may be formed from pimelic acid and CoA or acyl-CoA, and that an enzyme synthesizing pimelyl CoA from pimelic acid may be responsible for the first step in biotin biosynthesis by microorganisms. Eisenberg and Star (1968), using various microorganisms, reported that an enzyme activity catalyzing formation of pimelyl-CoA could not be detected by the hydroxamate method. They pointed out that one cause might be the high pimelyl-CoA deacylase activity in the cell extracts. Izumi et al. (1972a)were able to demonstrate enzyme activity catalyzing the formation of pimelyl-CoA from pimelic acid and CoA in microbial cell extracts, using the coupling reaction of the KAPA synthetase system described in Section II,B, where the KAPA formed from pimelyl-CoA was bioassayed with Saccharomyces cerevisiae as the test organism. On investigation the distribution of the activity of pimelyl-CoA synthesizing enzyme in cell extracts of about 100 strains of bacteria, Izumi et al. detected higher activity in Bacillus megaterium, Pseudomom jluorescens, and Micrococcus roseus. Izumi et al. (1972a)found that, other than pimelic acid and CoA, ATP and Mg2+ were necessary for this enzyme reaction. The enzyme was purified about 34-fold from the cell extract of B . megaterium by ammonium sulfate fractionation and DEAE-cellulose column chromatography (Izumi et al., 1974). K , values were 2.7 X lop4M for pimelic acid, 5.5 x M for CoA, 1.5 x lop3M for ATP, and 1.5 X M for M$+. Optimum temperature was 32"C, and optimum pH was 7.0 (phosphate buffer) or 8.0 (Tris-HC1 buffer). The pimelyl-CoA formed by this reaction was converted to its hydroxamate, and identified with the hydroxamate of authentic pimelylCoA by paper chromatography. Metal-chelating agents like EDTA, ophenanthroline, and a,a'-dipyridyl, and ferric ion, markedly inhibited this enzyme reaction. Further, Izumi et al. (1974) showed that Mnz+ and ADP could be substituted for Mg2+ and ATP, respectively. From these results, and the fact that dicarboxylic acids other than pimelic acid could not serve as a substrate, the pimelyl-CoA synthetase appears to be a new enzyme that should belong to EC 6.2.1 (acid-thiol ligases).

MICROBIAL PRODUCTION OF BIOTIN

149

B. KAPA SYNTHETASE Eisenberg and Star (1968), using crude cell extracts from a biotin auxotrophic mutant ofE. coli, reported that KAPA was synthesized from pimelylCoA and L-alanine through the participation of pyridoxal5’-phosphate (PLP). This was the first report to elucidate the biosynthetic pathway of biotin at the enzymic level. There were two main reasons which led them to consider the possibility of this reaction. One was the finding of Iwahara et al. (1966e) of the remarkable stirnulatory effect of L-alanine on the production of DTB from pimelic acid by resting cells of Bacillus sphaericus. The other was the close resemblance between the reaction of pimelic acid and alanine to form KAPA and the reaction of succinyl-Cob and glycine to form 6-aminolevulinic acid through the preparation of PLP (6-aminolevulinic acid synthetase involved in prophyrin biosynthesis) (Shemin et al., 1955). Izumi et al. (1972b, 1973a) partially purified the enzyme from the cell extract of B . sphaericus by ammonium sulfate fractionation, protamine treatment, and DEAE-cellulose column chromatography. This enzyme reaction required PLP as coenzyme and was strongly inhibited by various carbony1 reagents which are inhibitors peculiar to vitamin B6 enzymes. They investigated the amino acid specificity of the enzyme and established that the only amino acid that condensed with pimelyl-CoA was L-alanine; L-serine, which Eisenberg and Star (1968) demonstrated to replace Lalanine using a crude cell extract of E . coli, and L-cysteine, which Lezius et al. (1963) presumed to condense with pimelyl-CoA to form 9-mercapto8-amino-7-oxopelargonic acid, did not serve as substrate. The reaction suffered strong competitive inhibition by such amino acids as L-cysteine, D-alanine, L-serine, glycine, and D- and L-histidine, especially L-cysteine. Further, Izumi et al. (1973b) measured this enzyme activity in the cell extracts of about 100 strains of bacteria and found the enzyme present in many bacteria. Thus, KAPA synthetase, like 6-aminolevulinic acid synthetase, catalyzes the condensation reaction accompanying decarboxylation to form a ketoamino acid from acyl-CoA and amino acid with PLP as coenzyme. Therefore, Izumi et al. (1973b)proposed that KAPA synthetase should be regarded as a member of the acyltransferases (EC 2.3.1)and that it should be named pimelyl-CoA:L-danine 2-C-pimelyltransferase or L-danine pimelyltransferase. C. DAPA AMINOTF~ANSFERASE Pai (1971), using the cell extract from a biotin auxotrophic mutant ofE. coli which was deficient in DTB synthetase, found that DAPA was formed from

150

YOSHIKAZU IZUMI AND KOICHI OGATA

KAPA, and pointed out the existence of a DAPA aminotransferase. To measure the activity of this enzyme, DAPA was converted to DTB by the coupling reaction of the DTB synthetase system, as described in Section II,D, and DTB was bioassayed. He reported that methionine and PLP were amino donor and coenzyme, respectively, in this enzyme reaction. Later, Eisenberg and Stoner (1971) also found this enzyme activity using the crude cell extract of a biotin auxotrophic mutant of E. coli, but in this case the enzyme activity was measured directly by bioassay of the reaction product, DAPA. In the subsequent investigation of amino donors, L-methionine was found to be effective in the reaction with resting cells but required the addition of ATP and M 3 + with the cell extract. From this interesting result, they thought the true amino donor might be S-adenosyl-L-methionine (SAM). Izumi et al. (1975) investigated the activity of this enzyme in the cell extracts of about 100 strains of bacteria. Their method of measurement of the activity, like that of Pai (1971), was to convert the DAPA formed to DTB by the coupling reaction of DTB synthetase, and to determine DTB by bioassay with Bacillus subtilis. They found Brevibacterium divaricatum, Salmonella typhimurium, Bacillus roseus, Micrococcus roseus, and E . coli to have high activity. From the cell extract of B . divaricatum, which showed the highest activity, they purified the enzyme about 5000-fold by ammonium sulfate fractionation, acetone fractionation, DEAE-cellulose, and hydroxylapatite column chromatographies, and two applications to Sephadex G-100 gel filtration (Izumi et al., 1973c, 1975). The enzyme preparation gave a single band on disc gel electrophoresis. This enzyme was found to be a typical PLP enzyme, having absorption maxima in the region of 320 nm and 410 nm as well as 280 nm. It was confirmed to be a new aminotransferase showing specifcity only for SAM as amino donor. As amino acceptor, 7amino-8-ketopelargonic acid, an isomer of KAPA, as only one-hundredth of the activity of KAPA. Pyridoxamine 5'-phosphate (PMP), as well as PLP, can act as coenzyme. K , values were 0.69 X M for KAPA, 0.55 X M for SAM, and 0.83 X M for PLP. Stoner and Eisenberg (1975a,b)made a more detailed investigation of the properties of this enzyme purified 1000-fold from an extract of a regulatory mutant of E. coli. They were able to resolve the cofactor, PLP, from the enzyme in the presence of phosphate buffer after incubation with the amino donor, SAM. The molecular weight was estimated to be 94,OOO+- 10,OOOby gel filtration and sucrose gradient sedimentation. Furthermore, sodium dodecyl sulfate disc gel electrophoresis established the molecular weight of the enzyme subunit as 47,000 +- 3000, and therefore, the enzyme is considered to consist of two subunits of similar molecular weight. Since Stoner and Eisenberg (1975a) could not detect the expected keto product of SAM, S -adenosyl-2-oxo-4-methylthiobutyric acid, in the DAPA

MICROBIAL PRODUCTION OF BIOTIN

7-keto-&aminopdargonic acid (KAPA) PMP MenzymeP

I

151

7.8-diaminpdargonic acid

) PLPA enzyme

nonenzymic

2-0x0-3-butenoic acid

+

5'-methylthioadenosine

FIG. 2. Proposed mechanism for the DAPA aminotransferase reaction. From Stoner and Eisenberg (1975a).

aminotransferase reaction with the use of chemical reagents, they considered that the keto product might decompose nonezymically to yield 5'-methylthioadenosine and 2-0x0-Sbutenoic acid. This assumption was supported by the result that when the reaction mixture containing S-adenosyl-~-[2-'~C]methionine as amino donor was acidified and passed through a Dowex 50-H+ column, a radioactive neutral compound was detected in the effluent. The compound was confirmed to be enzymically produced in amounts equivalent to the DAPA produced. On the basis of this experimental evidence, the DAPA aminotransferase reaction was formulated as shown in Fig. 2 (Stoner and Eisenberg, 1975a). KAPA was found to show strong substrate inhibition (Pai, 1971; Eisenberg and Stoner, 1971; Izumi et al., 1975). The inhibition by KAPA is competitive with SAM (Stoner and Eisenberg, 1975b; Hotta et al., 1975). The enzyme activity was strongly inhibited by phenylhydrazine, semicarbazide, isoniazide, and hydroxylamine, which are typical inhibitors of vitamin B, enzymes (Izumi et al., 1975). Thus, because of the high specificity for SAM as amino donor, the enzyme should be classified as a new type of enzyme (S-adenosyl-~-methionine:7keto-8-aminopelargonate aminotransferase) of the group EC 2.6.1 (Izumi et al., 1975).

D. DTB SYNTHETASE Eisenberg and Krell (1969a) found that DTB was formed from DAPA by resting cells of a biotin auxotrophic mutant of E . coli. This reaction was accelerated 2- to 3-fold by addition of L-serine, NaHC03, and glucose. Sub-

152

YOSHIKAZU IZUMl A N D KOICHI OGATA

sequently, Eisenberg and Krell (1969b), Pai (1969b), and Cheeseman and Pai (1970), using E . coli, and Yang et al. (1970b), using Pseudmonas graueolens, observed the formation of DTB from DAPA by cell extracts, and clarified that HC03-, ATP, and M$+ as well as DAPA are necessary for this enzyme reaction. Krell and Eisenberg (1970) purified the enzyme (DTB synthetase or ureido ring synthetase) about 2OO-fold from the cell extract of E . coli and obtained an enzyme preparation of over 90% purity. The enzyme has a molecular weight of 42,000 and is composed of two subunits. Yang et al. (1970b, 1971c)also purified the enzyme about 2000-fold from the cell extract of P . graveolens by ammonium sulfate fractionation, DEAE-cellulose and hydroxylapatite column chromatographies, and Sephadex G-200 gel filtration, and obtained an enzyme preparation showing a nearly symmetric peak upon ultracentrifugation. The sedimentation coefficient (s,,,,~) was 3.496 x cm/sec. The optimum pH was 7.0-8.0 and the optimum temperature was almost 50°C. Yang et al. (1969a, 1971a) observed that biotin diaminocarboxylic acid (BDC), a compound lacking the ureido part of biotin, was converted to biotin by resting cells of Bacillus sphaericus and Rhodotorula rubra and proved that the reaction proceeds by means of DTB synthetase. However, the activity of BDC as substrate for this enzyme is about one-tenth that of DAPA, and pelargonic acid derivatives, KAPA and 7-amino-8-ketopelargonic acid, could not act as substrate (Yang et al., 1970b, 1971~). CO, had higher activity than HC03- (Krell and Eisenberg, 1970). Of metal ions tested, Mn2+ showed 95-136% and Fez+ 71-91% of the activity of M g + (Yang

H2N NHz -COOH H3C

*I

c02

9-0H~&COOti

H3C

I

ATP

FIG.3. Proposed mechanism for DTB synthesis from DAPA by DTB synthetase. From Krell and Eisenberg (1970).

MICROBIAL PRODUCTION OF BIOTIN

153

et al., 1971~).CTP, UTP, GTP, and ITP showed 10-20% of the activity of ATP (Ogata et al., 1973b). Ogata et al. (1973b) demonstrated that the enzyme reaction was strongly inhibited by chelating agents, such as EDTA, a,a’-dipyridyl, and o-phenanthroline. Moreover, it was established that ADP shows competitive inhibition toward ATP (Krell and Eisenberg, 1970; Ogata et al., 1973b) and that the substrates DAPA and BDC are competitive with each other (Ogata et al., 1973b). Investigation of the enzyme reaction stoichiometry proved that equimolar amounts of DTB and ADP are formed (Krell and Eisenberg, 1970; Ogata et al., 1973b). Based on this observation, Krell and Eisenberg (1970) proposed a reaction mechanism for DTB synthetase as shown in Fig. 3. DTB synthetase is a new kind of carboxylase (EC 6.3.3.aa) because it catalyzes carboxylation accompanying the formation of ureido ring (Barman, 1974).

E. BIOTINSYNTHESIZING REACTION It has been recognized that DTB is converted to biotin during growth of Saccharomyces cerevisiae (Dittmer et al., 1944), Aspergillus niger (Wright and Driscoll, 1954), E . coli (Pai and Lichstein, 1965c), and other microorganisms including yeasts, molds, actinomycetes, and bacteria (Iwahara et al., 1966~).Tepper et al. (1966) confirmed this conversion with A . niger using [14C]DTB, labeled in either the carbonyl group or the carboxyl group. Biotin synthesis from DTB using resting cells was demonstrated with E . coli (Pai and Lichstein, 1965c, 1966), S. cerevisiae (Niimura et al., 1964a,b,c), and Rhodotmula glutinis (Izumi et al., 1973d). Furthermore, Niimura and Shimada (1967) observed the conversion using protoplasts of Bacillus megaterium. However, there have been no studies on enzymic synthesis of biotin from DTB. In the investigation of sulfur sources for biotin biosynthesis from DTB using resting cells of S . cerevisiae, Niimura et al. (1964b) found methionine sulfoxide and methionine to be most effective, and that NazS03, NazS, NaZSO4,homocysteine, SAM, methylmercaptane were also effective. Niimura et al. (1964~) next used [35S]-methionineand detected the reaction products by radioautography: radioactive biotin, biotin d-sulfoxide, biotin 1-sulfoxide, biocytin, and biocytin sulfoxide. Izumi et al. (1973d) tested the effect of various sulfur compounds using resting cells of R . glutinis which forms appreciable amounts of biotin from DTB. In the presence of DTB, this organism formed hardly any biotin on addition of inorganic sulfates and sulfites or L-cysteine, but formed considerable amounts of biotin on addition of DTB and methionine, in particular the

154

YOSHIKAZU IZUMI A N D KOICHl OGATA

L-form of methionine. Next, after reaction using ~-[~~S]methionine, they isolated radioactive biotin by cation and anion exchange column chromatography, avidin treatment, and dialysis, and identified it by radiochromatography and bioautography. They also confirmed that S contained in 1 molecule of L-methionine is incorporated into 1 molecule of biotin. Li et al. (1968b) carried out experiments with A . niger on the incorporation into biotin of car~nyl-['~C]DTBand carboxyl-[14C]DTB randomly labeled with 3H in order to measure a change of the number of hydrogen atoms during the conversion of DTB to biotin. The ratio of 3H/'4C for DTB and for the biotin formed showed that the 3H radioactivity of biotin was 15 to 20% lower than that of DTB. Thus, they considered that DTB might be converted to biotin with the loss of 3 of 4 hydrogen atoms. More recently, Parry and Kunitani (1976) have developed a new, stereospecific synthesis of DTB and reexamined the mechanism of the conversion of DTB to biotin by using specifically labeled [3H]DTB. The samples of tritiated DTB synthesized were each mixed with dZ-[IO-'4C]DTB and the doubly labeled precursors were then administered to cultures of A . niger. After incubation, the biotin synthesized from each doubly labeled precursor was isolated as d-biotin sulfone, and converted to biotin sulfone methyl ester. The methyl esters were purified by chromatography and then recrystallized to constant activity and constant ratio of 3H/14C.From the results shown in Table I, it appears that the introduction of sulfur at C-1 and C-4 of DTB takes place without the loss of hydrogen from C-2 or C 3 , suggesting that unsaturation is not introduced at C-2 or C 3 during the biosynthesis of biotin from DTB. However, they consider that the possibility of enzymic removal of hydrogen from C-2 or C 3 followed by replacement of the hydroTABLE I TFUTIATED DETHIOBIOTIN INTO BIOTIN"

INCORPORATION OF SPECIFICALLY

0

Expt. no. 1

2 3 4

precursor

3H/"C for biotin sulfone methyl ester

Percent 3H retention

6.05 2.89 6.88 5.88

5.74 3.04 4.81 3.10

95 105 70 53

3H/14Cfor Precursor

dl-[2,3-3H;10-'4C]DTBb d-[3-3H;IO-14C]DTE dl-[1-3H;IO-'*C]DTB dl-[4(RS)-3H;10-14C]DTB

"From Parry and Kunitani (1976). *Precursor had 58% 3H at C-2, 42% at C-3. 'Precursor had 17% 3H at C-2, 83% at C-3.

MICROBIAL PRODUCTION OF BIOTIN

155

gen without exchange cannot be excluded. Moreover, the result that the incorporation of dl-[1-3H]DTB into biotin proceeds with 30% tritium loss is consistent with the removal of one hydrogen atom from the methyl group of DTB. The result that dl-[4(RS)-3H]DTBis incorporated into biotin with 47% tritium loss suggests that the stereospecific removal of one hydrogen atom from C-4 of DTB may occur during the formation of biotin. Thus, these results clearly demonstrate that two hydrogen atoms are removed from d-DTB during its conversion to d-biotin. The next step in elucidating the mechanism of the biosynthetic conversion of DTB to biotin should be to investigate how the methyl group at C-1 and the methylene group at C 4 of DTB are converted and to determine the order of fimctionalization of C-1 and C 4 in DTB during its conversion to biotin.

111. Regulation of Biotin Biosynthesis Pai and Lichstein (1962, 1965a) cultured E . coli in medium supplemented with various concentrations of biotin, and measured the amounts of the biotin vitamers formed. In contrast to the constant intracellular content, the amount of biotin vitamers formed extracellularly decreased remarkably with added biotin. This inhibition of biotin vitamer biosynthesis was specific for biotin, hardly occurring with DTB, oxybiotin, and biocytin. Using resting cells of E . coli, Pai and Lichstein (196513) investigated whether this regulation occurred via repression, via feedback inhibition, or via both. In the presence of chloramphenicol, which inhibited the growth of the organism, the cells grown in a biotin-free medium synthesized as much as 80 x p g of biotin vitamers per milligram of cells during a 90-minute incubation period, whereas those grown in the biotin-supplemented (50 x p g m l ) medium synthesized only 5 x lop4 p g of biotin vitamers per milligram of cells. Furthermore, the cells grown in the biotin-free medium synthesized as much biotin vitamers in the presence of both chloramphenicol and biotin as they did in the presence of chloramphenicol alone. These facts indicated that biotin biosynthesis in E . coli might be controlled via repression rather than via feedback inhibition. Moreover, as no conversion of DTB to biotin occurred in the resting cells of either the mutant or wild-type strain which had been grown in the presence of exogenous biotin, the repression by biotin was also found to be operative in this conversion step ofE. coli (Pai and Lichstein, 1966).Pai and Lichstein (1966)pointed out that there is a critical level of exogenous biotin and that the biotin synthesizing enzyme system is almost completely repressed only when the biotin content of the medium was raised above 10 to 20 x lop4pg/ml. This coincides with the concentration of intracellular biotin (15 x lop4pg/ml culture) found in the organism after growth had ceased (Pai and Lichstein, 1965a) and

156

YOSHIKAZU IZUMI A N D KOICHI OGATA

TABLE I1 DTB-SYNTHESIZING ABILITY OF RESTING CELLSOF Bacillus sphaericus a DTB synthesized Cells harvested

(/ALg/ml)

From basal medium (cells: 97.1 rng/rnl)

45.0

From DTB-supplemented (100 pg/ml) medium (cells: 95.0 mg/ml)

42.3

From biotin-supplemented (0.2 pg/ml) medium (cells: 98.7 mg/ml)

0.05

“From Iwahara (1968)

is close to the level giving optimum growth of a biotin-requiring mutant ofE. coli (Ferguson and Lichstein, 1957). Iwahara (1W)also found the accumulation of biotin vitamers to be almost completely inhibited by the addition of biotin to the culture medium of various bacteria. However, this inhibitive action was not observed at all with fungal species. Whether this is due to the impermeability of the organisms toward exogenous biotin or to the insusceptibility of their biotin biosynthesis to repression by biotin has not been determined. As shown in Table 11, resting cells of B . sphaericus harvested from the basal medium without

Biotin added (pg/ml) FIG. 4. Effect of biotin on the synthesis of 7-keto-8-aminopelargonic acid (KAPA) synthetase of Bacillus sphaericus (-0) and 7,8-diaminoplargonic acid (DAPA) aminotransferase of Breoibacterium divaricaturn (0-0). From Izumi et al. (197313, 1975).

157

MICROBIAL PRODUCTION OF BIOTIN

0

01

02

03

04

Q5

06

"

Ib

Biotin added (pqlml)

FIG. 5. Effect of biotin on the synthesis of pimelyl-CoA synthetase (0-0) of Bacillus inegaterium. From Izumi et al. (1974).

(-0)

and DTB syn-

thetase

added biotin or from the DTB-supplemented medium readily synthesized DTB from pimelic acid, whereas cells obtained from medium supplemented with biotin synthesized very little DTB. These facts also bear out the suggestion of Pai and Lichstein (1965b)that the inhibitive action of biotin depends on repression. From progress in the enzymic solution of the biosynthetic pathway of biotin, the biosynthetic regulation mechanism has also been elucidated at the enzymic level. Eisenberg and Krell (1969b) have reported that KAPA synthetase and DTB synthetase ofE. coli are almost completely repressed by the addition of 1ng/ml of biotin to the medium. Pai observed similar repression by biotin of DTB synthetase (Pai, 1969b) and DAPA aminotransferase (1971) ofE. coli. Izumi et al. (1973b)found KAPA synthetase ofB. sphaericus and B . subtilis was repressed by biotin. As shown in Fig. 4, they demonstrated that KAPA synthetase of B . sphaericus and DAPA aminotransferase of Brevibacterium divaricatum were repressed by the addition of 0.1 pg/ml of biotin to the medium (Izumi et al., 197313, 1975). Moreover, they found that, in contrast to the complete repression of DTB synthetase in B . megaterium by 0.25 pg/ml of biotin, as can be seen in Fig. 5, pimelyl-CoA synthetase was not repressed by even 1 pg/ml of biotin (Izumi et al., 1974). In this way, a strong repressive action of biotin has been demonstrated on all the enzymes between pimelyl-CoA and DTB and on part of the biosynthetic system between DTB and biotin. This repression is thought to be the main reason for the minute amounts of biotin produced by a large number of microorganisms.

158

YOSHIKAZU IZUMI AND KOICHI OGATA

IV. Preparation of Biotin and Its Vitamers

A. CHEMICALPROCESSES FOR PREPARATION OF BIOTIN In this section, the practical chemical processes for preparation of biotin are briefly described for the purpose of comparison with the microbial synthesis of biotin, although the latter is not yet competitive. Presently, the industrial preparation of biotin is being carried out by the method of Hoffmann-La Roche, Inc. (Goldberg and Sternbach, 1949;

0 d-Camphorsulfonate o f d- and Z - ( X I )

Resolution

(j)

(XII)

d-Camphorsulfonate o f &(XI)

I

(k) -y-y

HZC\s/CH-(CH

A00C2H5 > )-CH

(1)

&Biotin

‘COOC2H5

(XIII)

( X W

FIG. 6. Flow sheet of one chemical process for industrial preparation of biotin. (a) Benzylamine; (b) phosgene; (c) acetic anhydride; (d) Zn, a mixture of acetic acid and acetic anhydride; (e) H,S, HC1; (0 C,H,O(CH,),MgBr; (9) H,, Raney nickel; (h) HBr, acetic acid; (i) silver d-camphorsulfonate; (i) isopropanol; (k) sodium diethylmalonate; (1) HBr. From Coldberg and Sternhach (1949).

HoaOHCQ

R

e

C

H

3

0

0 x 0

O

HO

F

(1)

OB?

Ox'

Ez=benzyl

(11)

W

C

O

H

O

C

H

0

H R

R

Y

( I X ) R=H

(VIII)

(X)

R=CH3S02

(")

R=MCOOCH3

j H--

-H

N3

N3

(XI)

C

e

OR

HflCooCH3

S H ' H h 9 R

0

(111) R = w C O O C H ,

3 H@COOCH3

H -

0 OxO

062

(IV)

-

159

MICROBIAL PRODUCTION OF BIOTIN

( V I ) R=H

(VII) R=CH3S02

r3

+ H-

+I

AcNH

__$

&Biotin

HNAc

Ac=CH3C0

(XI11

FIG. 7. Stereospecific synthesis of d-biotin from D-mannose. From Ohrui and Emoto (1975).

Gyorgy and Langer, 1968). As illustrated in Fig. 6, the synthesis is characterized by the use of a meso-diaminosuccinic acid derivative as a starting material, which contains two groups in the same spatial arrangement as the two amino groups present (in substituted form) in the biotin molecule, i.e., the meso-configuration in diaminosuccinicacid derivatives corresponding to the cis-structure of the two amino groups in a ring compound such as biotin. Moreover, since the resolution is carried out at the intermediate stage (XII-XIII), it permits the direct production of the optically active biotin, d-biotin and I-biotin, of which the d-form is naturally occurring and physiologically active. Recently, another efficient stereospecific total synthesis of d-biotin has been achieved by Ohrui and Emoto (1975).They used D-mannose as starting material. As shown in Fig. 7, D-mannose is converted to the aldehyde (11) through isopropyridenylation, benzoylation, selective isopropyridenylation, and periodate oxidation. Wittig reaction and hydrogenation of the aldehyde give the compound (IV) which has the side chain like biotin. Treatment of (IV) with NaOCH, in methanol, followed by the reduction of the resulting aldehyde (V) with NaBH, affords 0711) via (VI). Treatment of (VII) with NaS affords a tetrahydrothiophene derivative (VIII) which is converted via (IX) to (X). Treatment of (X) with NaN3 gives a diazido compound (XI). Catalytic reduction of the azido groups of (XI) in a mixture of methanol and acetic anhydride gives a diacetoamido derivative (XII). Treatment of (XII) with Ba(OH),, followed by the treatment with phosgene affords d-biotin. Thus, d-biotin is synthesized from D-mannose in good yield, because a fivemembered ring consisting of isopropyridene, which protects the hydroxyl groups of the sugar, is used to fix the molecular conformation during the intramolecular substitution reactions.

160

YOSHIKAZU IZUMI A N D KOICHI OGATA

B. MICROBIALSYNTHESIS OF BIOTINAND ITS VITAMERS 1. Biotin

Ogata et al. (1965a) and Iwahara et al. (1966~) examined the accumuation of biotin from pimelic acid and DTB by about 800 strains of stocked and isolated microorganisms. As a result, the maximum amount of biotin accumulated in the culture medium with 50-500 pg/ml of pimelic acid or SO pg/ml of DTB was about 500 ng/ml. The molds and Streptomyces tested generally accumulated larger amounts of biotin in their culture filtrates (Ogata, 1970a). In contrast, most of the bacteria and yeasts tested accumulated little biotin. Iwahara and Oguni (1973)have demonstrated that peptone and yeast extract have a remarkable promotive effect on the formation of biotin from DTB by various bacteria isolated from soil, and that one active ingredient in yeast extract is an iron salt. They reasoned that iron plays an important role in the biosynthesis of biotin. Yang et al. (1969a, 1971a)found that biotin was synthesized from BDC by the resting cells of B. sphaericus and R . glutinis. The conversion was markedly stimulated by addition of amino acids, especially alanine and glutamic acid, and under aerobic conditions. Under optimal conditions, about 6 pg/ml of biotin was formed from 10 pg/ml of BDC. Izumi et al. (1973e)found that biotin was synthesized from bisnorbiotin, a compound with two less carbon atoms in its side chain than biotin, by various microorganisms. Pseudomonas iodinum was the most effective convertor, giving 0.55 pg/ml of biotin from 1.0 pglml of bisnorbiotin. Similarly, they also observed the efficient conversion of bisnordethiobiotin to DTB. Ogino et al. (1974a,b) have demonstrated a useful method for industrial biotin production using an n-pardin-utilizing bacterium from a new compound, d-cis-tetrahydro-2-oxo-4-n-pentylthieno-(3,4d)-imidazoline (dlTOPTI). TOPTI was chemically synthesized from N1,N3-dibenzyldl-cistetrahydrothieno-(3,4-d)-imidazoline-2,4-dione[compound (VII) in Fig. 61 through Grignard reaction with n-pentyl magnesium bromide, dehydration in the presence of acidic catalyst, catalytic hydrogenation, and debenzylation with concentrated HBr. In this method of producing biotin, n-pardin was used as carbon and energy source for cell synthesis with concurrent transformation (cooxidation) of TOPTI to biotin. First, n-pardin-utilizing microorganisms that cooxidize TOPTI were selected from natural sources. Of the 9 strains that could convert TOPTI to biotinol and biotin, 3 strains identified as Corynebacterium were the most excellent producers. The cooxidation products synthesized from d-TOFT1 were isolated from culture broth and identified as d-biotinol and d-biotin by infrared (IR), nuclear magnetic resonance (NMR), and mass spectrometry (MS). The time course ofthe transformation showed that biotinol was initially produced before biotin began to

161

MICROBIAL PRODUCTION OF BIOTIN

dl-TOPTI

dl-Biotinol

dl- B iot in

FIG.8. Transformation of dl-cis-tetrahydro-2-0~04-~-pentylthieno-(3,4d)-imidazoline (dTOPTI) to biotin.

appear. From these results, the conversion ofTOPTI to biotin was assumed to occur via w-oxidation such as that of aliphatic hydrocarbon (Heydeman, 1960), as shown in Fig. 8. In a medium containing 2% n-paraffin and 0.2% urea, with addition of 50 mg/100 ml of dl-TOPTI after 24 hours of cultivation, maximum conversion of about 60% (32 mg/100 ml of dl-biotin) was obtained 96 hours after the addition of TOPTI. However, selective degradation of d-biotin occurred on prolonged incubation, leaving the h o m e r . Thus, to avoid such degradation, mutants that were incapable of assimilating n-paraffin and of degrading biotin, but capable of utilizing acetate, were derived. A mutant having onethirtieth of the ability of the parent strain to degrade biotin was cultivated on a medium containing 0.5%n-paraffin, 0.02% urea, 0.1% corn steep liquor, and 1% sodium acetate, and 0.8 mg/ml of dl-TOPTI was added after 15 hours cultivation. Under this condition, 0.65 mg/ml of dl-biotin was synthesized, i.e., maximum conversion increased to 80.5%. Ogino et al. (1974b) suggested that the enzyme system involved in the transformation might be inducible, from the fact that all the mutants obtained oxidized dt-TOPTI only when grown on a medium containing n-paraffin but not when grown on a medium containing acetate. In this manner, the unique method of Ogino et al. is characterized by microbial production of biotin with the use of a chemically synthesized substrate. 2.

DTB and Other Biotin Vitamers

Schopfer (1943) reported that the maximal yield of total biotin (referred to as biotin vitamers assayed with Saccharomyces cerevisiae) was about 0.020.03 pg/ml of the culture medium ofPhycumyces blakesleeanus after 7 days of growth. Eisenberg (1963), using the same mold, demonstrated that the addition of pimelic acid and the aeration of the culture increased the biotin yield 10- to 12-fold, with the average yield varying between 0.35 and 0.60 pglml of culture medium after 10-14 days of growth. On screening about 700 microorganisms, Ogata et al. (1965a) found that large amount of biotin vitamers were accumulated by Streptomyces, molds, and bacteria, in a medium with pimelic acid or azelaic acid. The main com-

162

YOSHIKAZU IZUMI A N D KOlCHl OGATA

ponent (60-95%) of the vitamers accumulated by these microorganisms was identified as DTB by anion exchange column chromatography, paper chromatography, and chemical analysis (Ogata et al., 196513).In particular, Bacillus sphaericus accumulated the vitamers in large amounts (150-200 pg/ml), and the dominant component was DTB (Iwahara et al., 1966a). Optimization experiments for maximum production of DTB by the bacterium indicated that peptone (1%)-soybean meal (10%) mixture as nitrogen sources was most effective. When the bacterium was cultivated on a medium containing this mixture and 0.5% casamino acid as nitrogen source, 2% glycerol as carbon source, and 1mg/ml of pimelic acid, the maximum amount of total biotin was about 150 pg/ml after 5 days of growth. In contrast, the accumulation of true biotin (referred to as biotin vitamers assayed with Lactobacillus plantarum) was less than 0.5 pg/ml under the cultural conditions tested. The DTB accumulated in the culture filtrate was isolated by active carbon treatment, and Dowex 1-f formate column chromatography, and crystallized from hot water. As a result, 30 mg of pure crystalline DTB was obtained from 3 liters of the culture medium containing 3 gm of pimelic acid (Iwahara et al., 1966a; Ogata, 1970a). Iwahara et al. (1967) also found that a large amount (20 pg/ml) of avidinuncombinable biotin vitamer was accumulated by a soil isolate, Bacillus cereus, and they identified it as KAPA. Subsequently, Eisenberg and Maseda (1970) obtained about 50 mg of KAPA in crystalline form from 100 gallons of culture medium of Penicillium chrysogenum. Tsuboi et al. (1966a) have screened many hydrocarbon-utilizing bacteria which accumulate biotin vitamers in hydrocarbon medium without exogenous precursors. Of more than 600 isolated strains, 35 strains accumulated over 100 ng/ml of biotin vitamers. A strain 0fPseudornona.s sp. which showed good assimilation of kerosine accumulated large amount of biotin vitamers, mainly DTB. Of various n-alkanes, n-alkenes, and glucose tested as carbon source, n-undecane a o r d e d the highest accumulation of biotin vitamers by this bacterium, and at the same time pimelic acid and azelaic acid were accumulated in the undecane culture broth (Tsuboi et al., 1966b). From this fact and the result that these two acids promoted accumulation of biotin vitamers, they assumed that n-undecane (Cll) might be converted to biotin vitamers via pimelic acid (C,) and azelaic acid (C,) through diterminal oxidation and P-oxidation mechanisms. This bacterium accumulated about 20 pg/ml of total biotin from kerosine in the presence of adenine (Tsuboi et al., 1967). This action of adenine on the accumulation of biotin vitamers is described in Section V,E. Yang et al. (1971b) found that various bacteria and yeasts could convert DAPA to other biotin vitamers during cultivation. I n particular, Pseudomonas graveolens and Saccharomyces marxianus accumulated large

MICROBIAL PRODUCTION OF BIOTIN

163

amounts of biotin vitamers, which responded to B . subtilis, from DAPA added to the medium. Bioautograms of culture filtrates during cultivation revealed that DAPA was first converted into DTB and that the DTB thus formed was converted into bisnordethiobiotin on longer incubation. Pseudomonas graveolens, which can convert DAPA to biotin vitamers in 60% yield under optimal conditions, was cultured in 15 liters of medium containing 1.5 gm of DAPA. After 6 days of cultivation, the biotin vitamers formed in the culture filtrate were purified by Dowex 1-=-formate column chromatography. As a result, 295 mg of d-DTB in crystalline form, together with bisnordethiobiotin in powder form, were obtained. In investigating fatty acid metabolism by microorganisms, Ohsugi et al. found that some strains which were able to utilize oleic acid (Ohsugi et al., 1972a; Ohsugi and Ishikawa, 1975), salicylic acid (Ohsugi et al., 1972b), or pelargonic acid (Ohsugi and Baba, 1975) as sole carbon source formed biotin vitamers in the culture broth, and the amounts of the vitamers accumulated were some micrograms per milliliter in each case. Ogata et al. (1966) have reported the isolation of bacterial strains that accumulate pimelic acid from azelaic acid. They obtained several strains of bacteria which showed heavy growth in a medium containing azelaic acid or acetic acid as a sole carbon source, but feeble growth in a medium containing pimelic acid. These strains accumulated pimelic acid in good yield from azelaic acid. Among these strains, a bacterium identified as Micrococcus sp. accumulated about 7.5 gmAiter of pimelic acid from 10 gmniter of azelaic acid in the culture filtrate after 22 hours growth.

V. Biotin Antimetabolites and Their Actions on Biotin Biosynthesis In fermentative production of amino acids, nucleotides, and nucleosides, methods using modified regulatory mutants that are insensitive to end product inhibition or to end product repression have been intensively developed in recent years (Abe, 1972; Ogata, 1975). In isolating regulatory mutants, strains resistant to amino acid and nucleic base analogs are derived, as in, e.g., L-threonine production by an a-amino-P-hydroxyvaleric acid-resistant mutant of Brevibactaium jZuvum (Shiio and Nakamori, 1970), L-histidine production by a thiazolealanine resistant mutant of Corynebacterium glutamicus (Araki et al., 1974), and inosine production by an 8azaguanine-resistant mutant of Bacillus sp. (Nogami et al., 1968). Since biotin biosynthesis is controlled via strong feedback repression by biotin, as described in Section 111, this control must be overcome in order to microbially produce large quantities of biotin and its vitamers. Some potent biotin antimetabolites have already been found. Therefore, it has become

164

YOSHIKAZU IZUMI A N D KOICHI OGATA

possible to use these antimetabolites for the derivation of regulatory mutants producing biotin and its vitamers.

A. ACTITHIAZIC ACID Actithiazic acid, or acidomycin (ACM), is an antibiotic that was independently isolated from the culture filtrates of Streptomyces sp. (Sobin, 1952), S. uirginiae (Grundy et al., 1952), S. lavendulae (Tejera et al., 1952), S . cinnumonensis (Maeda et al., 1952; Umezawa et al., 1953), and S. acidomyceticus (Ogata and Igarashi, 1954). The chemical structure of ACM was established as 4-thiazolidone-2-caproicacid (McLamore et al., 1952; Schenck and DeRose, 1952; Miyake et al., 1953). Its chemical synthesis has also been performed (Clark and Schenck, 1952). This antibiotic is most active against mycobacteria (Tejera et al., 1952; Grundy et al., 1952; Maeda et al., 1952). M . phlei was inhibited at 2.5 bglrnl and M. tuberculosis at 0.0625-0.125 pg/ml (Grundy et al., 1952). However, it exhibited no activity against tuberculosis in uivo (Sobin, 1952; Umezawa et al., 1953). The in vitro antibiotic activity was completely lost on addition of biotin to the test medium. Consequently, it was thought that the lack of activity in uivo might be due to the presence of biotin in the tissue. This loss of activity of ACM at concentrations of from 0.25 to lo00 pg/ml was accomplished by the addition of 0.064 p g ofbiotin per milliliter with M. tuberculosis (Grundy et al., 1952). Hamada et al. (1953)demonstrated that the competition ratio is 4 x lop4by the cylinder plate method using M. tuberculosis as test organism. Miyake (1953) studied the relationship between antitubercular activity and chemical structures of various synthetic compounds related to ACM. This results may be summarized as follows: (1)the side chain attached to the 2-position of the thiazolidone ring must contain a carboxyl or a group derivable from carbonyl for expression of antitubercular activity; the alcohol derivative has some antitubercular activity, but when the -CH,OH group is reduced to a methyl group, the activity disappears; (3)the side chain must be a straight pentamethylene chain for antitubercular activity; (4) the side chain is not the sole determinant of the activity; (5) modifications at the 2- and 3-positions of the thiazolidone ring cause disappearance of the activity; (6) a carbonyl group at the 4-position and a bivalent sulfur at the 1-position are essential. Subsequently, the antagonistic activity of biotin to the antitubercular activity of the active ACM derivatives shown in Fig. 9 was examined (Kawashima et al., 1956). These compounds inhibited the growth of aviantype tubercle bacilli at 0.32 to 1.6pg/ml. The inhibition caused by 10pg/ml of these compounds was lost on addition of 3.2 ng/ml of biotin in all cases.

MICROBIAL PRODUCTION OF BIOTIN

(I)

li'

(11)

(111)

165

R=COOH : Actithiazic acid RzCH20H R=COOCH3

FIG. 9. Actithiazic acid and its derivatives having antibiotin activity. From Kawashima et al. (1956).

Ogata et al. (1970, 1973a) found that the accumulation of biotin vitamers affected by addition of ACM to the culture medium, and investigated the action of ACM in relation to the pathway of biosynthesis of biotin vitamers in microorganisms. When ACM was added to the medium, the growth of most yeasts, molds, bacteria, and actinomycetes was not inhibited at all, but the amounts of DTB accumulated in the medium on incubation with pimelic acid were remarkably enhanced. Conversely, the amount of biotin formed when ACM was added dropped considerably. In the case of Bacillus sphaericus, the amount of DTB formed increased about five-fold by addition of 200 pglml of ACM, and reached a maximum of 350 pg/ml as shown in Fig. 10. As a result of investigation of the action of ACM on the biosynthesis of biotin (Ogata et al., 1973a), it is thought that ACM is not incorporated into the DTB molecule but that, because it suppresses formation of biotin by W ~ S remarkably

ACM added (pglrnl) FIG. 10. Effect of actithiazic acid (ACM) on the accumulation of biotin vitamers by Bacillus sphawicus. From Ogata et al. (1973a).

166

YOSHIKAZU IZUMI A N D KOICHI OGATA

inhibition of a part of the biosynthetic system of biotin from DTB, it releases the repression by biotin of the biosynthetic system of DTB from pimelic acid. Eisenberg (1973)also investigated the action of ACM using resting cells of E. coli and confirmed that ACM showed competitive inhibition of the biosynthetic system of biotin from DTB. Further, he found the enzyme activity of both DAPA aminotransferase and DTB synthetase to be considerably higher from cells cultured in ACM-supplemented medium.

B. a-DEHYDROBIOTIN a-Dehydrobiotin (aDHB) is an antibiotic produced by Streptomyces lydicus (Hanka et al., 1966, 1969). The antibiotic is active against a variety of gram-positive and gram-negative bacteria and fungi, such as Staphylococcus aureus, Sarcina lutea, Streptococcus pyogenes, E . coh, Proteus vulgaris, Salmonella pullorum, Candida albicans, Saccharomyces cerevisiae, and Penicillium oxalicum (Hanka et al., 1966). Minimum inhibitory concentration (MIC) of aDHB is 0.78 pg/ml for E. coZi (Pai, 1975).This antibiotic has a double bond conjugated with a carboxyl group (Hanka et al., 1966)(Fig. 11). The amount of aDHB produced in the culture increased on addition of biotin. The conversion of [14C]biotinto [14C]aDHBhas also been demonstrated using a growing culture of Streptomyces lydicus. These results suggest that aDHB is a product of biotin catabolism in S. lydicus (Hanka et aZ., 1969). More recently, the synthesis of aDHB has been established with the cyclic sulfonium salt [compound (XI) in Fig. 61 as starting material (Field et al., 1970, 1976). The antibacterial properties of aDHB are lost in the presence of biotin in synthetic media (Hanka et al., 1966). Eisenberg (1975) investigated the action of aDHB on the synthesis of the biotin biosynthetic enzymes in E. coli. Repression of the synthesis of both the DAPA aminotransferase and DTB synthetase was observed with increasing concentrations of aDHB, reaching a maximum between 10 and 80 ng/ml. However, only 73 and 81%repression was attained with aDHB for the aminotransferase and DTB synthetase, respectively, while repression of the synthesis of the two enzymes was essentially complete with 5 ng/ml of biotin under the same conditions.

A

N

N H

CH~-CH~-F=C-COOH H

FIG. 11. Structure of a-dehydrobiotin. From Hanka et al. (1966).

MICROBIAL PRODUCTION OF BIOTIN

167

Pai (1975) has isolated aDHB-resistant mutants from strains ofE. coli and classified them into two groups: dhb A and dhb B . The dhb B mutants overproduced biotin, and the levels of their biotin biosynthetic enzymes were elevated in comparison with those of the parent strains. One of the mutants accumulated 25 times as much extracellular biotin and 38 times as much intracellular biotin, and the specific activity of the mutant's DTB synthetase was 11times as high. Furthermore the biotin biosynthetic enzymes of dhb B mutants were not represented by 10 ng/ml of biotin. In the dhb A mutants, biotin biosynthetic activity was normal in that the amount of biotin and its precursors excreted in the culture medium and the levels of the biosynthetic enzymes were similar to the wild-type strain, and that the enzyme was subject to repression by biotin. From the findings that the ability of the mutant to take up biotin into the cell was reduced significantly and that aDHB, a competitive inhibitor of biotin uptake, was much less inhibitory to biotin uptake in the mutants than in the wild strain, dhb A mutants were suggested to be deficient in aDHB transport. Eisenberg et al. (1975) have also isolated four classes of aDHB resistant mutants of E. coli. One mutant group showed enhanced excretion levels of biotin vitamers (73 to 110 ng/ml of total biotin and 40 to 88 ng/ml of true biotin, compared with 4 ng/ml and 0 ng/ml, respectively, in the parent strains), derepressed levels of the biotin biosynthetic enzymes, and resistance to repression by biotin. A second class of mutants showed derepressed levels of the DTB synthetase enzyme. The other two mutant groups showed alterations in permeability; biotin uptake was also affected and growth on minimal media was poor, suggesting a generalized permeability defect.

c. a-METHYLBIOTINAND a-METHYLDETHIOBIOTIN During the isolation of aDHB produced by S. Zydicus, Hanka et al. (1972) isolated two more biotin antimetabolites from the fermentation liquors of this microorganisms: a-methylbiotin (aMB) and a-methyldethiobiotin (aMDB) (Fig. 12). [14C]Biotinand [14C]pimelicacid were not incorporated

aMDB

aMB

FIG. 12. Structure of a-methyldethiobiotin (aMDB) and a-methylbiotin (aMB). From Hanka et al. (1972).

168

YOSHIKAZU IZUMI A N D KOICHI OGATA

into these metabolites by the growing culture, and neither metabolite could satisfy the biotin requirement in Saccharomyces cerevisiae. Both compounds had strong antimicrobial activity against mycobacteria (MIC of aMDB, 0.23.12 pg/ml; MIC of aMB, 1.25-200 pg/ml). Furthermore, aMDB inhibited E . coli (MIC, 8 pg/ml) and B . subtilis (MIC, less than 0.5 pg/ml) cultivated in synthetic media. The antibacterial activities of both aMDB and aMB were reversed strongly by biotin. DTB also reversed the former, although less than biotin, but not the latter. Pimelic acid had no effect even at lo00 times the concentration of biotin or DTB. Further studies have not been carried out on the action of these antimetabolites on biotin biosynthesis. The chemical synthesis of racemic aMB was achieved with racemic N-blocked thiophanium salt [compound (XI) in Fig. 61 as a starting material (Martin et al., 1971). D. AMICLENOMYCINAND STELAVIDIN Amiclenomycin (AM) is produced by Streptomyces lavendulae (Okami et al., 1974). Its chemical structure was identified as ~-2-amino-4-(4'-amino2',5'-cyclohexadienyl)butyric acid (Fig. 13).The antibiotic inhibits growth .of mycobacteria including various resistant strains and tubercle bacilli (MIC, 3.1-6.25 pglml), but not other bacteria and fungi. The MIC value was significantly reduced when AM and ACM were added together: growth was completely suppresed even at one-eighth of their MIC (Kitahara et al., 1975). The action of AM alone or in combination with ACM was reversed by biotin at a concentration of 0.01 pg/ml, but not at 0.001 pglml. This reversal was also observed with DTB (0.1 pg/ml) and DAPA (1 pg/ml), but not with KAPA, pimelic acid, and other compounds involved in biotin biosynthesis. Furthermore, when Mycobacterium smegmutis was cultured in a medium containing pimelic acid and AM (2-6 pg/ml), significant inhibition of growth coupled with an appreciable increase of KAPA accumulation was observed. On the other hand, DTB was detected in the medium without AM, but not in that containing AM. This was also confirmed with B . sphaericus. From

4

HzaCHrCHrr-XlOH AM

/C-c,Hz HzN-C\H CHrCHrCHrCHrCOOH CHa KAPA

FIG. 13. Structure of amiclenomycin (AM). From Okami et al. (1974)

MICROBIAL PRODUCTION OF BIOTIN

169

FIG. 14. Structure of stravidin. From Baggaley et al. (1969).

these results, AM was thought to be a strong inhibitor of the DAPA aminotransferas e reaction. Enzymic investigation was carried out on the action of AM on biotin biosynthesis (Hotta et al., 1975). As expected, the DAPA aminotransferase from B . divaricatum was inactivated by AM, while AM exhibited no significant activity against the DTB synthetase from P . gruveoZens. In the aminotransferase reaction, the activity was reduced remarkably even when AM was added at a concentration of one-tenth of that of KAPA as substrate. Preincubation of the enzyme with AM resulted in the abrupt inactivation of the enzyme. However, on dialysis 46.1%of the enzyme activity was recovered from the inactivated form. Moreover, inhibition by AM decreased in proportion to the amount of KAPA, while increased amounts of SAM exhibited a tendency to enhance inhibition by AM of DAPA aminotransferase. Thus, AM was considered to exert its inhibitory action against DAPA aminotransferase by coupling with the KAPA binding site of the enzyme. It was also revealed that AM derivatives such as aromatized AM and y-phenylbutyrine had no antimicrobial activity. Stravidin, an antibiotic produced by Streptomyces avidinii, has a chemical structure containing a 4-alkylcyclohexa-2,5-dienylamine residue similar to AM (Fig. 14) (Baggaley et al., 1969). This antibiotic also inhibited biotin synthesis by susceptible organisms (Stapely et al., 1963; Chaiet et al., 1963; Miller, 1964). Although the action mechanism of this antibiotic has not been studied, it may exert an inhibitory action on DAPA aminotransferase similar to that of AM, judging from its structural resemblance to KAPA.

E. ADENINE Iwahara and Yoshikawa (1974)found that the synthesis of biotin from DTB by a biotin-forming bacterium was greatly accelerated by the addition of adenine sulfate to the culture medium. They investigated the action of adenine on biotin biosynthesis. The growth of the bacterium was strongly inhibited when adenine or adenosine was added to the medium, while other purine and pyrimidine bases and nucleosides had no effect on the growth. In order to reduce the growth inhibition by adenine sulfate (4 mg/ml), it was necessary to add biotin as well as thiamine and a purine or pyrimidine base or nucleoside, such as uracil, uridine, cytidine, guanine, guanosine, or

170

YOSHIKAZU IZUMI A N D KOICHI OGATA

xanthine. Adenine added to the culture medium was partly converted to adenosine and hypoxanthine during the culture, and finally completely converted to hypoxanthine (Iwahara and Kanemaru, 1974). While adenine remained in the medium, a large amount of DTB (about40 pg/ml) was accumulated from pimelic acid, but biotin was not accumulated. After adenine had disappeared from the medium, the accumulation of biotin rapidly increased with culture time to reach about 400 ng/ml. Furthermore, Iwahara and Kanemaru (1975) investigated the mechanism of the function of adenine using intact cells. The biosynthesis of biotin from DTB by intact cells harvested from adenine-supplemented medium or from adenine-unsupplemented medium was strongly inhibited by the addition of adenine or adenosine. However, the intact cells harvested from adeninesupplemented medium had greater capacity to synthesize biotin from DTB than those harvested from adenine-unsupplemented medium. From these results, they suggested that adenine inhibits the biosynthesis of biotin from DTB, and that repression of biotin biosynthesis by biotin is released during the inhibition by adenine, resulting in the large accumulation of DTB. Subsequent disappearance of adenine in the late stage of culture results in normal biotin biosynthesis from DTB and in large accumulation of biotin. Tsuboi et al. (1967) have also found the accelerating effect of adenine on DTB accumulation by a hydrocarbon-utilizingPseudomonus.

VI. Biodegradation of Biotin and its Vitamers Brady et al. (1966)isolated a pseudomonad which grew on biotin as its sole carbon, nitrogen, and sulfur source and observed the total degradation of carbonyl-[l4C1biotinand ~arboxyl-['~C]biotin to 14C02by the cells and by a particulate preparation of this organism. In the particulate system, this degradation was promoted by addition of ATP, M g + , NAD, and CoA. Biotinrelated substances having a labeled carbonyl carbon atom were also degraded in the order biotin d-sulfoxide, most readily degraded, biotin, and biotin l-sulfoxide. Biotin sulfone, and oxybiotin were hardly degraded to C02. Those having a labeled carboxyl atom showed the same order of ease of degradation. Iwahara et al. (1966d)found that a large number of molds degrade DTB to form a product inactive toward S. cerevisiae, but having biotin activity toward B. subtilis. Next, they isolated the degradation product in crystalline form from the medium of Aspergillus oryzae, the organism showing the strongest degradation, and identified it as bisnordethiobiotin, a compound with two less carbon atoms in its side chain than DTB. Li et al. (1968a) also found that carbonyl-[14C]DTBwas degraded by A. niger; the degradation products were isolated in crystalline form and identified as bisnor-

171

MICROBIAL PRODUCTION OF BIOTIN

dethiobiotin and tetranordethiobiotin, a compound with side chain shorter by a further two carbon atoms. These results suggest that, as shown in Fig. 15, the degradation of DTB proceeds by P-oxidation. Yang et al. (1969b) tested a large number of yeasts and fungi, and found that several strains of genera Endomycopsis, Rhodotorula, and Penicillium showed strong degradation of biotin. Biotin was converted to compounds inactive toward Lactobacillus plantarum and S . cerevisiae and active toward B . subtilis. This conversion reached a maximum of 95%. Yang et al. (1969b) found by bioautography using B . subtilis that biotin d-sulfoxide and two unknown substances were formed from biotin. These were isolated in crystalline form by column chromatography (Yang et al., 1968, 1970% Ogata, 1970b). From physicochemical analyses, the two unknown compounds were identified as bisnorbiotin and bisnorbiotin sulfoxide, compounds having side chains shorter by two carbon atoms than biotin and biotin sulfoxide, respectively. Since biotin sulfoxide was not degraded by Endomycopsis and remained unchanged in the culture medium, biosnorbiotin sulfoxide is thought to be formed from bisnorbiotin but not from biotin sulfoxide. Ruis et al. (1968) examined the degradation of homobiotin and norbiotin labeled with I4C at the carbonyl or carboxyl group using a particulate preparation of a Pseudomonas sp. These compounds also underwent p-oxidation losing two carbon atoms each from the side chain; the degradation proceeds: homobiotin + norbiotin + trisnorbiotin. Similarly, Iwahara et al. (1968)also incubated a Pseudomonas sp. in medium supplemented with carbonyl[14C]biotin, and isolated radioactive bisnorbiotin, a-dehydrobisnorbiotin, tetranorbiotin, urea, and uracil from the culture filtrate. From these facts, they proposed that after two p-oxidations of the side chain of biotin (biotin + bisnorbiotin + a-dehydrobisnorbiotin + tetranorbiotin), urea is produced by degradation of the thiophane ring, and COz, and NH3 are finally produced by the action of urease. They considered that uracil is produced either from C 0 2 and NH3, or from further ring closure of a degradation product having a ureido fragment. Roth et al. (1970) and Im et al. (1970), using the same Pseudomonas sp., studied the degradation of ~arbonyl-[~~C]biotin I-sulfoxide and biotin d-sulfoxide, respectively. An unknown metabolite was isolated from the day 16 culture with biotin 1-sulfoxide and identified as

H3k

L(CHzbCOOH

Dethiobiotin

H3k

L(CH2)zCOOH

Bknordethiobiotin

&

LCOOH

Tetranordethobiotin

FIG. 15. Degradation of dethiobiotin by microorganisms.

172

YOSHIKAZU IZUMI A N D KOICHI OGATA

Biotin

Bisnorbiotin

Tet ranorbiotin

FIG. 16. Degradation of biotin by microorganisms.

P-hydroxybiotin 1-sulfoxide (Roth et al., 1970). Several metabolites were isolated from culture filtrates of the pseudomonad grown for 9 days on a medium containing biotin d -sulfoxide and identified as biotin, bisnorbiotin, tetranorbiotin, biotin 2-sulfoxide, biotin sulfone, and a new catabolite, bisnorbiotin sulfone, after purification (Im et al., 1970). Christner et al. (1964) observed the degradation of ~arboxyl-[~~C]biotin to 14C02by cell extracts of an isolated Pseudomonas sp. which grew on biotin as a sole carbon source, and by cell extracts of mammalian tissue. They found the presence of a biotin-activating enzyme (biotinyl-CoA synthetase), which is thought to participate in the first degradation reaction of biotin via p-oxidation. This enzyme forms biotinyl adenylate from biotin and ATP in the presence of M$+, and further forms biotinyl-CoA from biotinyl adenylate and CoA. All these results support thoroughly the presence of P-oxidation for the side chain of biotin in microorganisms as shown in Fig. 16.

VII. Conclusion Almost the complete picture of the enzyme system participating in the biosynthetic pathway from pimelic acid to DTB has been built up. Also the degradation of biotin has been elucidated. One of the most important tasks for future studies in biotin metabolism will be the elucidation of the enzymic system of biotin biosynthesis from DTB and its regulation mechanism. This elucidation may provide a clue to the problem of accumulating large quantities of biotin. In order to establish biotin fermentation, considerable efforts will have to be made to alter the strong regulation mechanism in biotin biosynthesis by mutation or by other means. Several effective biotin antimetabolites have been found and some mutants resistant to those antimetabolites show greater ability to synthesize biotin and its vitamers, although the amounts found are still very small. Therefore, further induction of such resistant mutants may make possible the microbial production of biotin and its vitamers. For this purpose, these antimetabolites have to be prepared effec-

MICROBIAL PRODUCTION OF BIOTIN

173

tively in large amounts, or other active antimetabolites which can be easily prepared have to be sought. One further means that might be effective for accumulating large quantities of biotin by alteration of the regulation mechanism is the induction of revertants from biotin auxotrophic mutants, similar to the revertants that can accumulate considerable amounts of amino acids. ACKNOWLEDGMENT

We are deeply indebted to Associate Professor Y. Tani of our laboratory for his helpful advice and encouragement during the course of this work and in the preparation of this manuscript.

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Martin, D. G . , Hanka, L. J., and Reineke, L. M. (1971). Tetrahedron Lett. No. 41, p. 3791. Melville, D. B., Moyer, A. W., Hoffmann, K., and du Vigneaud, V. (1942).J . Biol. Chem. 146, 487. Miller, A. K. (1964).Antimimob. Agents Chemother. p. 33. Miyake, A. (1953). Pharm. Bull. 1, 89. Miyake, A,, Morimoto, A., and Kinoshita, T. (1953). Pharm. Bull. 1, 84. Mueller, J. H. (1937a). Science 85, 502. Mueller, J. H. (193713).,I. Biol. Chem. 119, 121. Niimura, T., and Shimada, S. (1967). Vitamins 35, 444. Niimura, T., Suzuki, T., and Sahashi, Y. (1964a).J. Vitaminol. 10, 218. Niimura, T., Suzuki, T., and Sahashi, Y. (196413).J. Vitaminol. 10, 224. Niimura, T., Suzuki, T., and Sahashi, Y. (1964~). J. Vitaminol. 10, 231. Nogami, I., Kida, M., Iijirna, T . , and Yoneda, M. (1968).Agric. B i d . Chern. 32, 144. Ogata, K. (1970a). In “Methods in Enzymology” (D. B. McCormick and L. D. Wright, eds.), Vol. 18, Part A, pp. 390394. Academic Press, New York. Ogata, K. (1970b). In “Methods in Enzymology” (D. B. McCormick and L. D. Wright, eds.), Vol. 18, Part A, pp. 397400. Academic Press, New York. Ogata, K. (1975). Adti. Appl. Mimobiol. 19, 209-247. Ogata, K., and Igarashi, S. (1954).J . Takeda Res. Lab. 13, 78. Ogata, K . , Tochikura, T., Iwahara, S., Takasawa, S., Ikushima, K., Nishimura, A., and Kikuchi, M. (1965a). Agric. B i d . Chem. 29, 889. Ogata, K., Tochikura, T., Iwahara, S . , Ikushima, K . , Takasawa, S . , Kikuchi, M.,and Nishimura, A. (1965b). Agric. B i d Chem. 29, 895. Ogata, K., Tochikura, T., Osugi, M., and Iwahara, S. (1966). Agric. B i d . Chem. 30, 176. Ogata, K . , Izumi, Y., and Tani, Y. (1970). Agric. B i d . Chem. 34, 1872. Ogata, K., Izumi, Y., and Tani, Y. (1973a). Agric. B i d . Chem. 37, 1079. Ogata, K., Izumi, Y., Aoike, K . , and Tani, Y. (1973h). Agric. B i d . Chem. 37, 1093. Ogino, S., Fujimoto, S., and Aoki, Y. (1974a).Agric. Biol. Chem. 38, 275. Ogino, S., Fujimoto, S., and Aoki, Y. (1974h). Agric. B i d . Chem. 38, 707. Ohrui, H., and Emoto, S. (1975). Tetrahedron Lett. No. 32, p. 2765. Ohsugi, M., and Baha, A. (1975). Agric. Biol. Chern. 39, 731. Ohsugi, M., and Ishikawa, Y. (1975). Agric. B i d . Chern. 39, 559. Ohsugi, M., Yang, H. C., and Ogata, K. (1972a).Agric. Biol. Chem. 36, 1285. Ohsugi, M., Nakazawa, M., and Ogata, K . (1972b). Agric. B i d . Chern. 36, 1293. Okami, Y., Kitahara, T., Hamada, M., Naganawa, H., Kondo, S., Maeda, K . , Takeuchi, T., and Umezawa, H. (1974). J . Antibiot. 27, 656. Okumura, S . , Tsugawa, R., Tsunoda, T., and Kitai, A. (1962a).Nippon Nogei Kaguku Kaishi 36, 197.

Okumura, S . , Tsugawa, R., Tsunoda, T., and Kaishi 36, 204. Okumura, S . , Tsugawa, R., Tsunoda, T., and Kaishi 36, 506. Okumura, S . , Tsugawa, R . , Tsunoda, T., and Kaishi 36, 599. Okumura, S ., Tsugawa, R., Tsunoda, T., and Kaishi 36, 605. Pai, C. H. (1969a). C u n J . Microbiol. 15, 21. Pai, C . H. (1969b).J . Bacteriol. 99, 696. Pai, C. H. (1971).J. Bacteriol. 105, 793. Pai, C. H. (1975). Mol. Gen. Genet. 134, 345.

Motozaki, S. (196213). N i p p o n Nogei Kagaku Motozaki, S. (1962~).Nippon Nogei Kagaku Motozaki, S. (1962d). Nippon Nogei Kagaku Motozaki, S . (1962e). Nippon Nogei Kagaku

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Polyether Antibiotics: Versatile Carboxylic Acid lonophores Produced by Streptomyces J. W. WESTLEY Hoflrnann-La Roche Inc., Nutley, New Jersey I. Introduction

................ ................ .............. B. Proposed Numbering System for Polyether Antibiotics. . . IV. Microrganisms That Produce Polyether Antibiotics . . . . . . . . . . V. Biosynthesis of Polyether Antibiotics A. Lasalocid . . . . . . . . . . . . . , . . . . . . B. Monensin . . . . . . . . . . . . . . . . . . C. Microbial Transformation of Mo VI. Physical Constants of the Polyether ............. VII. In Vitro Antimicrobial Activity and Moiise Toxicity of the Polyether Antibiotics A. Antimicrobial Activity B. Toxicity of Polyether Antibiotics . . . . . . . . . . . . . . . . . . . . . . VIII. Coccidiostat Activity of the Polyether Antibiotics . . . . . . . . . . . IX. Antimicrobial and Coccidiostat Activity of Lasalocid Analogs. . A. Natural Homologs and Isomers . . . . . . . . . . . . . . . . . . . . . . . B. Synthetic Derivatives of Lasalocid . . . . . . . . . . . . . . . . . . . . X. Improved Ruminant Feed Utilization by Addition of Polyethers . . . . . . . . . , . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . XI. Pharmacology of the Polyether Antibiotics . . . . . . . . . . . . . . . . . A. Cardiovascular Activities of the Polyethers . . . . . . . . . . . . . B. Miscellaneous Effects References _ . _ _ . _ . _ _ _ . . _ . _ _ . _ . . _ . _ . _ . _

177 178 178 178 181 183 183 183 188 189 193 194 202 203 203 203 203 209 211 211 211 213 215 216 216 218 220

I. Introduction The polyether antibiotics are members of a large and growing group of compounds possessing the ability to form lipid-soluble complexes that provide a vehicle for a wide variety of cations to traverse lipid barriers. This characteristic ion-bearing property led to their being named ionophures (Moore and Pressman, 1964; Pressman et al., 1967). The ionophores range in molecular weight between 500 and 1500 and include synthetic compounds of which the most well known are the crown ethers (Pedersen, 1967). Naturally occurring ionophores include the octadecadienoic acid recently isolated from beef heart mitochondria (Green, 1975; Blondin, 1975) and four distinct classes of ionophore antibiotics, of

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which the most important in terms of potential clinical utility are the polyether antibiotics (Westley and Berger, 1974; Westley, 1975). Several reviews of the ionophores have appeared recently (Hodgson, 1974; McGlocklin and Eisenberg, 1975; Truter, 1975) and include a book (Ovchinnikov et al., 1974) in which ionophores are referred to as “complexones.” The most comprehensive recent review (Pressman, 1976) deals primarily with the biological applications of the ionophores. In order to accentuate the unique chemical and biological characteristics of the polyether antibiotics within this vast group of ionophores, a brief summary of the other major classes of ionophores will be presented first.

I I. Classification of lonophores Some of the problems associated with antibiotic classification (Bitrdy, 1974) also exist in attempting to classlfy ionophores. Among the potential bases for classification of this broad array of compounds are their origins, spectra of biological activity, physicochemical properties, and, in the case of the ionophore antibiotics, the biosynthetic routes employed by the producing organisms in assembling the antibiotics. However, the two most popular methods have been (1)by mechanism of action and (2) by chemical structure. The former has been preferred by Dr. Pressman and the latter by this reviewer.

A. BY MECHANISM OF ACTION The ionophores are subdivided into three groups in Pressman’s most recent review (1976) consisting of: (1) neutral ionophores, (2) carboxylic ionophores, (3)channel-forming quasi-ionophores. B. BY CHEMICALSTRUCTURE Two of the difficulties encountered using mechanism of action as the basis of classification are (1)determining whether a primary or secondary process is involved, and (2)frequent disparity between in vitro and in vivo results. Probably the biggest stumbling block, however, is the danger inherent in assuming simplistic theories of mechanisms that subsequently have to be modified and in some cases completely revised. For the reasons outlined above, chemical structure will be the primary consideration in classifying both the ionophores as a whole and the polyether antibiotics in particular. The unique property of chemical structure unambiguously distinguishes any compound from all others. The eight structural classes used in this review can be divided evenly as those of nonmicrobial origin and the ionophore antibiotics:

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Nonmicrobial ionophores: (1)crown polyethers, (2) synthetic cryptates, (3) noncyclic synthetic ionophores, (4)octadecadienoic acids isolated from beef heart mitochondria. lonophore antibiotics (Section II,C); (1)peptide ionophores, (2) cyclodepsipeptides, (3) macrotetrolides, (4)polyether antibiotics (Section I11 and following sections).

1. Crown Polyethers These synthetic, cyclic ethers (Pedersen, 1967) were given the name crown polyethers because of the characteristic conformation they assume in solution. Different-sized rings have been synthesized containing either two benzene or two cyclohexyl moieties. For example, dibenzo-18-crown-6 (Fig. 1)is an eighteen-membered ring containing two benzene molecules and six Svnthetic ionophores

Dibenzo-18- crown-6

PJ,N,PJ,N-Tetropropyl-

Dibinophthyl crown ether

1.2 -

phenylenedioxydiocetomide

Dibenro [222] cryptate

Mitochondriol oxyoctodecodienoic ocids

9-Hydroxy-lo-=. octodecodisnoic acid

12-c&-

13- Hydroxy-9-CiS-Il-=octodecodienoic acid

FIG. 1. Ionophores of nonmicrobial origin

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oxygen atoms, which by ion-dipole interactions act as six ligands for K+ ions (FrensdorfF, 1971; Christensen et al., 1971). A smaller crown polyether, dicyclohexyl-14-crown~,exhibits cation selectivity for Na+:K+by a factor of 7.6:1, whereas the larger dibenzo-21-crown-7 complexes Cs+ four times more tightly than K+. The atomic radius of the alkali and alkaline earth cations are (.hi):Li+ (0.60), Na+ (0.95), K+ (1.33),Rb+ (1.48),Cs+ (1.60), Mg2+ (0.65), Ca2+(0.99),Ba2+(1.35). Optically pure dibinaphythyl-crown-ether (Fig. 1)can resolve asymmetric amine salts by enantioselective extraction from an aqueous solution into chloroform. Using this technique, 1-amino-1-phenylethanewas obtained in the R(+) form with an optical purity of 34% (Cram and Cram, 1974).

2. Cryptates These molecules are similar to the crown polyethers except that they are more three-dimensional or cagelike in structure. A typical example, dibenzo [222] cryptate is illustrated in Fig. 1. The best-fitting cations for this compound are K+ and Ba2+, which are complexed equally strongly (T;uter, 1975). It is worth noting here the results of Lehn and co-workers (Dietrich et al., 1973)that the cryptate with the highest stability constant for complex formation did not give the most rapid transport of cations from one aqueous solution to another through a nonpolar solvent. 3. Noncyclic Synthetic Ionophores Like the crown ethers and cryptates, these synthetic 1:4 dioxy ethers are neutral compounds, but quite distinct in terms of structure. A typical example (illustrated in Fig. 1) is N,N,N,N-tetrapropyl-1,2-phenylenedioxy diacetamide. Collaboration between Borowitz's group at Yeshiva University, New York, and Simon and Eidgenossische Technische Hochschule, Zurich, clearly demonstrated that several of these compounds exhibited Ca2+and Na+ selectivity in liquid membrane electrodes (Ammann et al., 1975). 4 . Natural Divaknt Cation lonophores from Beef Heart Mitochondria

A unifying model for bioenergetics (Green, 1975), proposed recently, suggests that ionophores are the universal charge-separating species used by all energy-coupling systems. Some support for this proposal was provided by the isolation of a number of hydroxylated octadecadienoic acids (Fig. 1) and their ketonic analogs (Blondin, 1975). At least some of these acids are claimed to be ionophores for divalent cations. Being carboxylic acids, these compounds would be in the second of Pressman's three groups, listed earlier (Section 11,A).

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POLYETHER ANTIBIOTICS: CARBOXYLIC ACID IONOPHORES

C. IONOPHOREANTIBIOTICS There are four types of ionophore antibiotics including the polyethers. The other three classes are: (1)peptides, (2) cyclodepsipeptides, (3) macrotetrolides. These compounds differ from the surjiactunt-type antibiotics, such as the tyrocidins, polymyxins, and polyenes, which cause general loss of cell contents by complete disruption of microbial membranes. The ionophore antibiotics owe their antimicrobial activity to spec& effects they have on the permeability of certain cations (Gale et al., 1972) across cell membranes. 1. Peptides

Gramicidins A, B, and C (Fig. 2) isolated from cultures of Bacillus brevis are linear peptides (15 residues) with a formyl group at the N terminus and ethanolamine attached as an amide to the carboxyl-terminal amino acid. As is the case for alamethicin, another peptide ionophore produced by Trichoderma viride (Meyer and Reusser, 1967), these antibiotics have been STRUCTURE

ANT IB IOTlC PEPTIDE IONOPHORES ~

nco-

GI"-

VOI-

L-AIO-

D.W-

L-AIO-

VALINE-

D-VOI

7 L-VOI

GRAUlClDlN A

o-vo1

B C

ALAYETHICIN

DEPSIPEPTIDE IONOPHORES D-How D-VaI L - IOI VALINOMYCIN L-MIV~I-D-~~I-L-Y.V~I

I

ENNIATIN B

MACROTETROLIDE

I

D-nov-L-M~V~I-D-H~v

IONOPHORES

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J . W. WESTLEY

found to induce cationic permeability by inserting themselves in membranes as stationary ion-conducting channels (Urry et ul., 1971). As this mechanism difFers from the mobile cation carriers, these antibiotics make up the group called the quusi-ionophores by Pressman.

2. Cyclodepsipeptides Characterized by infrared absorption bands at 1735-1750 cm-’ (lactone) and 1650-1680 cm-’ (amide), the cyclodepsipeptides contain both amino and hydroxy acids. They are often produced by cultures as families of chemically related compounds. The best method for distinguishing these structurally similar compounds is mass spectrometry. Valinomycin (Fig. 2) is the most well investigated of the sixteen reported depsipeptides (Hook, 1973) and was isolated from cultures of Streptomyces sp. fulvissimus (Brockmann and Schmidt-Kastner, 1955). Found to be a powerful uncoupler of oxidative phosphorylation (McMurray and Begg, 1959), valinomycin was later shown to act (Pressman, 1965)by catalyzing an energy-linked K+ for H+ exchange across mitochondria1 membranes. The antibiotic is very selective toward alkali cations and can discriminate between K+ and Na+ to the extent of 10,OOO:l. Valinomycin assumes a braceletlike conformation in solution (Pinkerton et ul., 1969; Ivanov et al., 1969) with the ester carbonyls forming a rigid sphere at the center. This sphere can easily accommodate K+ or Rb+ ions, but the carbonyl ligands cannot focus on ions as small as Na+ or Li+ (for atomic radii, see Section II,B,l). Over 80 valinomycin analogs have been synthesized and the Na+ and K+ stability constants carefully determined. Some analogs complexed K+ ions only very weakly whereas one analog displayed stability constants that were two orders of magnitude greater than valinomycin (Ovchinnikov et al., 1974). Although this latter compound and many other analogs inhibit microbial growth, none were more potent than valinomycin. The enniatins produced by Fusurium sp. are only half the size of valinomycin and cannot fold into the characteristic “bracelet” conformation of valinomycin. The selectivity as a result is much less (K+:Na+-lO:l, compared to 10,OOO:lfor valinomycin), but a broader range of cations are complexed, including all the alkali metals. The order of stability constants for enniatin A is K + > Na+, RB+, Cs+ > Li+; and for enniatin B, it is Rb+ > K + > Cs+ > Na+ > Li+ (Ovchinnikov et al., 1974). 3. Macrotetrolides Whereas the peptide ionophores are made up entirely of amino acids and the depsipeptides of alternating hydroxy and amino acids, the macrotetrolides are all constructed from f m r tetrahydrofurunyl hydroxy acids linked together through lactone bonds. An interesting aspect of the stereochemistry

POLYETHER ANTIBIOTICS: CARBOXYLIC ACID IONOPHORES

183

of this particular class of ionophore antibiotics is that, in all cases, two of the hydroxy acids are of opposite configuration to the alternating pair of acids. Nonactin and one or more of its homologs (Fig. 2) have been isolated from many Streptomyces cultures, including Streptomyces werraensis. Like the depsipeptides, they are neutral ionophores causing active uptake of cations such as K+ into mitochondria at the expense of energy generated by electron transport. This active uptake of cations causes swelling of the mitochondrial particle due to the concomitant uptake of water to hydrate the cations within the matrix. If at this point, a polyether antibiotic is added, the swollen particles begin to shrink owing to passioe d$fusion of excess K+ down the concentration gradient, a phenomenon first reported by Lardy’s group (Graven et al., 1966; Lardy, 1968).This passive diffusion of excess cations across membranes must be the primary effect of the many different biological activities reported for the polyethers.

D. ANTIMICROBIALACTIVITY OF NONPOLYETHER ANTIBIOTICS Alamethicin is only weakly active in vitro against various streptococci at 30 pg/ml and Staphylococcus aureus at 60 pg/ml. By comparison, gramicidins are relatively potent against Streptomyces hemolyticus at 2 pg/ml and Pneumococcus at 0.01 pgIml. Gramicidin is used topically for treatment of gram-positive infections. Valinomycin is active in vitro against gram-positive bacteria, Mycobacterium phlei, and Candida albicans. A similar spectrum of activity, but at much higher concentrations, is exhibited by the enniatins. The macrotetrolides are active against gram-positive bacteria and mycobacteria and have also been found to be cytotoxic agents (Meyers et al., 1965). A problem in testing ionophores in general and macrotetrolides in particular is their lack of water solubility. Japanese workers (Suzuki et al., 1971) have reported insecticidal activity for the macrotetrolides against mites, such as Tetranychus kanzawa. The antimicrobial activities discussed here probably result from loss of K+ ions across microbial membranes, and in support of this, inhibition of Streptococcus faecalis by gramicidin or valinomycin is reversed by addition of excess K+ ion (Harald and Baarda, 1967).

Ill. Polyether Antibiotics A. CLASSIFICATION OF POLYETHEM In addition to the ionophore reviews mentioned earlier, the polyether antibiotics have been reviewed as a class (Westley and Berger, 1974;

184

J . W . WESTLEY

Westley, 1975). Called carboxylic acid ionophores by Pressman, this name emphasizes an essential moiety of all polyethers, but one shared by many other natural products. The name "polyether" refers to a more unusual aspect of their structure, namely, the considerable number of tetrahydropyrans and -furans found in these compounds, and it has now become the name of choice in the antibiotic literature. The definition of polyethers as ionophores indicates that these antibiotics effect the transport of monovalent cations. They can be further classified by whether or not they have the ability to transport divalent cations. The polyethers not possessing this property will be referred to as monovalent-cation polyethers (Figs. 3 and 4), and those with the ability to CARBON BACKBONE

ANTIBIOTIC

STRUCTURE

STRUCTURE REFERENCE

la c26 -

MONENSIN

R , = CH ( Me1CO2H,R2 = E l

FACTOR B

R , =Cn(Me)c02n.R2=Me

Agtarop (1967)

R, = ( C H 2 ) 3 c 0 2 n . R 2 . ~ e

Gorman ef ut. (1968)

FACTOR C

LAIDLOMYCIN

R , = C H I Me )C02H.R2 = Me

etul.

Kitame and lshida (1976)

OMe

NlGERlClN

R = OH

GRlSORlXlN

R: n

SALINOMYCIN

FIG.3. Monovalent polyether antibiotics.

Steinrauf e f d (1968) Gachon eful. ( 1970)

Kinashi etoL (1973)

185

POLYETHER ANTIBIOTICS: CARBOXYLIC ACID IONOPHORES

transport dibasic cations as divalent cation polyethers (Figs. 5 and 6). The cation-binding sequences of the polyethers (Table I) are based on the measurement of equilibrium complexation affinities by optical rotatory dispersion (ORD), fluorescence, and conductivity or distribution coefficients in two-phase systems (Pressman, 1968; Pfeiffer et al., 1974). As a change in solvent system is known to change selectivity sequences (Pressman, 1973; Ovchinnikov et al., 1974), the more reliable chemical structures (see Section II,B) of the compounds are used to further classify the polyethers. The four classes of polyethers illustrated in Figs. 3, 4, 5, and 6 are, respectively: la: Monovalent polyethers (e.g., monensin, nigericin) Ib: Monovalent monoglycoside polyethers (e.g., dianemycin) CARBON BACKBONE

ANTIBIOTIC

STRUCTURE

STRUCTURE REFERENCE

Berg eta/. (1975)

NARASIN ( A 2 8 0 8 6 )

LONOMYCIN

Omuro (1976)

eta,!

ANTIBIOTIC X - 2 0 6

Blaunt and Westley (1975)

ALBORlXlN

Allaoume eta/. (1975)

FIG.3. (continued)

186

J . W . WESTLEY

STRUCTURE REFERENCE

STRUCTURE

ANT IB IOTlC

Ib

DIANEMYCIN

OH

Bl0""I

LENOREMYCIN (Ro21-6150)

OH

SEPTAMYCIN

ANTIBIOTIC

COzH

C02H

A204A

".'

1975

OH

Pctcher and Weber. 1974

e.

Jones 1973

fi.

ANTIBIOTIC CP 38295

'OZH

FIG.4. Monovalent monoglycoside polyethers.

Celmer 1976

187

POLYETHER ANTIBIOTICS: CARBOXYLIC ACID IONOPHORES

A N T I B lO T l C

STRUCTURE REFERENCE

STRUCTURE

20 Me

Westley eta/ (1970a) Johnson eta/ (1970)

LASALOCID A R, :EI.R2=R3=R4=Me

LASALOCID B C 0 E

IS0

R2:E!. R, = R 3 = R 4 = M e

Westley eta/. ( I 9 7 4 b)

R3=E1. R , : R 2 = R 4 ' M e R.=El.R, =R2:R,=Me

- LASALOCID

A OH

westley (1974 c)

FIG.5. Divalent polyether antibiotics.

2b

ANTIBIOTIC

STRUCTURE

STRUCTURE REFERENCE

Me

p

M

pN

$

p

Me

McNH

C02H

ANTIBIOTIC A 2 3 1 8 7

Choney eta,: (1974)

FIG.6. Divalent pyrrole ether antibiotic.

eta/

188

J . W . WESTLEY

TABLE I IONSELECTIVITYOF POLYETHERS Polyether antibiotic Class

la

Antibiotic Monensin Nigericin, Antibiotic X-206

Cation binding sequence' Monobasic cations

Dibasic cation

Na>> K > Rb> Li> Cs

-

K> Rb> Na> Cs> Li

-

Ib

Dianemycin

Na> K> Rb, Cs> Li

-

2a

Lasalocid (X-537A)

Cs> Rb, K> Na> Li

Ba> Ca> Mg

2b

Calcimycin (A-23187)

Li> Na> K

Ca> Mg> Ba

Size sequence of ionic radii (A):Cs+(1.69)>Rb+(1.48)>Ba2+(1.35)>K+(1.33) >Caz+(0.99)>Na+(0.95)>M$+(0.65).

2a: Divalent polyethers (e.g., lasalocid, lysocellin) 2b: Divalent pyrrole ethers (e.g., antibiotic A-23187) The distinction between polyethers of class la and Ib rests on whether the antibiotic contains the 2,3,6-trideoxy-4-0-methyl-~-erythrohexapyranose moiety, and the difference between 2a and 2b is self-explanatory. B.

PROPOSED

NUMBERING SYSTEMFOR ANTIBIOTICS

POLYETHER

A common feature of the polyether antibiotics is the presence, at one end of the molecule, of a single carboxylic acid function which represents the last carbon added during the biosynthesis of the antibiotics' polyketide precursors (Section V). In the proposed system, this carbon is designated C-1 and the carbon backbone of the molecule is numbered consecutively to the terminal carbon. In the case of lasalocid, this terminal carbon (C-24) is derived from the methyl of the acetate unit involved in initiation of the compound's biosynthesis. Another characteristic of the polyether antibiotics is the prevalence of C-alkyl groups. In the case of lasalocid, the four branched methyls present (in contrast to C-24) are propionate derived, and the three ethyls are derived from the C3,4carbons of butyric acid. The system proposed for numbering these branched alkyl groups follows the steroid model as illustrated for Ro 21-6150.The presence of a sugarlike function in Ro 21-6150 necessitates continuing the carbon numbering into the 2,3,6-trideoxy-4-O-methyl-~erythrohexapyranose moiety, present in all class Ib polyethers (Fig. 4). Oxygen and carbon atoms are differentiated in the proposed system with the oxygen numbers in parentheses as illustrated for antibiotic X-206.This

189

POLYETHER ANTIBIOTICS: CARBOXYLIC ACID IONOPHORES

46

MeQ,

38

Me

37

30

Ro 21-6150

Antibiotic X-206

will simplify assignment of trivial names for O-alkyl and deoxy analogs and can be used to distinguish methoxyls in those polyether antibiotics containing four or five - 0 M e groups, such as septamycin and antibiotic A204A (Fig. 4). As proposed for the carbon system, the oxygen numbering would be continued in antibiotics having the hexapyranose moiety (Ro 21-6150), in which the glycoside ether between C-41 and C-45 is 0-12 and the methoxyl group is at 0-13 (Westley, 1976). In antibiotic X-206, a convenient reference system for the rings (A, B, C, etc.) is indicated.

IV. Microorganisms That Produce Polyether Antibiotics All the polyether antibiotics listed in Table I1 were isolated from Streptomyces species. Up to 1976, no other microorganism had been demonstrated to be a polyether antibiotic producer. The Streptomyces cultures are listed in Table I1 in the order in which the antibiotics they produce are presented in Figs. 3-6. In addition to the four classes discussed in Section IIIa, a miscellaneous category (3) is added to accommodate cultures producing antibiotics that at the time of writing had not been fully characterized. When microanalysis is the only property defined, the empirical formula for a polyether antibiotic should be within the limits of the general formula (C3--4H5-,0L,and some evidence of a solvent-soluble alkaline salt, such as Na+ or K+, is good supporting evidence. If the salt has also been analyzed, then the subscript n of the general formula can be determined and based on compounds isolated to date (1976);n should be between 9 and 15.

TABLE I1 ORGANISMS PRODUCING POLYETHER ANTIBIOTICS

-8

Class la

Antibiotic Monensin (A-3823) and homologs Laidlomycin Nigericin [X-464 Polyetherin A Helixin C K- 178 Azalomycin hl] Grisorixin Salinomycin Narasin (A-28086) Lonomycin [Emericid (31559RP) DE-3936 A-2181 Antibiotic X-206

Producing organism Streptoinyces cinnainonensis Streptoinyces eurocidicus var. eurocidicus Streptomyces violaceoniger Streptomyces sp. X-464 Streptomyces hygroscopicus Actinomycete isolate A158 Streptoinyces albus Streptoinyces hygroscopicus Streptomyces griseus Streptomyces albus 80614 Streptomyces uureofuciens Streptoinyces ribosidificus TM Streptoinyces hygroscopicus Streptomyces hygroscopicus 9735-1 Streptomyces hygroscopicus A-218 Streptoinyces sp. X-206

Culture collection no. ATCC 15413 NRRL B1588 NRRL B1356 ATCC 21368

ATCC 13810 ATCC 21838 NRRL 5758, 8092 ATCC 31051 DS 24367 FERM-P 3159 FERM-P 928

Reference Haney and Hoehn (1968) Kitame et al. (1974) Harned et al. (1951) Berger et al. (1951) Kubota et ul. (1968) Smeby et u1. (1952) Horvath et al. (1964) Okazaki and Arai (1966) Gachon et al. (1970) Mitani et al. (1975) Eli Lilly (1974) Omura et al. (1976) Ninet et al. (1576) Ohshima et al. (1976) Tsuji et a1. (1976) Blount and Westley (1975)

Ib

2a

Alborixin

Streptoinyces ulbus

Dianemycin Lenoremycin (Ro 21-6150) [A- 130A] Septamycin [A-286951 Antibiotic A204 Antibiotic CP 38295

Streptoiiiyces hygroscopicus Streptom!yces h!ygroscopicus X-14563 Streptoiiiyces hygroscopicus Streptoinyces hygroscopicus Streptomyces albus S treptoinyces albus Streptoin!yces hygroscopicus

Lasalocids A, B, C, D, E and iso-lasalocid Lysocellin (K-5619)

Streptoinyces lasuliensis

[X-145371

2b -

'0

3

Streptoinyces cacaoi war. asoerisis K-9 Met- mutant S. longwoodenis

AllCaume et a / . (1975) NRRL 3444 ATCC 21840 NRRL 5678 NRRL 3883 NRRL 3384 ATCC 31050

Czerwinski and Steinrauf (1971) Liu et ul. (1976) Kubota et al. (1975) Keller-Juskn et a / . (1975) Hamill and Hoehn (1974) Jones et ul. (1973) Celmer et al. (1975)

NRRL 3382 and A'ITC 31180

Berger et ul. (1951) Westley el a / . (1974b,c) Ehata et ul. (1975)

Calcimycin (A-23187)

S. chartreusensis

ATCC 20251 NRRL 3882

Prosser and Palleroni (1976) Gale et a / . (1975)

BL-580a and /3 Ionomycin K-41

Streptoinyces hygroscopicus Streptoin!yces congoblatus Streptoinyces hygroscopicus

NRRL 5647 ATCC 31005 FERM-P 1342

Martin and Kantor (1974) Meyers et ul. (1975) Tsuji et ul. (1976)

192

J

. W. WESTLEY

The polyether antibiotics were first isolated 25 years ago (Berger et al., 1951; Harned et al., 1951), but 16 years elapsed before the first structure was solved (Agtarap et al., 1967). Monensin was the first polyether antibiotic whose structure was solved, and the following year, monensin, nigericin @-464),and antibiotic X-206 (Fig. 3) together with dianemycin (Fig. 4) were reported to be orally effective against poultry coccidiosis (Shumard and Callender, 1968). This report aroused considerable interest in the polyethers (see Section VIII), and approximately 30 had been reported (Table VI) by mid-1976. Of the polyethers reported, the structures of 24 have been solved in the 9 years since the first monensin paper (Agtarap et al., 1967). The structures of almost all these polyethers were elucidated by X-ray analysis of heavy-atom salts or derivatives, but in the case of the monensin (Fig. 3) and lasalocid (Fig. 5) homologs, mass spectrometry was the technique that distinguished these closely related compounds (Chamberlin and Agtarap, 1970; Westley et al., 1974~). In the crystalline state, the molecules exist in a cyclic conformation, with the two ends held together by a hydrogen bond between the carboxyl group and a tertiary hydroxyl on the terminal tetrahydropyran ring (hydrogen bond indicated by arrow in Fig. 7). The oxygen functions are concentrated in the center of the molecule, and the hydrophobic alkyl groups are all on the surface. This accounts for the unusual solubility properties of the antibiotic salts. They are virtually insoluble in water, but soluble in solvents such as benzene, ether, and chloroform. This characteristic conformation (Fig. 7) of the polyethers antibiotic salts also accounts for their ionophoric activity. The structures of nigericin (Steinrauf et al., 1968) and polyetherin A (Kubota et al., 1968) were reported simultaneously, and soon afterward both antibiotics, together with X-464 were shown conclusively (Stempel et al., 1969) to be identical. The following year, the structure of lasalocid (X-537A) was reported (Westley et a l . , 1970); Johnson et al., 1970), and shown to be the first polyether antibiotic possessing an aromatic chromophore (Fig. 5). X-ray crystallographic analysis of a number of different inorganic cation salts of lasalocid, nitrolasalocid, and bromolasalocid free acid revealed that in all cases two antibiotic molecules exist in a nonsymmetrical dimeric conformation in each independent unit of the crystal. The polar sides of the two lasalocid molecules (Fig. 7) face each other, forming a sandwich structure around a dibasic cation such as Baz+ and a water molecule (Johnson et a l . , 1970), two monobasic cations (Maier and Paul, 1971; Bissell et al., 1971) or two water molecules (Bissell and Paul, 1972). The versatility of the lasalocid molecule is demonstrated in the different combinations of oxygens 0 - 1 , -5, -6, -7, and -8 employed in ligand formation with cations and water molecules in the various complexes. The lipophilic sides of the two lasalocid molecules

193

POLYETHER ANTIBIOTICS: CARBOXYLIC ACID IONOPHORES

Polar side, with complexed cation (MI

2

Lipophilic side of Lasalocid

1

Me

Me

Me

-

Et

-

OH 8

4 Numbering System for Oxygens in Lasalocid FIG. 7. CPK model of a lasalocid salt showing the conformation in the crystalline state from both the polar and lipophilic sides of the molecule.

involved in the dimer form the outer coating of the complex providing a lipid-soluble surface. The existence of this dimeric complex for lasalocid barium and sodium salts in solution has recently been demonstrated by high-resolution proton NMR (Pate1 and Shen, 1976). A unique structural feature of lasalocid is the presence of three C-ethyl groups in the molecule, which prompted an investigation of the antibiotic’s biosynthesis. The results of this investigation are described in Section V.

V. Biosynthesis Of Polyether Antibiotic The only polyether antibiotics whose biosynthesis have been reported up to 1976 are lasalocid and monensin. The agreement between these two independent studies, however, is good enough to suggest that all polyethers (not including the pyrrole ethers, 2b) are assembled by similar biosynthetic routes.

194

J . W . WESTLEY

A. LASALOCID

An early proof of the polyacetate rule resulted from a study of the assembly of the carbon skeleton of 6-methylsalicylic acid (6-MSA) from four acetate units, and the biosynthesis of 6-MSA is now thought to be accomplished on a multienzyme complex. The similarity of the chromophores of (I) and 6-MSA suggested that the antibiotic is probably also assembled by a malonyl coenzyme A mechanism involving the initial formation of a polyketide chain on a multienzyme complex. The five C-methyls could arise through transmethylation of the polyketide precursor or by the insertion of propionic acid units into the polyketide skeleton. Likewise, the C-ethyl groups could be formed from successive C-methylation steps, by transethylation or by insertion of butyrate units. 1. 14C Incorporation Experiments

The first publication on the biosynthesis of a polyether antibiotic (Westley et al., 1970b) reported the testing of a number of 14C-labeledcompounds for incorporation into lasalocid to determine which of the possible pathways discussed above was being utilized by Streptomyces lasaliensis to assemble the antibiotic. Of the 14 substrates tested in this study and a later one (Westley et al., 1974a), eight were incorporated into the antibiotic (Table IIIa). Negative results with [14C]formate,[Me-14C]methionine,[Me-14C]ethionine, and [2-'4C]mevalonate ruled out transmethylation, transethylation, or the TABLE IIIa INCORPORATION OF I4C-LABELED SUBSTRATES

INTO

LASALOCID

Substrate for lasalocid [1-l4C]Acetate [ 1-14C]Propionate [2-I4C]Propionate [3-14C]Propionate [ LL4C]Butyrate [2-I4C]Butyrate [3,4-I4C]Butyrate [2-14C]Malonate

[Me-14C]2-Methylmal~nate [ 2-14C]2-Ethylmalonate [ l-L4C]3-Hydroxybutyrate [ l-14C]Stearate

Percentage incorporation

0.2 1-4 0.2 3.7 2-10 1.6

8.5 0.4

0.5 1.8 11.1 0.8

POLYETHER ANTIBIOTICS: CARBOXYLIC ACID IONOPHORES

195

introduction of terpenoid units as possible mechanisms leading to the branched alkyl groups in lasalocid A. The other two I4C-labeled acids not incorporated into (I) were salicylic and shikimic acid. The effective utilization of sodium [l-14C]acetate, sodium [I-, 2-, or 3-14C]propionate, and sodium [I-, 2-, or 3,4-14C]butyrate showed that the antibiotic was derived from these three units probably after conversion to malonate, 2-methylmalonate, and 2-ethylmalonate, respectively. This mechanism is well established in the case of acetate and propionate (Birch, 1957; Lynen, 1967; Kaneda et al., 1962), but the involvement of butyrate and 2-ethylmalonate had no precedent in the literature. Degradation experiments and I3C NMR (next two sections) showed incorporation pattern to be as in (VIII) (Scheme 1).

2 . Chemical Degradation of %-Labeled Lasalocid A (Scheme 1 ) Pyrolytic decarboxylation of lasalocid A at 220°C gave radioactive COZ (11) only in the case of [l-14C]acetate-derived antibiotic, showing that C-1 in (I) is derived from an acetate carboxyl. Retro-aldol cleavage of (I) followed by oxidation of (VI) and hydrolysis gave acetic acid (VII) from the C-methyl group at C-23 in (I). This C-methyl was shown to originate as an acetate unit by obtaining radioactive (VII) from [ l-I4C]acetate-derived (I) (Table IIIb). Kuhn-Roth oxidation of (I) produced acetic acid (111) from the eight C-methyls and propionic acid (IV) from the three C-ethyls in the molecule. These acids were separated by silica gel chromatography as their p-bromophenacyl esters. If all the I4C atoms in a labeled (I) preparation are situated in the C-methyl groups, the theoretical relative molar activity of (IV) would be 12.5%. Similarly, if all the 14C atoms are in C-ethyl groups, the theoretical activity of (IV) would be 33.3%.When this reaction was carried out on [2- or 3-14C]propionate-derived(I), the resulting acetic acid had activity of 112 % and 10.5%,respectively, whereas [ l-'4C]propionate-derived (I) gave inactive acetic acid. The propionic acid (IV) produced in the oxidation was virtually inactive in all three cases. These results show clearly that the propionate units incorporated into (I) are only at C-methyl positions in the antibiotic. Retro-aldol cleavage of the three [14C]propionate-derived samples of (I) demonstrated that there are four propionate units in the molecule and that one is situated at the C-11, C-12 bond, the site of this cleavage. This results in a (V):(VI)ratio of 1:1(or 2 2 ) in the [2-and 3-14C]propionate experiments, but 3:l in the [ l-14C]propionatecase. The four propionate-derived C-methyls must be at C 4 , C-10, C-12, and C-16 as the C-23 methyl is acetate-derived. Kuhn-Roth oxidation of the three [14C]butyrate-derived samples clearly established that the butyrate units incorporated into (I) are only at the

196

1. W . WESTLEY

MeCOZH

c o z cni

t 220-

Iml

f Kuhn-Roth

I

+ EtC02H IlYI oxidation

OH Me

Relro-aldol

CO2H

Et*Et

OH

IYI

OH

Me

IYII

1

Jones o x i d a t i o n

Me’

MeCOzH

M~CHZCOIH

MeCHZCti2co2H

SCHEME1. Chemical degradation of 14C-labeled lasalocid A.

C-ethyl positions. Thus, [ 1-’4C]butyrate-derived (I) gave virtually inactive acetic and propionic acid, [2-14C]butyrate-derived(I) gave inactive acetic, but active propionic acid (26.696, theoretical 33.3%) and [3,4-’4C]butyratederived (I) gave active acetic (9.3%, theoretical 12.5%)and active propionic acid (27.3%). The results from retro-aldol cleavage showed that all butyrate incorporation was taking place in the ketone (VI) part of the molecule, but could not be used to determine whether one, two, or all three of the C-ethyls were butyrate-derived. This question was subsequently answered by 13C incorporation experiments described in the next section.

197

W L Y E T H E R ANTIBIOTICS: CARBOXYLIC ACID IONOPHORES

TABLE IIIb RESULTS OF CHEMICAL DEGRADATION OF LASALOCID DERIVED FROM DIFFERENT SUBSTRATES~ Percentage relative molar activity of Lasalocid derived from following substrates

[ l-I4C]Acetate [ l-14C]Propionate [2-14C]Propionate [3-"C]Propionate [ l-I4C]Butyrate [2-14C]Butyrate [3,4-14C]Butyrate [2-14C]Malonate [Me-14C]2-Methylmalonate [2-I4C]2-Ethylmalonate [ l-'4C]3-Hydroxybutyrate [ l-14C]Stearate

(11)

(111)

(IV)

(V)

(VI)

(VII)

8.9 0

4.1 0.8 0.7

-

-

-

-

-

-

-

-

-

-

-

45.7 75.3 47.1 45.3 2.6 9.5 3.5 48.0 50.4 1.6 12.6 40.7

56.4 25.1 50.7 50.8 97.4 91.2 96.5 52.0 49.5 98.4 87.4 57.9

10.5

-

3.4 0 11.2 10.5 0.1 0.3 9.3 -

-

0 -

-

1.0 0.2

26.6 27.3

-

-

-

-

"The degradation reactions and products are shown in Scheme 1.

3 . I3C lncorpmation Experiments (Westley et al., 1972) The I3C enrichments considered most significant were those that resulted in at least a doubling of the natural abundance signal as determined by I3C NMR. In the case of [l-'3C]butyrate-derived lasalocid (I), three carbons were enriched consistent with the C-ethyls at C-14, -18, and -22 being butyrate-derived. The results with [ l-'3C]propionate-derived (I) were consistent with the conclusions from the earlier 14C experiments that the four C-methyl groups at C 4 , -10, -12, and -16 were propionate-derived (Scheme 1). Sodium [l-I3C]acetate at 0.5 gmfliter in S . lasaliensis fermentations gave antibiotic in which five carbons were enriched (Table IV). The enrichments at C-1 and C-23 confirmed earlier results with [14C]acetate-derived and the other three enrichments at C-5, -7, and -19 accounted for the biosynthetic origin of the remaining six carbon atoms in the molecule [(VIII) in Scheme

11. Following the work of Lynen on the biosynthesis of natural products from acetate (Lynen, 1967), Scheme 2 was proposed for the formation of the carbon skeleton of lasalocid (Westley et al., 1974a). The subsequent discovery of iso-lasalocid A in cultures of Streptomyces lasaliensis (Westley et al., 1974b) led to speculation on the possible cyclization reactions involved in completing the biosynthesis of lasalocid A.

INCORPORATION

OF

TABLE IV [1-I3C]ACETATE,[ l-'3C]PROPIONATE, AND [1-13C]BUTYFiATEINTO LASALOCID % Abundance" of I3C in (I) produced from

Multiplicitf S

S S S

d S

d s S

d d S

d d

I3C Shift in CH,CI: (ppm) 219.9 176.4 161.3 143.5 131.5 123.0 119.6 118.2 87.6 83.1 77.2 71.5 71.0 68.6

Carbon no. in (I)

13 1 3 7 5 4 6 2 18 15 23 22 11 19

Functional group

-0 COzH =C-OH =C-CHz

=CH =C- CH, =CH =C-CO,H C(Et)-0 CH-O CH(Me)-0 C(Et)-OH CH-OH CH-O

CH,13COzNa 1 2 1 2.5

3 1 1 1 1 1.5 3 1 1.5 3

CH&Hz13COZNa 1 1 4.5

1 1 1

1 1 1 4

1 1 4

1

CHzCHzCHz'3COzNa 4

1 1 1 1.5 1 1 1 1 1.5 1.5 1 1.5 1

d d t t d d t t t t t t 8 x q

e

W \o

55.9 49.0 38.1 37.9 34.6 34.2 33.6 31.3 29.9 29.3 19.7 16.2 16.1, 15.4, 13.6, 13.4 12.7, 12.6, 9.6, 6 . 9

14 12 9 17 10,16 10,16

1 1 1 1 1 1

-

1 1 4 1 1 1

-

1 1 1

5 1

1

-

4

a Multiplicity of l3C signals was obtained from off-resonance spectra of lasalocid sodium. Concentration of natural abundance and I3C-enrichedlasalocid sodium was 0.76M. The 'H decoupled I3C NMR Fourier transform spectra were recorded in 10 m m spinning sample tubes on a Bruker HFX-W/6 spectrometer at 22.63 MHz, using an internal "F lock of C6F6 at 84.66 MHz. A Fabritek FT-1083, computer was used for accumulation of free induction decays and Fourier transformation. s, Singlet; d, doublet; t, triplet; q, quartet. *Downfield from internal Me&. Shifts are for the sodium salt form of lasalocid. 'Corrected to nearest 0.5%.

200

J . W . WESTLEY

\

\

-

2- mOIh~lmm10001~ 11101on01.

/

SCHEME2. Hypothetical hiosynthetic scheme outlining the formation of the carbon skeleton of lasalocid A on a multienzyme complex. After Lynen (1967).

The structures of lasalocid A and iso-lasalocid A dif€er only in the size of the terminal ring and the configuration at carbons C-22 and C-23, suggesting that cyclization of the tetrahydropyran moiety is the final step in the biosynthesis of lasalocid (Scheme 3). The possible origin of the R,R-epoxide in Scheme 3 and cyclization routes to the other polyether antibiotics have been recently discussed (Westley et al., 1974b). 4 . Lasalocid Homologs

The four homologs, after long and tedious countercurrent distribution experiments, were finally separated and shown, mostly by mass spectrometry (Westley et al., 1974c), to have the structures illustrated in Fig. 8. These structures were subsequently confirmed by high-resolution (270 MHz and 360 MHz) proton nuclear magnetic resonance (NMR) spectroscopy (Pate1 and Shen, 1976). The homologs of lasalocid A each arise most probably by replacement of one of the four propionates in the antibiotic precursor molecule by a butyrate

POLYETHER ANTIBIOTICS: CARBOXYLIC ACID IONOPHORES

20 1

Et

he I

n

P

LASALOCID A

i s 0 - LASALOCID -

A

Me

Me

Me

Et

SCHEME3. Proposed final step in the biosynthesis of lasalocid A (and iso-lasalocid A)

Lasolocid A

R, =R2=Rj=R4 = Me

Lasolocid 8 C

D E

FIG.8 . Structures of lasalocid A and the four isomeric homologs B, C, D, and E.

OH

202

1. W . WESTLEY

OH

RI

Me

Me

Me

MONENSIN

R I =CH ( Me)C02H.

FACTOR B

R I =CH(Me)C02H.R2 = M e

FACTOR C

RI = (CH2)3C02H.R2= Me

R p =E l

FIG. 9. Structure of monensin and factors B and C.

unit. This replacement in turn results in the formation of the four distinct isomeric homologs of lasalocid A, each containing four C-ethyl groups (Fig. 8). As the total homolog content in lasalocid fermentation varies from 1% up to 25%, it appears that partial replacement of each propionate by butyrate occurs from one to six times per hundred molecules of lasalocid generated by S. lasaliensis under the fermentation conditions employed. We found no evidence of an ethyl homolog at C-23, which supports the thesis that this particular C-methyl arises from acetate. B. MONENSIN Studies of the biosynthesis of monensin were first published in 1973 (Day et al., 1973), and six ''C-precursors were shown to be incorporated into the antibiotic (Table V). By methods similar to those employed for ['4C]lasalocid, monensin was shown to be synthesized by Streptomyces cinnamonensis from five acetate molecules, seven propionate molecules, and one butyrate molecule. These three building blocks had previously been shown to be involved in lasalocid biosynthesis, but one of the differences in structure of monensin compared TABLE V INCORPORATION OF ''C-PRECURSORS INTO MONENSIN

Incorporation Precursor

[ U-'4C]Glucose Sodium [l-I4C]acetate Sodium [2-I4C]acetate Sodium [2-14C]propionate Sodium [2-I4C]butyrate ~-[Me-'~C]methionine

(%I 0.70 1.11 1.86 19.80 3.18 6.40

Molar incorporation 0.025 0.074 0.124 1.320 0.189 0.280

POLYETHER ANTIBIOTICS: CARBOXYLIC ACID IONOPHORES

203

FIG. 10. Incorporation pattern in monensin biosynthesis.

to lasalocid is the presence o f a methoxyl group, which was shown to be derived from methionine. Kuhn-Roth oxidation showed again that the C-ethyl was butyrate derived and the seven C-methyls were propionate derived as indicated in Fig. 10. C. MICROBIALTRANSFORMATION OF MONENSIN The reaction of glucose and monensin to form the glucoside metabolite

A-27106 (C42H72016) is claimed in the patent literature (Brannon and Horton, 1976). This transformation is catalyzed by an enzyme from submerged aerobic cultures of Streptornyces candidus, NRRL 5449. The glucose moiety is postulated to be attached to the monensin through a glycosidic bond to the -CH20H group of the terminal ring (for the monensin structure, see Section V,B above). Both anticoccidial and increased feed utilization in ruminants are claimed for A-27106.

VI. Physical Constants of the Polyether Antibiotics The data listed in Table VI follow the antibiotics, class by class, in the identical order to that presented in Table 11. For that reason, the list of references is omitted.

VII.

In Vitro Antimicrobial Activity and Mouse Toxicity of the Polyether Antibiotics A. ANTIMICROBIAL ACTIVITY

Although the polyether antibiotics exhibit virtually no activity against gram-negative organisms, the results of in uitro testing against gram-positive

TABLE VI THE POLYETHER ANTIBIOTICS a

PHYSICAL CONSTANTS OF

[aID Class

la

N

0 0

Antibiotic

Molecular formula

Molecular weight

Melting point ("C)

CHCZ

Monensin Free acid Sodium salt

C36H62011 ' HzO C36H61Ol1Na.HzO

688.9 728.9

103-105 267-269

Factor B

c 3 5 H6,OI I

656.8

227-228

Factor C

C37H64011

684.9

212-214

Factor D

C37H64011

684.9

25 1-252

Laidlom ycin Free acid Sodium salt

C37H62012 C37H6iOizNa

698.9 720.8

151-153 277-279

+51.3" +78.9"

Nigericin (X-464) Free acid Sodium salt

C4nH.56011 C~CIHWOIIN~

724.9 746.9

183-185 245-255

f35.2" +Zob

Grisorixin Free acid Sodium salt

C4oH~sOin C4nH67010Na

708.9 730.9

75-78 242-246

16" (acetone)

+75"b

+

CH3OH

+47.7" +57.3"

f9.2" +7.8"

Salinomycin Free acid Sodium salt

750.9 772.9

113 140-142

-63"

98-100 150-153 96-98

-54"

C44H74011

764.9 762.9 778.9

CdMhNa

850.2

188-189

+67"

+47"

&O C47Hs1014Na

889.2 893.1

133-145 189-190

-1.9" +21"

+17.7" + 15"

884.9 922.1

100-115 209-210

-7" (acetone)

C47H77014Na

885.1 889.1

156-157 212

Lenoremycin(Ro 21-6150) Free acid C47H78013 Sodium Salt C47H7~013Na

851.1 873.1

235

Septamycin (A28695A) Sodium salt

937.2

Narasin (A-28086A) Free acid A-28086B A-28086C Lonomycin (emericid, DE-3936, A-218) Sodium salt

X-206 Free acid Sodium salt

N

C42H70011

C~ZHBSOII Na

C43H72011

C43H70011

C47H82014

-37"

- 56"

8

Alborixin Free acid Potassium salt

1b

Dianemycin Free acid Sodium salt

C48H84OI4

C4sH83014K

C47H78014 '

HzO

C48H8101 G

N ~

+39.9" +37.1" +71" +95"

+45"* +94"* + 19.1" (continued)

TABLE VI (continued)

[alD Class

Ib contd.

N

8

2a

Molecular formula

Molecular weight

Melting point ("C)

A-204A Free acid Sodium salt

C49H84017 C49H83017Na. C3H60

945.2 1025.2

96-98 144-145

+55"

A-204B Sodium Salt

C50Hs,017Na

981.2

177-179

+42.3"

CP-38295 Free acid Sodium salt

C47H80015 C47H79015Na

883.1 905.1

197-198

+38"

Lasalocid A (X-537A) Free acid Sodium salt

CsHs408 C&O Hd8Na

636.9 612.8

100-109 168-171

-39.8" -84.6"

Lasalocid B

C35H5608

604.8

85-87

-36.3"

Lasalocid D

C35H5608 'C3H8O

664.9

102-104

-63.6"

Lasalocid E Free acid Sodium salt

C35HsOs C35Hs08Na

604.8 626.8

181-182

Antibiotic

90

CHC1,

CH,OH

+68.1"

-42.1" -79.5"

-7.6" -30"

N -4 0

iso-Lasalocid A Free acid Sodium salt

C~H5408 Cd5,0sNa

590.8 612.8

203 183-185

Lysocellin Sodium, hemihydrate

C34H59010Na. 1/2H20

659.8

158-160

2b

(Calcimycin) A-23187

C29H37N306

523.6

181-182

-Mob

3

BL-SOa, sodium BL-SOP, sodium

C3&5OuNs

711 641

173-175'

C36H57012Nac

+16" fl.1"

Ionomycin Calcium salt

C41H7009Ca

746

K-41 Sodium salt

C~IHB&~~N~

857

-39.2" -93.9"

+11.5"

196- 198

aAll the data are from the literature except for those indicated by b . bNew data from the reviewer's laboratory. CMolecularformula calculated from microanalytical data reported in the patent literature

+15.6"

+ 1.v

208

J . W. WESTLEY

TABLE

IN VZTRO ANTIMICROBIAL ACTIVITY AND Minimum inhibitory

Polyether Class

antibiotic

la

Monensin (A3823) Nigericin (X-464) Grisorixin Salinomycin Narasin (A-28086A) Lonomycin (DE-3936)

-

X-206 1b

Dianemycin

Ro 21-6150 Septamycin (A28695A)

A-204A CP 3895-2 2a

Lasalocid (X-537A) is0 -Lasalocid

Lvsocellin

ATCC No:

Staphylococcus aureus

Sarcina lutea

Bacillus sp. E

Bacillus subtilis

6538P

9341

27859

558‘

3.1 0.2 0.4 3.1 0.8 1.6 0.2

12.5 0.1 0.2 3.1 1.6 12.5 0.8

0.4 0.004 0.1 0.2 0.2 1.6 0.02

1.6 0. I 0.2 0.8 0.9 3.1 0.4

1.6 0.2 0.8 3.1 12.5

3.1 0.2 1.6 12.5 6.3

0.2 0.02 0.006 0.8 1.6

3.1

1.6 12.6 0.8

3.1 6.3 0.4

0.2 1.6 0.03

1.6 1.6 0.2

0.4

1.6 6.3 6.3

2b

A-23187

0.2

0.04

0.2

0.1

3

BL-580a

0.8

1.6

0.2

0.4

aLowest 2-fold dilution giving zone of inhibition in agar-well diffusion ass.ay by Mrs. L. Fern and Ms. P. Gunter (HLR). *Results of Dr. W. Pool and Miss D. Hane (HLR); these are %-hour acute toxicities unless reference given. “NRRL collection number. “Indicates no activity up to 50 figiml.

bacilli, cocci, and filamentous microorganisms as presented in Table VII show that all the polyethers exhibit activity against either eight or all nine organisms tested. In general, the bacilli appear to be the most sensitive, but no correlation between the antimicrobial activity and the valency-chemical structure classifications, la, Ib, 2a, and 2b, discussed earlier was apparent. The conclusion from these results is that the microbial inhibition is most likely due to losses of essential monovalent cations such as K+ (with resulting uncoupling of oxidative phosphorylation)as all the polyethers are capable of

209

POLYETHER ANTIBIOTICS: CARBOXYLIC ACID IONOPHORES

VII TOXICITY OF THE POLYETHERANTIBIOTICS concentration (/&mi)"

Bacillus

Bacillus

Mycobacteriuin

Streptoinyces

Paecilomyces

inegateriuin

sp. TA

varwti

27860

phlei 355

cellulosae

8011

3313

26820 -d 0.8 3.1 3.1 -

Toxicity (mg/kdb mouse LD,, i.p.

p.0.

16.8 65 18 13 1.2

43.8' 190

3.1 0.1 0.1 0.2 0.4 0.8 0.2

1.6 0.02 0.4 0.8 0.4 1.6 0.2

12.5 0.4 0.2 6.3 3.1 12.5 0.2

6.3 0.4 0.2 3.1 3.1 6.3 0.8

3.1 0.1 1.6 12.5 3.1

1.6

0.4 0.2 1.6 3.1

6.3 0.2 1.6 12.5 3.1

6.3 1.6 3.1 12.5 12.5

6.3 0.8 6.3 25 12.5

3.1 1.6 0.4

1.6 3.1 0.4

12.5 6.3 3.1

6.3 25 1.6

12.5 1.6

64 250

0.8

0.04

1.6

6.3

-

10.'

0.4

0.4

3.1

3.1

-

14

50

0.8

-

w -

45.8" 17

-

-

-

55 41.1h 8' -

-

-

65

146 lo00 350 220

'Haney et (11. (1970). 'Miyazaki et al. (1974). gOshima et al. (1976). hHamill and Hoehn (1974). 'Worth et al. (1970). 'Gale et al. (1975).

transporting such cations (Section 111). This conclusion is analogous to the results of Harald and Baarda (1967)for valinomycin and gramicidins (Section II,B,4) that demonstrated the antibiotic activity of those particular ionophores was reversed by the addition of excess K+ ions.

B. TOXICITY OF POLYETHER ANTIBIOTICS The acute toxicity of the polyether antibiotics in the mouse (Table VII) ranges from LDS0(i.p.) 1to 250 mg/kg and (p.0.) 17 to lo00 mgkg. The most

TABLE VIII COCCIDIOSTAT ACTIVITY OF THE POLYETHER ANTIBIOTICS

Class la

lb

Polyether antibiotic Monensin Nigericin Lonomycin Antibiotic X-206 Alborixin Dianemycin

RO 21-6150 Septamycin Antibiotic A-204A

Effective level (ppm) in feed vs. Eirneria tenella 110 200 50-125 80 -100 40 50- 100 50-100 60

References Shumard and Callender (1968) Shumard and Callender (1968) Oshima et al. (1976) Shumard and Callender (1968) Delhomme et al. (1976) Shumard and Callender (1968) M. Mitrovic and E. G. Schildknecht, private communication (1976) M. Mitrovic and E. G. Schildknecht, private communication (1976) M. Mitrovic and E. G. Schildknecht, private communication (1976)

2a

Lasalocid Ly socellin

75 300

Mitrovic and Schildknecht (1975) M. Mitrovic and E. G . Schildknecht, private communication (1976)

3

Antibiotic BW80a

100

Martin and Kantor (1974)

POLYETHER ANTIBIOTICS: CARBOXYLIC ACID IONOPHORES

21 1

toxic of the antibiotics (LDJo< 20 mg/kg i.p. and 6 50 mgkg p. 0 . ) are X-206, A-204A, A-23187, lonomycin, salinomycin, and monensin. Lasalocid, nigericin, and lysocellin have virtually identical LD50 (i.p.) at 65 mg/kg, and the least toxic of those reported is iso-lasalocid with an LD,o (i.p.) of 250 mg/kg. In most cases, the drugs are only moderately absorbed; using the ratio of LDW(p.o.):LD5, (i.p.) as a measure, they fall between 3 and 5 : l .

VIII. Coccidiostat Activity of the Polyether Antibiotics Coccidia, an order of protozoa of the subphylum Sporozoa is parasitic in the epithelial cells of the internal tract of birds and mammals. Included among the genera found most commonly in the avian species such as poultry are Eimeria. Coccidiostats are agents that, when mixed in feed, control infections of coccidia and are particularly important agents to the poultry industry. Therefore, the first report in 1968 that monensin, nigericin, dianemycin, and antibiotic X-206 all exhibited coccidiostat activity (Shumard and Callender, 1968) stimulated considerable efforts to find other (polyether) antibiotics with the same activity. Many claims have been made for coccidiostat activity and the data known by this reviewer concerning activity vs. Eimeria tenella are summarized in Table VIII. The patent (Haney et al., 1970) for the use of monensin as a coccidiostat was issued in 1970, and the drug was put on the market the following year. Since that time, monensin has acquired -80% of the coccidiostat market. The patent for lasalocids use as a coccidiostat (Berger, 1973) was issued 3 years after the monensin patent and the drug was approved by the FDA in October. 1976.

IX. Antimicrobial and Coccidiostat Activity of Lasalocid Analogs A. NATURAL HOMOLOGS AND ISOMERS As described in the biosynthesis section (V,A), lasalocid is actually produced as a complex by Streptomyces lasaliensis consisting of approximately 96% lasalocid A, 1%each of lasalocids B, C, D, and E, and less than 0.1% iso-lasalocid A. The antimicrobial spectra of lasalocid and iso-lasalocid against 9 gram-positive organisms were presented in Table VIII (Section VI1,A) and the relative in uitro antimicrobial activity of these natural analogs and two bromo derivatives (Westley et al., 1974c) are compared vs. Bacillus TA

RELATIVEin Vit7-0 ACTIVITY

TABLE IX NATURALANALOGS OF LASALOCID

O F THE

Compound

X

RI

Rz

R3

h

iso-Lasalocid A Bromo derivative of iso-lasalocid A

H Br

Me Me

Me Me

Me Me

Me Me

R5

48

75 OH

Lasalocid A Bromolasalocid A Lasalocid B Lasalocid C Lasalocid D Lasalocid E

Relative in oitro activity (9%) vs. Bacillus TA (lasalocid A = 100)

100 Br

Me

H H H H

Et

Me Me Me

Me Me Et Me Me

Me Me Me Et Me

Me Me Me Me Et

124 90 180 160 170

POLYETHER ANTIBIOTICS: CARBOXYLIC ACID IONOPHORES

213

(ATCC 27860) by cup-plate agar diffusion assay (lasalocid A = 100)in Table

Ix.

The four homologs oflasalocid A were equally active to the parent antibiotic as coccidiostats, whereas iso-lasalocid A exhibited no activity vs. Eimeria teneUa at concentrations up to 300 ppm in feed. €3. SYNTHETICDERIVATIVES OF LASALOCID

Approximately 50 compounds derived from lasalocid have been described in the literature (Westley et al., 1970a, 1971, 1973a,b), and it would not be appropriate to discuss the chemistry here. In general, the most active compounds were the esters of the phenolic hydroxyl groups (Table X) and derivatives substituted para to the phenol (Table XI). As Table XI demonstrates, there appeared a much closer relationship between the partition coefficient (octanol-water) than apparent pK,' with the in vitro activity of lasalocid derivatives (Westley et al., 1973a).

TABLE X COMPARISON OF PARTITIONCOEFFICIENT AND in Vitro ACTIVITYOF SOME ACYLAND ALKYLDERIVATIVES OF LASALOCID MP

Relative in oitro activity R

Partition coefficient 705 349 9

120

Bacillus TA

Bacillus E

100 1 1

100 2 2 2 4

1 1

-

-

163 557 3031

16 28

35

9 23 19 14 4 10

214

1. W . WESTLEY

TABLE XI COMPARISON OF PARTITION COEFFICIENT AND APPARENT pK,' VitrO ACTIVITY OF LASALOCID DERIVATIVES

WITH

in

Me

Relative in uitro activity vs.

pK.' in 60%

R3

&

RI H

H

Na Na Na Na Na Na Na Na

H

H CH,CO H H H

H H

Partition coetficient

aqueous methanol

1.9 7.3 11 31 291 705 1330 1556 1775

6.0

NH* N==CHC6H5 NH . COCH,

Br N9 H

CI I Br

Bacillus TA <1 1 1 27 24 100 140 112 124

4.3 4.25 2.4 4.4 4.0 3.9 3.9

Bacillus E

<2 2 2 3 26 100 73 55 67

All the derivatives listed in Tables X and XI (except the methyl ether, and the four nitrogen-substituted derivatives in the second table) were active vs. Eimeria tenella at 300 ppm (or less) in feed. From these results, all compounds related to lasalocid which fit into the general structure defined in Fig. 11 exhibit in vitro antimicrobial activity

I I

I

Me,,2,3,4

can be reploced by Et

R can be H or acyl group X can be H or halogen

3%. I

OH

I

FIG. 11. Structural requirements for in oiho antimicrobial activity of lasalocid antibiotics.

POLYETHER ANTIBIOTICS: CARBOXYLIC ACID IONOPHORES

215

against gram-positive bacteria. The only compound of this general structure (Fig. 11) which is not active at 300 ppm vs. Eimeria tenella is iso-lasalocid A.

X. Improved Ruminant Feed Utilization By Addition of Polyet hers Chemical analysis of the fermentation occurring in the rumen of animals such as cows, sheep, and goats has revealed a correlation between improved efficiency of feed utilization by the ruminant and an increase in the amount of propionic acid produced in the rumen. The volatile fatty acids (VFA) are produced in the rumen fermentation by microorganisms that degrade carbohydrate present in the diet as outlined in Scheme 4. Utilization of the VFA occurs after absorption from the gut of the animal. One of the major inefficiencies in rumen fermentation is the production of acetate (and hence butyrate), which is accomplished with the sacrifice of one of the pyruvate carbons as methane and loss of the associated energy (route a of Scheme 4). Any agent, therefore, which tends to direct the fermentation toward propionate production (route b ) will also result in more efficient utilization of the cellulose, which is the major component of the diet of most ruminants (Leng, 1970). Several polyether antibiotics have recently been claimed (Raun, 1976) to exhibit the ability of changing the levels of VFA production in favor of propionate as demonstrated by gas-liquid chromatographic analysis of in uitro fermentations of rumen fluid (from steers) treated with these antibiotics. Some data selected from the patent are presented in Table XII. The antibiotics are also claimed to benefit monogastric animals such as horses, swine, and rabbits, which ferment fibrous vegetable matter in the C a r boh y dra t e )

CH4 ( m e t h a n e )

mono s a c c h a r ide

CH3CH2CH2C02H

butyrate

SCHEME4. Chemical degradation of carbohydrate in rumen.

216

J . W . WESTLEY

TABLE XI1 RELATIVELEVELSOF VOLATILE FATTY ACIDS(VFA) PRODUCED IN FERMENTATIONS OF RUMENFLUIDFROM STEERSin Vitro,TREATED WITH POLYETHER ANTIBIOTICS AT 10 pg/ml' VFA ratio in treated:untreated fermentations Polyether antibiotic

Acetate

Propionate

Butyrate

la

Monensin Nigericin Antibiotic X-206

0.94 0.84 0.93

1.56 1.48 1.32

0.80 0.88 0.70

1b

Dianemycin Antibiotic A-204

0.92 0.90

1.56 1.55

0.79 0.86

2a

Lasalocid

0.83

1.80

0.73

Class

"All data were selected from Raun (1976).

cecum. The cecal fermentation is claimed to follow a similar pathway to Scheme 4.

XI. Pharmacology of the Polyether Antibiotics Once the ability to mobilize Ca2+ ions was demonstrated for antibiotic A-23187 (Reed and Lardy, 1972) and lasalocid (Pressman, 1972), these two polyether antibiotics became the focus of attention for many biologists (Pressman, 1976). Although both possess aromatic chromophores (Figs. 5 and 6) which have not been found in any other polyethers reported up to mid-1976, the structures of lasalocid and A-23187 are actually very Merent. There are also distinctions in their selectivity toward cations. Lasalocid forms complexes readily with both monovalent and divalent cations in the sequence Ca2+, K+ > Li+, Na', and Mg2+ (Schadt and Haeusler, 1974) whereas antibiotic A-23187 is more specific toward the divalent cations, Ca2+ and M$+. Many effects caused by these antibiotics have been ascribed to calcium release, but potentially the most important are the effects reported for the cardiovascular system.

A. CARDIOVASCULAR ACTIVITIES

OF THE

POLYETHERS

Working first with isolated rabbit hearts (Pressman, 1972) and perfused working heart rat preparations (Levy et al., 1973), the discovery was made

POLYETHER ANTIBIOTICS: CARBOXYLIC ACID IONOPHORES

217

that in these isolated systems the divalent cation polyethers are very potent inotropic agents. Inotropic agents such as digitalis are compounds which stimulate myocardial contractility. The reason for this inotropic effect was ascribed to the release by A-23187 and lasalocid of myocardial Ca2+ions. Investigations then progressed to the normal, anesthetized dog (de Guzman et al., 1973; Schwartz et al., 1974). A single dose of lasalocid (2 mgkg i.v.) was found to cause: (1)yofold increase in contractility; (2)doubling of the cardiac output; (3) slight increase in blood pressure and heart rate; (4) decrease in peripheral resistance; (5) decrease in coronary resistance. The combination of these effects reach a peak after 20 minutes when the coronary flow was reported (Pressman and de Guzman, 1975) to be ten times higher than the control value. The effects of lasalocid persist for several hours and are consistent with the effects on rat heart preparations (SchaEeret al., 1974) and strips of human atrial muscle (Levy and Inesi, 1974). Antibiotic A-23187, however, although active in isolated hearts (Holland et al., 1975), produced eccentric effects when administered to the intact animal, often depressing cardiovascular function (Pressman, 1976). As A-23187 is much more selective (PfeiEer et al., 1974) toward divalent over monovalent cations than is the case for lasalocid, this cast some doubt as to the cardiovascular effects in vivo being mediated simply by Ca2+release. The ability of a number of lasalocid derivatives to transport biogenic amines (serotonin, dopamine, norepinephrine, and epinephrine) across artificial lipid bilayers has been measured (Schadt and Haeusler, 1974) and compared with the inotropic and antimicrobial activity of the derivatives (Westley et al., 1973a). The results (Table XIII) show good correlation between the inotropic effect and permeability coefficients induced by the different derivatives in the membrane. These results tend to favor the mobilization of a catecholamineby lasalocid (and derivatives such as bromolasalocid) and are consistent with their adrenergic nature, since they are prevented by p-adrenergic blockage (Schwartz et al., 1974) from exhibiting inotropic activity. During 1973, a number of monovalent cation polyether antibiotics were found to be active also as inotropic agents, and some, like antibiotic X-206 (Fig. 3), appeared to be more potent cardiovascular agents than lasalocid (L. B. Czyzewski, A. Gooden, and A. Hooper, private communication, 1973). Subsequently, these results were confirmed by Pressman and de Guzman (1975), who reported the inotropic activity of six polyethers. The order of potency they found was: A-204A > X-206 > monensin > dianemycin > nigericin > lasalocid. The mechanism of action now appears to be considerably more complex than earlier hypotheses concerning the simple mobilization of either Ca2+or

218

J . W . WESTLEY

TABLE XI11 PERMEABILITY COEFFICIENTS ( X I@) IN DOPED(0.1%) LIPIDMEMBRANES= RI r 3 h

I

.

Me

Me

-

Et Me - L E

t

Et

u-+ 7

Me

//

Me

C02Na H C0,Na H C0,Na H C02Na COCH, NO2 Na

H Br

0 0 H NOH H 0 NOz 0

750 190 18 1 X lo-' 750 190 18 1 X lo-' 210 100 - 2 x 14 2.2 - 4 X 3.8 - -

"Schadt and Haeusler (1974). bDA, dopamine; NE, norepinephrine; E, epinephrine; HA, molarity required to increase contractile force of cat hearts by 50%.

catecholamines because antibiotics such as X-206 or monensin do not transport either species across membranes in vitro. The foregoing discussion clearly illustrates the tenuous nature of most simplistic theories concerning the mechanism of action of drugs. In this particular field, the mechanism is far from being settled (Pressman, 1976).

B. MISCELLANEOUSEFFECTS Many other physiological effects ascribed to the calcium-mobilizingproperty of lasalocid, including coagulation of platelets (Massini and Luscher, 1974), release of histamine from mast cells (Forman et al., 1973; Cochrane and Douglas, 1974), fertilization of eggs (Steinhardt et al., 1974; Chambers et al., 1974), and various exocytotic processes (Douglas, 1974) should now be checked using monovalent cation polyethers in view of the cardiovascular activities discussed in Section XI,A. Direct evidence for the release of catecholamines by lasalocid from chromafh granules (Johnson and Scarpa, 1974) definitely points to the formation of lipid-soluble lasalocid-catecholaminecomplexes in these processes, at least (Thoa et al., 1974). As a result of these findings, we recently attempted to crystallize some

POLYETHER ANTIBIOTICS: CARBOXYLIC ACID IONOPHORES

219

0=c e -H FIG.12. Model of the R-(+)4-bromophenethylamine salt of lasalocid (open bonds) illustrating the conformation and hydrogen bonding of the salt in the crystalline state as elucidated by X-ray analysis. The star indicates the asymmetric center of the amine.

lasalocid-catecholamine complexes. Both dopamine and norepinephrine gave highly crystalline 1:1, molar antibiotic:amine salts, although the same technique failed completely with epinephrine (Westley et al., 1976). In addition, a number of substituted 2-aminoalkanes were resolved, yielding the optically pure enantiomer, in the case of R ( +)-l-amino-l-(4-bromopheny1)ethane after three crystallizations, which compares well with Cram’s results (Section II,B,l). The crystalline complex of the bromophenethylamine salt of lasalocid has been structurally defined by X-ray analysis, and a ball-and-stick model built from the X-ray data is illustrated in Fig. 12.

R(f)- 1-amino- 1- (4-bromopheny1)ethane salt of lasalocid. The conformation of this salt is illustrated in Fig. 12.

220

.

J W. WESTLEY

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The Microbiology of Aquatic Oil Spills R. BARTHA Department of Biochemistry and Microbiology, Rutgers University, New Brunswick, New Jersey AND

R. M. ATLAS Department of Biology, University of Louisville, Louisville, Kentucky I. Sources and Behavior of Oil Pollutants .................... A. Sources of Oil Pollutants B. Behavior of Oil Spills ............................... 11. Effects of Petroleum Hydrocarbons on Microorganisms . . A. Effects on Population Size ....................... B. Effects on Microbial Diversity. . C. Sublethal Effects ................................... 111. Microbial Emulsification and Degradation of Petroleum Hydrocarbons .......................................... A. Biological Constraints . . . B. Environmental Constraints. C. Interplay of Physicochemi Self-Purification of Aquatic Environments . . . . . . . . . . . . . . IV. Microorganisms and Oil Pollution Abatement . . . . Cleanup Techniques on Oil

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

gradation ........................ C. Environmental Impact of Stimulated Oil Biodegradation D. Role of Stimulated Biodegradation in Integrated Oil Pollution Control Programs .......................... References

.

226 226 228 230 230 234 234 236 236 246 253 253 254 255 258 260 261

This review is concerned with the microbiology of accidental or routine oil discharges into aquatic environments only. Oil discharges onto land surfaces will not be discussed here. A separate treatment is justified by the considerable differences in the environmental and nutritional parameters as well as in the microbial communities of the two environments. In addition, the physical behavior of oil pollutants in the two environments is quite distinct. Horizontal movement of a land spill is restricted and containment is accomplished with relative ease, though vertical and subsurface movements are often troublesome to control. The loading per surface area tends to be high, and subsurface oil weathers slowly or not at all. Conversely, in aquatic environments horizontal spreading is very rapid and extremely hard to control, while vertical movement is initially restricted. In consequence, the bulk of an aquatic oil spill is subject to extensive weathering changes which 225

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influence its composition and ultimate fate. A quantitative recovery of an oil slick is almost never feasible, and for some or most of the spill microbial degradation remains the principal mechanism of removal.

1. Sources and Behavior of Oil Pollutants

The sources and behavior of oil pollutants have been previously reviewed by Atlas and Bartha (1973~) and more recently by the National Academy of Sciences (1975). This section will rely in part on these reviews, and the reader should consult the bibliographies of these works for a more comprehensive listing of pertinent literature. Only more recent papers not cited in the above reviews will be referenced individually. A. SOURCESOF OIL POLLUTANTS A great public awareness of the problems of oil pollution of the Oceans has come about following a series of catastrophic tanker accidents from the mid 1960s through the early 1970s. The wrecks of the tankers Tampico Maru near Baja, California, the Torrey Canyon southwest of England, the Ocean Eagle in San Juan (Puerto Rico) harbor, the Florida at West Falmouth, Massachusetts, and the Arrow at Chedabucto Bay, Nova Scotia, and the blowout of wells in the Gulf of Mexico and Santa Barbara channel, all have brought to public attention the ecologic consequences of oil pollution. The death of coastal organisms, especially shorebirds, following these incidents were dramatically shown in the popular press. In spite of the high visibility of these spillages, it is estimated that such accidents account for only a small percentage of the annual input of petroleum pollutants into the biosphere (Table I). It is likely that future safety improvements will further reduce the input of petroleum pollutants from accidental sources. A comparison of the estimated inputs in 1970 and predicted inputs for 1980 is shown in Table II. As shown in the table, it is estimated that input of petroleum pollutants into the ocean is declining, largely owing to changes in marine transportation practices. The prediction for 1980 is that the main inputs of petroleum hydrocarbons into the worlds oceans will continue to be from natural or routine operations rather than from catastrophic accidental spillages. Principal concern is with the input of petroleum hydrocarbons into the oceans, since the oceans are the ultimate recipients of such pollutants and since catastrophic oil spills occur more frequently in the marine realm. Oil spills on land or in quiescent inland waters can be contained and collected more readily, while oceanic spillages often defy collection.

TABLE I BUDGETOF PETROLEUM HYDROCARBONS INTRODUCEDINTO THE OCEANS~ Millions of tons per year

Source Natural seeps Offshore production Transportation LOT tankers Non-LOT tankers Drydocking Terminal operations Bilges hunkering Tanker accidents Nontanker accidents Coastal refineries Atmosphere Municipal wastes Industrial wastes Urban runoff River runoff

0.6 0.08 0.31 0.77 0.25 0.003 0.5 0.2 0.1 0.2 0.6 0.3 0.3 0.3 1.6 6.113

-~

aNationd Academy of Sciences (1975)

TABLE I1

ESTIMATES OF PETROLEUM HYDROCARBONS ENTERINGTHE OCEANAN NU ALLY^ Millions of tons per year Source Marine transportation Offshore oil production Coastal refineries Industrial waste Municipal waste Urban runoff River runoff Natural seeps Atmosphere

1970

1980

2.133

0.8 0.2 0.02

0.08

0.2 0.3 0.3 0.3 1.6 0.6 0.6

6.113

0.45

0.3 1.6 0.6 0.6

4.57

(IBased upon estimates by National Academy of Sciences Workshop (1975).

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The routine petroleum inputs to the oceans are of two types: continuous low-level chronic inputs and discontinuous small spillages. The ecologic problems associated with chronic inputs of oil are quite different than those associated with small routine or large accidental spillage. Chronic inputs allow for adaptation by the biological community under the stress of polluting petroleum hydrocarbons. Chronic inputs also allow for accumulations of large amounts of hydrocarbons in affected ecosystems. The sudden input of large amounts of petroleum hydrocarbons, as occurs with spillages, stresses the environment in a way not imposed by natural hydrocarbon inputs. The ecologic effects of oil spillages generally depend upon the location of the spill, the type of oil spilled, and the amount of oil spilled. Catastrophic oil spillages often occur in nearshore regions, affecting both marine and coastal ecosystems. Such spillages often involve a relatively large amount of oil within a small region. The eventual fate and ecologic effects of oil spills, large or small, depend upon many factors. B. BEHAVIOROF OIL SPILLS Immediately upon spilling, oil begins to undergo a series of physical and chemical changes. The processes causing these changes include spreading, emulsification, dissolution, evaporation, sedimentation, and adsorption. Collectively, the oil is weathered by these processes. The weathering of oil depends upon the amount and type of oil spilled and on environmental conditions. Petroleum hydrocarbons have only a very limited solubility in water. Therefore, most oil spillages initially form a surfice slick. The surface slick can be moved by wind, wave, and current action. A surface oil slick immediately begins to spread, initially owing to gravitational forces, resulting in a thinner layer of oil covering a larger area. The viscosity of the spilled oil will, to some extent, influence the rate of spreading and, since viscosity is temperature dependent, water temperature will also influence the area covered by a surface slick. Volatile hydrocarbons in the oil will also immediately begin to evaporate. As the oil spreads and the slick thins, the rate of evaporation will increase. Evaporation of the lighter hydrocarbons increases the density and viscosity of remaining oil slick. The increased viscosity decreases the rate of further spreading. The increased density of the oil may make it denser than water, causing it to sink. Dissolution also results in removal of light hydrocarbons and some polar components from the oil slick. As with evaporation, dissolution processes leave behind a denser, more viscous oil slick. The water-soluble components of the oil are of special concern because of their potential toxicity to or-

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ganisms in the water beneath the oil slick. The relative importance of dissolution versus evaporation was recently studied by Sutton and Calder (1974), Harrison et al. (1975), and Regnier and Scott (1975). Both water-in-oil and oil-in-water emulsions may form following an oil spillage. Oil emulsified in the latter fashion can move down into the water column. Emulsions of oil may persist for extended periods of time. Eventually, the emulsified oil may dissolve, be incorporated into sediment, or be deposited on the shore. Particulate matter in the water column may adsorb spilled oil. The adsorbed materid, when denser than water, may then sink and become incorporated into the sediment. Emulsified oil may also become adsorbed to particulate matter, and sink. Oil that has sunk owing to adsorption may later be desorbed and resurface, forming a new oil slick. The above weathering processes, coupled with partial oxidation of the oil, can result in the formation of dense tar balls. Tarry materials tend to accumulate in sediment and along the shoreline. Tar balls can be found along many beaches and tar crusts can be seen adhering to rocky surfaces. Pelagic tar lumps have been found in many regions of the worlds oceans. The measurement of pelagic tar is generally accomplished by collection with a calibrated neuston net. Pelagic tar lumps appear to be in highest concentrations in the vicinity of major shipping routes. Pelagic tar is subject to further oxidation and weathering processes, resulting in its ultimate sedimentation. A recent monograph on pelagic tar from Bermuda and the Sargasso sea was prepared by Butler et al. (1973). The standing stock of floating tar in the Northwest Atlantic was estimated to be at least 86,000 metic tons, the highest concentrations ( 2 4 0 mg/m2)being found in the Sargasso Sea. The apparent lifetime of floating tar was estimated to be one year or more. The transport of oil from the original slick to other locations and the chemical modification of oil can be quite important in determining the persistence of spilled oil. The formation of tar balls with limited surface area retards other degradative processes. The movement of oil into oxygenlimited sediments severely limits biological degradative processes that require molecular oxygen. Movement of oil onto beaches and rocks may also increase the persistence of spilled petroleum hydrocarbons. It is often necessary to determine the source of an oil spill. Various methods have been developed to fingerprint spilled oil. These methods are based on the appearance of the oil following weathering. Fingerprinting analysis may involve determination of metal content and hydrocarbon distribution. The environment in which spilled oil undergoes weathering and whether the oil has been sedimented or stranded along the shore may affect the appearance of the residual weathered oil. Brown and Lynch (1976)found that treatment with warm salt water and vacuum quickly and accurately

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reproduces the early weathering changes that af€ect an oil spill in the ocean and thus allows the more accurate matching of an oil spill with its source.

II. Effects of Petroleum Hydrocarbons on Microorganisms

A. EFFEC~S ON POPULATION SIZE The addition of hydrocarbons to an ecosystem, as occurs from oil spillages, may result in selective increases or decreases in the sizes of microbial populations. The effect of petroleum hydrocarbons on the size of microbial populations will depend upon the chemical composition of the contaminating hydrocarbons and on the species of microorganisms present within the microbial community of the particular ecosystem.

1 . Enrichment of Microbial Populations by Petroleum Hydrocarbons In some c s e s the addition of hydrocarbons to an ecosystem will enrich primarily for microorganisms capable of utilizing the hydrocarbons and secondarily for microorganisms capable of utilizing metabolites produced by the hydrocarbon-utilizingmicroorganisms. Such enrichment results in increased numbers of hydrocarbon-utilizing microorganisms and associated secondary colonizers, and generally also results in an overall increase in the total number of microorganisms present within the ecosystem. There are numerous reports of increases in microbial numbers following experimental addition of hydrocarbons to a variety of microbial communities. For example, Atlas and Bartha (1972a), ZoBell (1973), Traxler (1973), Miget (1973), Soli (1973), Kator (1973), Perry and Cerniglia (1973), and Pritchard and Starr (1973) have all reported large increases in total microbial populations and numbers of hydrocarbon-degrading microorganisms in laboratory experiments when a variety of individual hydrocabons or mixtures of hydrocarbons, including crude and refined oils, were added to marine or estuarine waters. Selective enrichment by particular petroleum hydrocarbons may result in increases in the population of a limited number of microorganisms or even a single species. Horowitz et al. (1975) reported that addition of different fractions of the same oil to seawater resulted in increases in the populations of different bacterial species. Zajic and Daugulis (1975) also cited the selective enrichment of particular species of hydrocarbonoclastic microorganisms by petroleum hydrocarbons. Westlake et al. (1974) found that both oil type and incubation temperature were of importance in causing differential popu-

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23 1

lation increases. Various microbial genera were enriched in response to various crude oils. Also, the same crude oil favored different genera at various incubation temperatures. Increases in numbers of microorganisms have also been observed in situ underlying artificial oil slicks. For example, Atlas and Schofield (1975) reported that a large increase in numbers of a hydrocarbon-degrading Pseudomonas sp. and of a nonhydrocarbon-utilizing Staphylococcus sp. occurred in Prudhoe Bay, Alaska, in the water column beneath experimental oil spills. Selective enrichment for populations of hydrocarbon-utilizing microorganisms has also been reported in several ecosystems receiving petroleum hydrocarbon pollutants from nonexperimental sources. Atlas and Bartha (1973a) found that the distribution of hydrocarbon-utilizing microbial populations in Raritan Bay were directly correlated with presumed patterns of oil influx into the Bay. Colwell et al. (1973) and Walker and Colwell (1974a) also found that the distribution of hydrocarbon-utilizing microorganisms in Chesapeake Bay was related to sources of oil pollutants; numbers of petroleum-degrading microorganisms were higher in natural environments exposed to oil. Colwell et al. (1973) further found that populations of Cladosporium resinae and actinomycetes were predominant among the hydrocarbon-utilizing isolates. In the Indian Ocean and the Black Sea populations of hydrocarbon-oxidizing microorganisms were found to be highest near shipping zones (Mironov, 1970). Similar distributions of hydrocarbonoxidizing microorganisms were reported in Neva Bay, U. S.S.R. (Polyakova, 1962). Numbers of hydrocarbon-oxidizing microorganisms in sediment samples from Barataria Bay also have been found to be correlated with sources of oil pollutants (ZoBell and Prokop, 1966). Floodgate (1976) found that along the English coast tar balls contained populations of penicillia but that similar populations did not occur in the adjacent unpolluted beach. Most of the above increases in microbial populations were associated with chronic inputs of petroleum hydrocarbons. Increases in numbers of h ydrocarbon-degrading microorganisms following major catastrophic oil spillages have not been well documented. Gunkel (1968) did, however, report on the occurrence of high numbers of oil-degrading microorganisms in sediments believed to be contaminated by oil from the T m e y Canyon spill. Some investigators have examined the microbial populations found in oily surface films. R. J. Miget (personal communication) has readily isolated hydrocarbonoclastic microorganisms from such films. Crow et al. (1976) reported that surface films contained microbial populations 10-100 times greater than occurred in underlying waters and that the predominant species were proteolytic or amylolytic but not hydrocarbonoclastic. In addition to finding an increase in the population of hydrocarbonoclastic microorganisms following experimental oiling of a salt marsh and in the

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vicinity of an accidental spill in Barataria Bay, Crow et al. (1975) found an increase in the population of proteolytic microorganisms. Following addition of South Louisiana crude oil, Walker et al. (1975d)also found an increase in absolute numbers of proteolytic bacteria as well as lipolytic, chitinolytic, and cellulolytic bacteria, autochthonous to a subestuary of Chesapeake Bay.

2. Suppression of Microbial Populations by Petroleum Hydrocarbons In contrast to the favorable effects listed in the foregoing section, Crow et al. (1975)found a decrease in numbers of cellulolytic microorganisms in salt marsh estuaries exposed to petroleum hydrocarbons. Walker et al. (1975d) did find that the percent of total microorganisms showing proteolytic, lipolytic, chitinolytic, or cellulolytic activity were lower in samples to which South Louisiana crude oil was added as compared to control samples. The addition of a No. 2 fuel oil only resulted in an absolute increase in lipolytic bacteria and caused a decrease in the percent of the total microbial population showing lipolytic, cellulolytic, proteolytic, and chitinolytic activity. The author’s conclusion that the decrease in the percent of total population of these bacterial populations represents toxicity of crude or fuel oil is unsupported in light of the lack of decreases in absolute numbers of these microorganisms and without substantiating metabolic measurements. Decreases in microbial populations exposed to certain petroleum components are, however, well known. Compounds such as toluene and phenol, which are found in petroleum, have been used for many years as disinfectants. Liu (1973) reported that toluene, ethylbenzene, and benzene were inhibitory to microorganisms, but that aromatic compounds with longer aliphatic side chains stimulated metabolic activity of the same microorganisms. The inhibitory action of petroleum components on microorganisms may be bacteriostatic or bacteriocidal. Atlas and Bartha (1972b) and Atlas (1975) reported that certain crude oils contain volatile toxic components that are bacteriostatic, delaying hydrocarbon biodegradation and increases in populations of hydrocarbon-degradingmicroorganisms until they evaporate. Kauss et al. (1973) similarly found that the size of an algal population was reduced in the presence of water-soluble petroleum components, that the toxicity was associated with volatile components, and that once the toxic components had evaporated there was a significant increase in algal population size. Benzene, toluene, xylene, and naphthalene had a similar effect as the soluble petroleum components in low concentrations, but high concentrations resulted in death of algae. The inhibitory effects of petroleum components are often highly dependent upon solubility and concentration. A hydrocarbon such as toluene may stimulate growth of microorganisms at low concentrations, but show bacteriocidal action at high concentration (Davis,

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1967). Phenol is another example of a petroleum component that can be utilized by some microorganisms but is bacteriocidal at high concentrations. Calder and Lader (1976) found that dissolved aromatic hydrocarbons decreased the growth rates and maximal cell densities of marine bacterial isolates. The degree of inhibition was found to be a function of concentration and solubility of the specific hydrocarbon. In addition to direct toxicity to microbial populations by petroleum hydrocarbons, products formed by microbial hydrocarbon degradation may be toxic. Liu (1973) found that C5-C9 alkanes were not toxic to a population of bacteria, but that the alcohols of these hydrocarbons were inhibitory. Atlas and Bartha (1973b)found that fatty acids formed during petroleum biodegradation exhibited synergistic toxicity with the substrate hydrocarbons toward autochthonous and single species populations of estuarine microorganisms. Oxidation products of aromatic hydrocarbons have been found to be more toxic than the original hydrocarbon (Calder and Lader, 1976). The inhibitory effects of petroleum hydrocarbons not only depend on the chemical nature of the hydrocarbons but also on the particular microbial population. Walker and Colwell(l975) found that bacterial populations from a polluted area increased in the presence of hydrocarbons, but that bacterial populations from a nonpolluted area decreased when exposed to the same hydrocarbons. Walker and Colwell(1976e)found that petroleum hydrocarbons can limit the growth of bacteria in sediment and water in nonoil contaminated estuarine ecosystems. They used a mixture of hydrocarbons, including aromatic and paraffinic compounds. Bacteria exposed to chronic inputs of petroleum in the area of their collection did not show decreased growth rates when exposed to the model hydrocarbon mixture, indicating adaptation to the presence of hydrocarbons. Petroleum hydrocarbons may also result in selective decreases in algal and protozoan populations. Adas et al. (1976)found that coccoid green algae and amoeboid protozoan populations disappeared when Prudhoe crude oil was added. Mironov (1971) found that low concentrations of oil prevented cell division of dinoflagellates and diatoms. Nuzzi (1973), Nelson-Smith (1973), Kauss et al. (1973), and Strand et al. (1971), among others, reported that a variety of refined and crude oils inhibited growth of a variety of algae. Not all microbial populations increase or decrease in response to the addition of petroleum hydrocarbons; some microorganisms show a neutral response to certain hydrocarbons, and, in these cases, population size of these organisms remains unchanged. Walker et al. (1975d), for example, found that while certain bacterial populations increased or decreased in response to addition of No. 2 fuel oil or South Louisiana crude oil, fungal populations remained unchanged under the same conditions.

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B. EFFECTSON MICROBIALDIVERSITY In addition to altering population size addition of petroleum hydrocarbons to an ecosystem may alter species diversity. Early work indicated that addition of oil would decrease species diversity (Baldwin, 1922). The isolation of only a limited number of bacteria from enrichment cultures has also been taken as an indication of decreased species diversity under the environmental stress of petroleum hydrocarbons. Recently, however, there have been several reports indicating that petroleum hydrocarbons may not alter, or may even increase, species diversity. Cobet and Guard (1973) found no change in the bacterial diversity of beach sand contaminated with Bunker C fuel oil. Crow et al. (1975) found that chronic pollution resulted in increased species diversity in contrast to decreased species diversity following incidental oil spillages. More studies are needed to determine the effects of oil on species diversity and the possible influence of chronic or incidental pollution by petroleum hydrocarbons on ecosystem stability.

C. SUBLETHALEFFECTS Petroleum hydrocarbons may have a variety of sublethal effects on microorganisms. Since microorganisms play an essential role in biogeochemical cycling, interference with microbial metabolic activities can have farreaching ecological consequences. Atlas et al. (1975)found that rates of algal photosynthesis and nitrogen fixation decreased in the presence of crude oil. Strand et al. (1971) and Kauss et al. (1973) also found that petroleum hydrocarbons inhibited algal photosynthesis. Gordon and Prouse (1973) found inhibition of phytoplankton photosynthesis at high concentrations of petroleum hydrocarbons, but that low levels of oil stimulated photosynthetic activities. Direct measurement of other metabolic activities involved in nitrogen and carbon recycling remains to be reported. Some indirect indications of changes in microbial metabolic activites, based upon population measurements, have been discussed above. The effects of petroleum addition on the mineralization of glutamic acid by bacteria in Beaufort Sea water have recently examined by Bunch and Harling (1976) and by Morita and Griffiths (1976). Both groups found that mineralization of amino acids was not affected by addition of oil. Morita and Griffiths found that mineralization of acetate, however, did increase in media with yeast extract but not in media lacking yeast extract. The implication of these preliminary reports is that oil contamination will not adversely affect normal microbial metabolism.

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An important sublethal effect of petroleum hydrocarbons on microorganisms is interference with chemoreception. Interference with chemoreception will result in behavioral changes in microorganismsand may interfere with the essential ecological role of affected microorganisms with respect to predatodprey relations and population balance. Walsh and Mitchell (1973)found that a variety of petroleum components inhibited chemotaxis by a motile marine bacterial isolate. The inhibition was reversible. When the hydrocarbon was diluted, normal chemotaxis was reestablished. Furthermore, the hydrocarbon concentrations needed to inhibit chemotaxis were higher than would occur in a natural ecosystem, but were within the range of concentrations that could occur around a major oil spillage. The inhibition of chemotactic response appears to involve a blockage of the chemoreceptor sites. The affected organisms retain motility, but movement is random and the organisms do not respond to available food sources (Mitchell et al., 1972). The presence of hydrocarbons does not appear to inhibit enzyme activities of the affected microorganisms. Young and Mitchell (1973) found that some marine bacteria also show negative chemotaxis toward some petroleum hydrocarbons, i.e., they actually move out of the region of hydrocarbon concentration. In contrast to the above findings, Chet and Mitchell (1975) found that marine bacteria showed increased positive chemotactic response to coral exposed to petroleum hydrocarbons. The increased chemotaxis was associated with secretion of a polymer by the coral. A consequence of such increased chemotaxis is overpredation by the bacteria, disappearance of the coral, and ultimate destruction of the ecosystems. Another possible sublethal effect of petroleum hydrocarbons on microorganisms involves the bioconcentration of such pollutants. Finnerty et al. (1973) found that an Acinetobacter sp. grown on hydrocarbons accumulated hydrocarbons in the cytoplasm. In addition to this bacterium they reported that yeasts and filamentous fungi also sequestered hydrocarbons in the cytoplasm. Andrews and Floodgate (1974)also reported that protozoa are capable of accumulating hydrocarbons. The consequences of such microbial accumulation of hydrocarbons is not known. Many petroleum hydrocarbons are carcinogenic and it would be a most serious situation if these compounds could be concentrated and passed through the food web. In addition to petroleum hydrocarbons other toxic substances may be concentrated within oil slicks and sequestered by microorganisms. Such substances include polychlorinated biphenyls and DDT (Study of Critical Environment Probe, 1970). Walker and Colwell(1974b)found that mercury is concentrated in oil. They also found that hydrocarbon-degrading bacteria in Chesapeake Bay exhibited a high resistance to mercury toxicity. It is not known whether such toxic substances can be sequestered by microorganisms

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along with petroleum hydrocarbons or can be passed through the food web. Such possible cobiomagnification of toxic compounds needs thorough examination.

Ill. Microbial Emulsification and Degradation of Petroleum Hydrocarbons Petroleum and its hydrocarbon components have low solubility in water and in case of a spill form a separate liquid phase or “oil slick.” Under metabolic attack by microorganisms, an oil slick undergoes characteristic changes. Especially when agitated, surface-active metabolic products of some oil-degrading microorganisms break up a part or all of the oil slick into more or less stable emulsions. Such emulsification may lead to the partial or complete disappearance of the surface oil slick and to a reduced recovery by solvent extraction of the emulsified oil. There is little doubt that losses by emulsification sometimes lead to overly high estimates of biodegradation, especially in open test systems. For the purpose of this discussion, emulsification is defined as physical dispersion of the oil promoted and stabilized by surface-active metabolic products, but without a reduction in biological oxygen demand (BOD). In contrast, biodegradation involves chemical changes in the parent hydrocarbons, usually accompanied by a reduction of BOD, but the products of biodegradation are not necessarily simple or harmless. “Mineralization” implies a complete recycling to harmless inorganic end products (C02 and H 2 0 for hydrocarbons) and a complete disappearance of the BOD. Typically, emulsification, biodegradation and mineralization processes affect an oil slick simultaneously, and form a logical sequence in the recycling of individual hydrocarbon molecules. The environmental impact of each of these processes is, however, distinct, and sometimes diametrically opposed. For an example, the emulsificationof oil tends to increase the exposure of vertebrates and invertebrates in the water column and in the benthic environment, while mineralization will decrease the overall extent of oil contamination. The ecological damage caused by an oil slick is influenced by the relative rate and importance of each of the discussed processes. These are, in turn, determined by intrinsic biological and external environmental constraints.

A. BIOLOGICAL CONSTRAINTS 1. Aquatic Hydrocarbon Degraders From the large number of hydrocarbon oxidizers listed in various books and reviews (Beerstecher, 1954; Davis, 1967; Fuhs, 1961; Friede et al.,

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1972) only a limited number are found on a regular basis in aquatic environments. Most of the isolation and determination work was carried out in coastal seawater or sediments, and in seawater taken along shipping lanes. All these environments are likely to be exposed to hydrocarbon pollutants on a regular or intermittent basis. In unpolluted pelagic seawater, the occurrence of hydrocarbon degraders varies from very rare to undetectable (ZoBell, 1969; Mironov, 1970). The genera of aquatic hydrocarbon degraders reported by various workers are listed in Table 111. Although some of the authors carried the identifications to the species level, owing to shifts in bacterial systematics and the general uncertainties involved, this information was judged to be of little additional value and was omitted. Differing isolation techniques and research goals are bound to have skewed the type and range of organisms reported by the various authors, but the number of citations for each genus serves to differentiate the abundant and presumably autochthonous organisms from the rare and/or allochthonous ones. Based on Table I11 and on additional information in the cited papers, the most prevalent hydrocarbondegrading bacteria in aquatic environments are Pseudomonus, Achromobacter, Arthrobacter, Micrococcus, Nocardia, Vibrio, Acinetobacter, Brevibacterium, Curynebacterium, and Flavobacterium. In case of the fungi, the scarcity of the reports render generalizations very tenuous, but Candida, Rhodotorula, Spurobolomyces, and Cladosporium resinae appear to be the most frequently encountered and most active forms. The relative importance of bacteria versus fungi in aquatic hydrocarbon degradation is a matter of controversy. In agitated enrichments bacteria tend to take over, but some investigators feel that fungal forms can be significant or even predominant in undisturbed surface slicks. Hydrocarbon degradation by eukaryotic organisms other than fungi is a largely unexplored area. The achlorophyllous algae Prototheca h ydrocarbonea and Prototheca zopfii were recently reported to degrade hydrocarbons. Scenedesmus strains utilized n-heptadecane mixotrophically, but were unable to grow on this substrate in the dark (Masters and Zajic, 1971). The numbers of oil degraders in a natural body of water is chiefly determined by its pollution history. In polluted sediments hydrocarbon degraders reached lOe CFU/ml (Zobell and Prokop, 1966), in polluted seawater up to 106 CFU/ml were reported (Polyakova, 1962). Comparison of data from various investigators is difficult because of the widely varying enumeration techniques. Walker and Colwell (197613) undertook to optimize and standardize the counting techniques for hydrocarbon-degrading microorganisms. They obtained the highest counts using a silica gel medium with 0.5% oil and 0.003% phenol red. The selective isolation of oil-degrading bacteria was promoted by addition of fungizone, the isolation of yeasts and

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R. BARTHA A N D R. M. ATLAS

TABLE I11 ISOLATED HYDROCARBON-DEGRADING MICROORGANISMS ENVIRONMENTS~ Bacteria Achromobacter Acineto bacter Actinomyces Aeromonas Alcaligenes Arthrobacter Bacillus Bacterium Beneckea Brevi bacterium Corynebacterium Flavobacterium Mierococcus Mieromonospwa M ycobacteriuni Nocardia Proactinomyces Pseudobacterium Pseudomonm Sarcina Spirillum Vibrio

References 3, 5, 8, 9, 12 3, 13, 14 15 13 3 5, 11, 12, 14 5, 8 8, 10

5 2, 3, 5 5, 12, 13 2, 5, 9 5, 8, 10, 14 15

10, 15 5, 9, 13, 14 15 8, 9 2, 5, 8-10, 13-15

FROM

Fungi Aspergillus Aureo basidium Candida Cephalospwium Cladosporium Cunninghamella Hansenula Penicillium Rhodosporidium Rhodotorula Saccharomyces Sporobolomyces Torulopsis Trichosporon

Algae Prototheca

AQUATIC

References 4, 13 13 1, 7, 13 4 I, 13 4 13 4, 13 1

I, 13 1

1, 13 7 1

6, 13

8, 10 8 5, 8, 9, 13

aReference (14)describes isolates from a freshwater lake; all other references pertain to marine or brakish environments. Isolations by one research group are covered under a single reference number, even if reported in separate papers. Key to references: (1)Ahearn and Meyers, 1971; Ahearn et al., 1971; ( 2 )Atlas and Bartha, 1972a; Dean-Raymond and Bartha, 1975; (3)Byrom et al., 1970; (4) Cerniglia and Perry, 1973; (5) Cundell and Traxler, 1973a,b; (6) Kockova-Kratochvilovaand Havelkova, 1974; (7)LePetit et a l . , 1970; Mironov, 1970; (9) Mulkins-Phillips and Stewart, 1974; (10)Polyakova,1962;(11)Reisfeld et a l . , 1972; (12) Soli and Bens, 1972; (13)Walker and Colwell, 1974a; Walker et a l . , 1975a,b,c; (14) Ward and Brock, personal communication; (15) ZoBell et a l . , 1943.

other fungi by the addition of streptomycin and tetracycline. The authors found meaningful correlations of benzene-extractable material in sediments with the percentage of oil degraders in the total microbial population, but not necessarily with the absolute numbers of the oil degraders. In freshwater ecosystems, Cooney and Summers (1976) found that only 0.1% of the total heterotrophic population of bacteria or yeast and fungi could grow on hydrocarbons as sole carbon source. There was a correlation

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between numbers of hydrocarbon degrading bacteria and fungi which the authors interpreted as indicating that both groups were important in hydrocarbon polluted ecosystems.

2. Ability to Emulsijy Oil In a two-phase liquid medium where the bulk of the carbon and energy source is water insoluble and all other mineral nutrients are dissolved in the water phase, microbial growth typically occurs at the interface of the two liquids. Within the geometric constraints imposed by the environment and the relative amounts of the two liquids, the interfacial tension works to reduce this interface to a minimal area. An ability by the microorganisms to lower the interfacial tension will increase the interface and thus the accessibility of the hydrocarbon substrate. It is a general observation that microbial growth reduces the original surface tension of culture media (LaRiviere, 1955a), as well as their interfacial tension measured against oil. This phenomenon, apparently due to incidental leakage of fatty acids and other metabolic intermediates, is of little conceivable benefit to a microorganism that metabolizes dissolved carbon sources. For a hydrocarbon-utilizingmicroorganism, however, an ability to lower the interfacial tension and thus to emulslfy the oil is an important selective advantage that keeps providing new habitat space as the microbes are multiplying. Common emulsifying agents are the long-chain fatty acids, derived from the partial biodegradation of parent hydrocarbons. In experiments by Reisfeld et al. (1972) an Arthrobacter strain designated as RAG 1 proved to be a highly efficient emulsifier when growing on crude oil, and the low cell yield coupled with extensive decrease of benzene-extractable hydrocarbons suggests the copious production of degradation intermediates, most likely fatty acids. The role of fatty acids and other biogenic dispersants has been well established in fermentation studies that used various alkanes as substrates (Iguchi et al., 1969; Hisatsuka et al., 1971; Whitworth et al., 1975). The microbial dispersion of petroleum products by microorganisms was extensively studied by Zajic and co-workers (Zajic and Knetting, 1972; Zajic and Suplisson, 1972; Zajic et al., 1974). High molecular weight extracellular polymers that were anthrone positive and were precipitated by 95% ethanol were produced by several pseudomonads and by Curynebacterium hydrocarboclastus. The polymers were found to be effective dispersants also in the absence of the bacteria, and acted also as flocculants (Zajic and Knetting, 1971; Knetting and Zajic, 1972). The majority of the workers in the oil biodegradation field appear to agree that hydrocarbons can be taken up by the microbial cell through direct contact with the hydrocarbon phase, and dispersion increases the surface for such contact. Others argue, partly on the basis of kinetic models, that only

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dmolved hydrocarbons are utilized (Valenkar et al., 1975;,Chackravartyet a l . , 1975). Nevertheless, these authors also assign a key role to microbial dispersants as promoters of hydrocarbon dissolution. A continued lively interest in microbial oil disperants is motivated in part by a search for low-toxicity surfactants for cleanup of oil with a minimum of environmental damage (Reisfeld et al., 1972; Zajic and Knetting, 1972). In addition, the current energy shortage has rekindled interest in earlier suggestions (LaRiviere, 1955b) for the use of microbial dispersants in the tertiary recovery of petroleum (Engineering Foundation Workshop, “The Role of Microorganisms in the Recovery of Oil,” November 9-14, 1975, Easton, Maryland).

3. Oil Biodegradation Potential The fact that hydrocarbon-degrading microorganisms are isolated with great regularity from oil-polluted aquatic environments is strongly suggestive of their significance in the self-purification process of these environments. The enumeration of hydrocarbon oxidizers and their densities in polluted and nonpolluted environments were discussed in a foregoing section (III,A,l). At a given density of the oil-degrading microorganisms, their actual contribution to the elimination of oil depends on their inherent metabolic capability, i.e., “heterotrophic potential,” and the degree to which environmental conditions allow this potential to be expressed. Of course, heterotrophic potential cannot be measured independently from environmental conditions. These are either selected arbitrarily, are kept constant for comparison of various environmental samples, or are designed to approximatein situ conditions as closely as practicable. Ideally, the results in the latter case should indicate at what initial rate the introduced substrates (e.g., hydrocarbons) will be biodegraded in the environment under study. Experiments aimed at single cell protein production demonstrated that under favorable conditions selected yeasts and bacteria metabolize n-paraffin mixtures at high rates. Kanazawa (1975)reported production of 64-78 kg dry yeast/m3/day, Coty and Leavitt (1971) obtained 31 kg of a Brevibacteriuml m3/day on n-alkanes. Conversion efficiencies being in both cases close to loo%, these values indicate the degradation of corresponding amounts of hydrocarbons. Unfortunately, the performance of these high-density monocultures grown under ideal conditions is unlikely to be even approached in natural environments. To obtain a reliable estimate for an in situ oil biodegradation potential is Wicult for the following reasons: (1)Oil biodegradation is a relatively slow process, and during the time a measurable amount of oil is consumed, the original potential has shifted due to enrichment. (2) Linearity is further distorted by lag phases due to toxic components (Atlas and Bartha, 1972b)

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24 1

and diauxic phenomena (Pirnik et al., 1974). (3)In situ situations are apparently often mineral nutrient-limited (Atlas and Bartha, 1972~).(4) Careful containment measures tend to alter in situ biodegradation rates, while a lack of such containment leads to erroneous results due to a loss of oil by uncontrolled mechanisms (dissolution, sinking, emulsification, and evaporation). Most oil biodegradation studies performed to date suffer from some or all of these deficiencies and have only limited value in calculation of the in situ biodegradation potentials. Based on metabolic rates measured under laboratory conditions and on the numbers of such oil degraders in some marine environments ZoBell (1964) estimated that polluting oil in the sea might be biodegraded at rates as high as 100-960 mg/m3/day. By some experts, this estimate was regarded to be overoptimistic (National Academy of Sciences, 1975). In experiments not designed to simulate in situ conditions, South Louisiana crude oil was degraded in moderately polluted coastal sea water samples at 2000 gm/m3/day. This approximate rate was calculated from results (Dibble and Bartha, 1976)of a 3-day incubation period at 28°C. Mineral nutrients were added and the flasks were agitated. A relatively unpolluted coastal seawater sample under similar conditions degraded only 500 gm/m3/ day. These results are representative of the near-maximal performance of naturally occurring marine populations under artificial conditions and in no way reflect the in situ biodegradation rates. Since the work of Parsons and Strickland (1962), the most widely accepted technique for determination of heterotrophic potential involves the use of a radiolabeled substrate. This approach was applied to hydrocarbon biodegradation by Caparello and LaRock (1975) using [14Cl]hexadecane.From their data n-alkane biodegradation rates of 2500 gm/m3/day to 25 gm/m3/day can be calculated for a polluted inshore and a relatively unpolluted offshore water sample, respectively. Their experiments also demonstrated that the rate of n-hexadecane biodegradation is representative of the biodegradation rate of n-alkanes in the Clo-C25range, and that the lag periods preceding measurable biodegradation can be used for estimation of the population size. However, these experiments were not designed to measure in situ rates, since the collected samples were diluted in a mineral salts medium and were incubated at the rather high temperature of 35°C. Lee and Ryan (1976) also utilized 14C-labeledhydrocarbons as substrates to study petroleum biodegradation. Their results indicated that microbes are important in degrading petroleum hydrocarbons in ocean waters. Polynuclear aromatic hydrocarbons were degraded at much lower rates than monoand dinuclear aromatics and n-paraffins. Turnover times of the order of months were found for paraffins and light aromatics and of the order of several years for polynuclear aromatics. Degradation rates were high in spring and low in winter.

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Atlas and Bartha (1973d) floated miniature slicks of Sweden (Texas) crude oil in flow-through seawater tanks. The slicks were contained within vertical glass columns. Some of these were open at their bottom, but others were closed with dialysis membranes, thus eliminating the possibility of escape for undegraded oil. Rate calculations were complicated by an initial toxicity of the oil that caused a 2-week lag period. However, from day 14 to 42, biodegradation was linear at the approximate rate of 10 gm/m3/day.For comparison purposes in this calculation, oil biodegradation was averaged for the total water volume of the columns (15cm deep, 300 ml), though, in a nonagitated system, it may be more meaningfbl to calculate biodegradation per surface area rather than per volume. The average seawater temperature during this experiment was 18"C, and biodegradation was not temperature, but nitrogen and phosphorus-limited. The long-term nature of this study renders this an in situ enrichment situation, and it is clear that initial biodegradation rates should be substantially slower. Walker and Colwell (1976a) used [14C]hexadecanein short-term (1hour) in situ experiments. Water temperature was 24.5"C. They obtained V, values for carbon mineralization at two coastal locations of 0.3 and 1.0 pg of C per liter per hour, respectively. Assuming that 50% of the degraded hydrocarbon was converted to COB, these rates translate to approximately 0.015 and 0.050 gm of hydrocarbon degraded per 1m3 per day, respectively. These figures appear to be the best currently available experimental data for a moderately polluted coastal environment at summer temperatures. In good agreement with the above figures, Seki (1976) measured up to 0.015 gm of radiolabeled n-hexadecane biodegradation in polluted Tokyo Bay at summer temperatures. In the much

THE O R D E R

OF

TABLE IV MAGNITUDEOF HYDROCARBON BIODEGRADATION RATES

System High-density monocultures in fermentors, optimal conditions Seawater samples under artificially optimized conditions I n situ marine enrichment I n situ marine potential

Order of magnitude (gm/m3/day)

Referencesa

10,000-100,000

3, 5

25-2500

2, 4

1-10 0.001-0.05

1 6-8

aThe cited references have served as a basis for our calculations, but the above-listed figures do not appear in the original papers. Key to references: (1) Atlas and Bartha, 1973d; (2) Caparello and LaRock, 1975; (3) Coty and Leavitt, 1971; (4) Dibble and Bartha, 1976; (5) Kanazawa, 1975; (6) Robertson et uZ., 1973; (7) Seki, 1976; (8) Walker and Colwell, 1976a.

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colder waters of Port Valdez, Alaska, Robertson et al. (1973) measured by essentially the same technique an in situ hydrocarbon biodegradation rate of 0.001 gm/m3/day. The same authors inferred a rate even lower than this in the open waters of the Arctic Ocean. Table IV lists the orders of magnitude of hydrocarbon biodegradation in various systems. It should be recognized that these values pertain to biodegradable n-alkanes. Recalcitrant and/or toxic hydrocarbons represent special situations not covered in Table IV. 4 . Substrate Range As microbial growth or the lack of it can be detected without advanced instrumentations, the early periods of hydrocarbon microbiology are prolific in substrate range studies. Both the early and the more contemporary work in this area has been repeatedly and extensively reviewed (ZoBell, 1946, 1950; Beerstecher, 1954; Fuhs, 1961;Foster, 1962; Gibson, 1968; Nyns and Wiaux, 1969; Evans, 1969; Kallio, 1969; Einsele and Fiechter, 1971; Markowetz, 1971; McKenna, 1961;Friede et al., 1972; Atlas and Bartha, 1973c; National Academy of Sciences, 1975). In the more recent reviews, information pertaining to substrate ranges is intimately interwoven with the discussion of metabolic pathways. Rather than review here the metabolic pathways of hydrocarbon biodegradation, we wish to present here only some concise and, by necessity, simplified, conclusions as they relate to the relative availability of the hydrocarbon components of an oil spill. The following generalizations are based on the above-listed reviews. (1) n-Alkanes are the most widely and readily utilized hydrocarbons. In terms of chain length, n-alkanes between Clo and CZsare most suitable as substrates for microorganisms. (2) Iso-alkanes are generally inferior to n-alkanes as growth substrates, especially if the branching is extensive or creates quaternary carbon atoms. (3) Olefins are more toxic and are less readily utilized than corresponding alkanes. (4) Low-molecular-weight aromatic hydrocarbons are quite toxic to microorganisms, but can be metabolized when present in low concentrations. Condensed polynuclear hydrocarbons are less toxic to microorganisms, but are metabolized only rarely and at slow rates. (5) Cycloalkanes are highly toxic and serve as growth substrates for isolated organisms only in exceptional cases. Some are readily degraded, however, by the cometabolic attack of mixed microbial communities. The above generalizations are based on work with organisms from culture collections, or from isolations where the substrate and procedure was often quite selective. Nevertheless, these conclusions are supported by a nutritional classification study of Soli and Bens (1973). From seawater agar plates incubated with a mixture of 28 hydrocarbons including normal- and iso-

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alkanes, aromatic and alicyclic hydrocarbons, these authors picked colonies randomly and subsequently tested their growth on the individual hydrocarbon components. The majority of the tested strains grew on normal alkanes only. Some isolates were able to use compounds from several or all tested hydrocarbon classes. Only rarely did an isolate utilize aromatic or cycloparaffinic compounds in preference to n-alkanes. Fredericks (1966) found that bacteria isolated on a relatively recalcitrant hydrocarbon such as cyclohexane could be induced to grow on the less recalcitrant n- and iso-alkanes, but the reverse was not observed. Walker et al. (1975e)found that in an oil-polluted harbor, bacteria from the water column rather than from the sediment had the greater capability to degrade polycyclic cycloalkanes. The practical consequences of the above-discussed substrate preferences are two-fold: (1)the rate and extent of the biodegradability of an oil spill will depend on the relative proportions of susceptible and recalcitrant hydrocarbons in the spilled oil; and (2) biodegradation will alter the composition of the spilled oil by decreasing the relative abundance of the more susceptible hydrocarbons as compared to the recalcitrant ones.

5. Diauxic Effects and Sequential Biodegradation When a microorganism with a broad substrate range is offered more than one type of organic substrate, it is likely that it will not attack the substrates simultaneously, but rather in a definite sequence. Diauxie, a phenomenon of metabolic regulation first described by Monod (1942) may determine whether or not the hydrocarbon components of an oil spill are degraded, and if so, in what order. The presence of nonhydrocarbon substrates may repress the inductive synthesis of enzymes required for hydrocarbon oxidation (van Eyk and Bartels, 1968).Ward and Brock (1976~) reported that the addition of glucose to lake water repressed hexadecane utilization by its microbial community in a diauxic manner. Pirnik et aZ. (1974) described a Brevibacterium erythrogenes strain capable of utilizing pristane and other branched alkanes only in the absence of n-alkanes. B. erythrogenes utilizes n-alkanes by a monoterminal P-oxidation sequence, but degrades iso-alkanes by diterminal oxidation. The two pathways are, apparently, catalyzed by the differing sets of enzymes, and the operation of the monoterminal pathway apparently shuts down the synthesis of enzymes required for the diterminal oxidation pathway. The commonly observed phenomenon that the n-alkane components of an oil spill disappear before the iso-alkanes and other hydrocarbon classes show substantial biodegradative change strongly suggests that such diauxic regulatory mechanisms apply not only to some pure cultures, but most likely also to the mixed microbial community of the environment. In addition to diauxie, the sequence of hydrocarbon degradation in an oil spill is likely to be determined by the ecological succession of the degrading

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microorganisms. n-Alkane degraders with rapid growth rates are likely to out-compete the slow-growing decomposers of the more recalcitrant hydrocarbons for scarce nutritional resources such as oxygen, nitrogen, phosphorus, etc. As the n-alkanes are depleted and most of their decomposers lack the metabolic ability to switch to the more recalcitrant hydrocarbons (Fredericks, 1966) they die off and are replaced by organisms with slower growth rate but greater metabolic flexibility. Some experimental evidence for such a succession was presented by Horowitz et al. (1975).These workers used crude oil, depleted by one microbial strain for the enrichment and isolation of the next. When four isolated hydrocarbon degraders were simultaneously inoculated into fresh crude oil, and the numbers of each population were monitored on differential counting media, successional changes in population sizes were evident. Gas chromatographic measurement of the crude oil degradation pattern by each strain indicated that the two strains that reached high population sizes initially were degrading n-alkanes, whereas the two strains that reached high population sizes after the decline of the former ones degraded mainly the more recalcitrant and unresolved components of the “envelope.” 6 . Product Inhibition The ultimate products of hydrocarbon biodegradation are carbon dioxide and water, but the biodegradation of higher molecular weight hydrocarbons involves many intermediates, some of which may accumulate to inhibitory levels. Aida and Yamaguchi (1969) found a dialyzable “growth-inhibitory factor” to be produced in yeast cultures growing on n-hexadecane, and lauric acid proved to be the principal inhibitory agent. Lauric acid proved to be inhibitory only for growth on alkanes but did not affect growth on glucose. Atlas and Bartha (1973b,c) found that biodegradation of a crude oil by a marine Breuibacterium and Flavobacterium strain was inhibited by a variety of accumulating fatty acids. In most cases, the presence of hydrocarbons was necessary for the manifestation of the inhibition, but the effect went beyond the mere repression of hydrocarbon oxidation, since alternative carbon sources did not relieve the inhibition. The biodegradation of aromatic hydrocarbons yields phenolics and benzoic acid intermediates. The accumulation of salicylic derivatives limited the yields of marine Pseudomonas strains utilizing naphthalene and methylnaphthalenes (D. D. Raymond and R. Bartha, unpublished). The significance of these product inhibitions, observed in pure cultures growing in a limited liquid volume, is dubious when projected to the biodegradation of oil slicks. Under ordinary circumstances, the presence of a mixed microbial community and larger amounts of water is expected to prevent the accumulation of metabolic intermediates to inhibitory levels. In tar globules and in water-

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in-oil emulsions, however, the trapped degradation intermediates may well contribute to the resistance of these residues to further microbial attack. An intriguing phenomenon is the appearance of new long-chain n-alkanes (“waxes”)during biodegradation of at least some crude oils (R. Kallio, personal communication; Walker and Colwell, 1976~). These waxes, either not present in the original crude oil, or only at lower concentrations, appear as a result of microbial activity but not as a consequence of weathering. Such “reverse biodegradation” appears paradoxical but is not without precedent. Reactive biodegradation intermediates, e.g., free radicals may undergo spontaneous head-to-head condensation reactions, resulting in very long n-alkane chains that are largely immune to further enzymic attack. Analogous synthetic reactions were reported in the microbial transformation of some herbicides (Bartha and Pramer, 1967). The mechanism of the latter reaction has been experimentally documented (Bordeleau et al., 1972) but is, at this time, entirely speculative for long-chain n-alkane formation. A clear need exists for further experimental work in this area. The long-chain n-alkanes (C25-C45)are not likely to inhibit microbial activity directly, but they contribute to the formation of tar balls which, owing to their physical properties, resist subsequent biodegradation.

B. ENVIRONMENTAL CONSTRAINTS 1 . The Nature of the Spilled Oil

A foregoing section (III,A,4) on substrate specificity has already touched upon oil composition and the suitability of the various hydrocarbon classes as microbial substrates. The presence of inhibitory substances in oil can, however, delay or prevent the biodegradation of otherwise suitable hydrocarbon substrates. Crude oils are mixtures of tremendous complexity, containing hundreds of hydrocarbon and nonhydrocarbon components, many of them still unidentified (Atlas and Bartha, 1973~).That some of these compounds have considerable toxicity toward microorganisms is illustrated by their historical and contemporary uses: tar and creosote are widely used in the preservation of wood, rope, sailcloth, etc., against fungal attack. Surgical quality petroleum ether is used in some countries as an alternative to ethanol for skin disinfection prior to minor surgery. The prime mode of action of the low molecular weight hydrocarbons is the solvation and hence destruction of the lipid-containing pericellular and intracellular membrane structures. Liquid hydrocarbons of the n-alkane, isoalkane, cycloalkane, and aromatic type with carbon numbers under ten all share this property to varying degrees. The mode of action of other toxic hydrocarbon and nonhydrocarbon components of petroleum is too complex and varied to be discussed here effectively. Some fresh crude oils have

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definite bacteriostatic properties that decrease or disappear during “weathering” (Atlas and Bartha, 1972b; Atlas, 1975). It was demonstrated that the bacteriostatic agents are volatile and their disappearance is mainly a function of the water temperature. In warm water the presence of such volatile bacteriostatic agents in an oil spill has little practical effect on biodegradation, since they evaporate within hours. In cold waters, however, they may delay the start of biodegradation for several weeks. Aromatic hydrocarbons are more polar and hence more water soluble than their saturated counterparts. The relative proportion of nonvolatile aromatics in the oil slick may be reduced by dissolution (Blumer et al., 1973). Brown and Huffman (1976) reported a proportion of aromatic hydrocarbons in weathered oil from open ocean waters that was considerably lower than in the suspected parent crudes. The cause for this finding is a matter of speculation (the authors invoke dilution by biogenic alkanes), but a greater solubility of aromatics could contribute to the observed phenomenon. Since aromatics are generally regarded as being more toxic than corresponding normul- and iso-alkanes, this aging process may be regarded as conducive to subsequent microbial activity. Not all weathering changes do necessarily promote biodegradation. Definitive studies in this area are unfortunately lacking, but it is likely that the free-radical intermediates and some of the oxygenated products of hydrocarbon photooxidation are harmful to oil-degrading microorganisms and, along with other physical and chemical factors, contribute to the recalcitrance of tar residues. The fact that floating oil is able to concentrate pollutants with hydrophobic character may also influence its biodegradability. Pesticides (Seba and Corcoran, 1969; Hartung and Klinger, 1970), polychlorinated biphenyls (Sayler and Colwell, 1976), and mercury (Walker and Colwell, 1976d; Sayler and Colwell, 1976)were shown to be concentrated in oil from several hundred to several hundred thousandfold of their ambient concentrations in water. Such toxic contaminants are likely to interfere with microbial degradation and will increase the persistence of oil, though microorganisms on occasion may adapt to such contaminants as long as their concentration stays within reasonable limits. Walker and Colwell(1974b)demonstrated mercury-resistant populations of oil-degrading bacteria in the polluted Colgate Creek area. Petroleum biodegradation proceeded when mercury in the oil was present in the low ppm concentration range, but was absent at 85 ppm (Walker and Colwell, 1976d).

2 . Temperature Temperature has a profound influence on the rate of all biochemical processes, and a€fects the biodegradation of hydrocarbons directly as well as in indirect ways. Biodegradation of hydrocarbons was reported to take place

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under temperature conditions that range from thermophilic to psychrophilic. Klug and Markowetz (1967) and Mateles et al. (1967) reported bacterial growth on alkanes up to 70°C. In thermophilic situations metabolic rates tend to be high but growth rates and cell yields are low, since much energy is expended for repair of thermal damage to cell constituents (Campbelland Pace, 1968).The results of Sukatch and Johnson (1972)on cell yields from n-hexadecane in the mesophilic versus thermophilic temperature range are fully consistent with this general picture. The significance of thermophilic hydrocarbon degraders in aquatic environments is low and is probably restricted to the thermal effluents of industrial and power plants. Current plans for exploitation of geothermal energy may create intriguing future environments for the study of thermophilic oil degraders. Recent developments in production and transport of petroleum in the Arctic Ocean stimulated a great deal of interest in psychrophilic hydrocarbon degraders. Ninety percent of the oceanic water mass has a temperature of 4°C or below (Morita, 1966), and even the surface water temperatures during the temperate zone winter season may select for psychrophilic microorganisms. ZoBell and Agosti (1972) reported growth and oxygen consumption by bacteria on mineral oil at temperatures as low as -1°C. The organisms also grew at 4", 8", and 25°C and thus represented facultative psychrophiles. Oxygen consumption approximately doubled in each case when the incubation temperature was raised from 4" to 8" and then from 8" to 25°C. Walker and Colwell (1974a) exposed a "model petroleum" consisting mainly of n-parains (86%)but also containing some branched, aromatic and alicylcic hydrocarbons to biodegradation in winter-collected enriched seawater at 0.5 and 10°C. Predictably, the stationary growth phase was reached more slowly at the colder temperatures but, paradoxically, hydrocarbon biodegradation was more extensive at 0°C than at the higher temperatures. Decreased toxicity of some hydrocarbon components at lower temperatures was proposed as a possible explanation. Only bacteria were active at 0" and 5"C, but at 10°C the participation of yeasts and filamentous fungi was noted. Atlas and Bartha (1972b) measured the mineralization and biodegradation of Sweden (Texas) crude oil in summer- and winter-collected enriched seawater samples at 5", lo", 15", and 20°C. No mineralization was observed in 60 days in summer-collected seawater at 5"C, but some mineralization did occur in winter-collected seawater, suggesting a population shift to psychrophilic forms during the winter. Although the mineralization curves are complicated by initial lag periods, slopes of best fit drawn through the linear phase of mineralization indicate that the rates roughly doubled with each 5°C temperature increase in the 5" to 20°C range. The lag periods were shown to be caused by inhibitory volatile components of Sweden crude oil that were lost increasingly slowly at decreasing water temperatures. Atlas (1975) ex-

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tended these observations to 6 additional crude oils and found that “light” crude oils having a higher proportion of low-molecular-weight volatile components are more completely biodegraded than “heavy” crudes, but at low temperatures the volatile components of light crudes cause long lag periods while heavy crudes are attacked without such delay. Temperate zone lakes have in general more extreme water temperature variations than the oceans in the same climatic region. Ward and Brock (1976a) studying limiting factors of oil biodegradation in temperate lakes of Wisconsin found water temperature to be the predominant limiting factor throughout the fall, winter, and spring, and only during the summer did nutrient limitation take precedence. 3 . Pressure

Polluting petroleum through gradual oxidation and incorporation of sediment particles may exceed the density of water and sink to the deep sea floor. The recent realization that high hydrostatic pressures in combination with low temperatures drastically reduce the rate of microbial metabolism (Jannasch and Wirsen, 1973)stimulated interest in hydrocarbon biodegradation under high pressure conditions. Schwarz et al. (1974a,b, 1975) obtained an enrichment culture from a sediment core taken at 4940 m depth and exposed to it radiolabeled n-tetradecane and n-hexadecane under both a m bient and in situ pressure and temperature conditions. At 20” and 25”C, 500 atm pressure delayed hydrocarbon biodegradation by the enrichment culture only moderately, but at 4°C the same pressure reduced biodegradation rate as compared to the atmospheric control by more than an order of magnitude. Experiments of this type have many technical limitations, e.g., it is not possible currently to retrieve deep sea samples without decompression and drastic temperature change. In consequence, obligate psychrophiles and barophiles that may be dominant in deep sea sediments are certain to be lost. Nevertheless, the experience with other organic substrates that were actually exposed on the deep sea floor (Jannasch and Wirsen, 1973) supports Schwarz et al. (1975) in their conclusion that petroleum degradation in the deep sea environment will be extremely slow. 4 . Oxygen

Hydrocarbons are completely reduced organic substrates that can be metabolized only in an oxidative manner. Anaerobic oxidation with nitrate or sulfate serving as electron sinks has been demonstrated in the laboratory, but experience has shown that from the practical point of view this type of biodegradation has a negligible effect on oil pollutants (Davis, 1967; Friede et al., 1972). Therefore, the availability of molecular oxygen is considered to be an important limiting parameter of hydrocarbon biodegradation.

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Limitation by oxygen deficiency is least likely to occur in case of thin, floating oil slicks, but the interior of floating water-in-oil emulsions (“chocolate mousse”) and tar globules of substantial size may well become anaerobic. When emulsified oil becomes dispersed in the water column, the high 0, demand of hydrocarbons ( 3 4 gm per gram of hydrocarbon) and the low solubility of oxygen (6-11 mg per liter of seawater, depending on temperature) combine to render O2 supply critical. Whether or not oxygen actually becomes the main limiting factor depends on a number of circumstances. In general, easily degradable hydrocarbons, high water temperatures (which increase metabolic rates and decrease O2 solubility), poor water circulation, additional carbon sources, and availability of mineral nutrients will all work toward making oxygen the main biodegradation-limitingfactor, whereas contrary conditions are likely to cause a shift to some other limiting factor. Extreme conditions of oxygen limitation evolve in stratified bodies of water where the hypolimnion becomes permanently or temporarily anaerobic. For example, such conditions exist permanently in meromictic lakes, in the Black Sea, and in some holomictic lakes during the summer stagnation period. Ward and Brock (unpublished) studied the biodegradation of radiolabeled n-hexadecane in the anaerobic hypolimnion of a Wisconsin lake. No significant biodegradation was recorded even with this highly sensitive technique as long as anaerobic conditions were maintained. Addition of sulfate or nitrate as electron acceptors did not alter the result. Hydrocarbon degraders were present, however, and degradation occurred if oxygen was permitted to enter the system. Calculation of free energy balances indicated that hexadecane oxidation coupled with sulfate reduction is probably not sufficiently exergonic to support microbial metabolism. With nitrate as electron acceptor the theoretical energy balance is more favorable, but nitrate is rapidly depleted in anaerobic environments by competing denitdcation processes. In aquatic sediments water movement is restricted by the small size of pore spaces and heterotrophic activity generally renders all but the surface layer anaerobic. In such environments oil pollutants are expected to persist for very long periods of time. Actual monitoring of oil-contaminated marine sediments confirm this expectation (Blumer and Sass, 1972). 5. Mineral Nutrients

Refined hydrocarbons supply only carbon and energy to degrading microorganisms, but crude petroleum contains in small amounts some other nutrient elements, e.g., sulfur, nitrogen, and heavy metals. With the possible exception of sulfur from thiol compounds, these elements are firmly bound in heterocyclic rings (e.g., thiophene for sulfur, pyridine and pyrrole compounds for nitrogen) or organometallic complexes, respectively, and are

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25 1

considered unavailable to most hydrocarbon degrading bacteria. Consequently, we have to look to the environment in which the biodegradation takes place to provide the other required nutrients. Some of these, e.g., nitrogen and phosphorus, are notoriously limiting in natural environments owing to their general scarcity and the competition for them by photosynthetic organisms. Therefore, the failure of an environment to provide adequate amounts of nitrogen, phosphorus and perhaps some other mineral nutrients can severely limit the biodegradation of polluting oil. Early observations on the need to supplement environmental samples with nitrogen and phosphorus salts were reviewed previously (Atlas and Bartha, 1973c), and here we wish to summarize only the work of the last 5 years (1971-1976). Bridie and Bos (1971) studied the biodegradation of topped Kuwait crude oil in freshly collected water samples from the North Sea. Typical nitrogen and phosphorus concentrations for these water samples in the winter season were quoted as 150 and 20 pg/liter, respectively. At 70 mditer oil addition, 3.2 mg of nitrogen (as ammonium) and 0.6 mg of phosphorus (as phosphate) allowed maximal biodegradation rates. At 30"C, the theoretical oxygen demand (TOD) of the oil was reduced by 34%in 10 days. Without supplementation, reduction was 4% in the same time period. Atlas and Bartha (1972~) measured the effect of nitrogen and phosphorus supplements on Sweden (Texas) crude oil in New Jersey coastal seawater that had natural nitrogen and phosphorus concentrations of 980 and 70 pg/liter, respectively. The crude oil was added at 10 ml(8 gm) per liter and incubation temperature was 28°C. Addition of 140 mg of nitrogen and 11 mg of phosphorus per liter was necessary to obtain optimal biodegradation (70% of the oil biodegraded, 42% mineralized in 18 days). Without mineral supplements, biodegradation reached only 3%. Reisfeld et al. (1972) working with Mediterranean seawater of undetermined nitrogen and phosphorus content found 10.6 mg of nitrogen and 1.8 mg of phosphorus supplement per liter optimal for biodegradation of up to 1 gm per liter of Iranian crude oil. In this case not the natural population of the seawater, but a selected Arthrobacter strain served as inoculum. In 4 days at 32°C about 65% of the oil was converted to material not extractable by benzene. Gibbs (1975) measured the biodegradation of topped Kuwait crude oil in seawater at 14°C in an apparatus allowing continuous addition of nutrients and the monitoring of O2 consumption. Under his experimental conditions nitrogen was the most limiting nutrient. Four micrograms of nitrogen were consumed during the complete oxidation of 1 mg oil. The above studies all pertain to seawater, but the situation seems rather similar in oligotrophic freshwater lakes. Ward and Brock (1976b)reported up to 20-fold increase in mineral oil oxidation upon supplementation of the

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water by 300 p g of nitrogen and 100 p g of phosphorus per liter. In this case phosphorus rather than nitrogen limitation was more acute. An imbalance of organic carbon versus available combined nitrogen often encourages nitrogen fixation activity. Coty (1967) explored the possibility that this occurs also in cases of hydrocarbon utilization in media that are deficient in combined nitrogen. On methane and butane, Pseudomonus and Mycobacterium strains were isolated that were able to fix molecular nitrogen. Several Azotobacter strains were isolated that utilized tetradecane, toluene and naphthenic acids while fixing atmospheric nitrogen. In 2 weeks at 30°C, 20-130 p g of nitrogen per milliliter was fixed by the various cultures. The intriguing possibility that the biodegradation of polluting oil in nitrogen-deficient natural environments is aided by nitrogen fixation remains to be explored, but the strong response to nitrogen supplements indicates that this process alone does not overcome the rate limitation of oil metabolism by nitrogen. Sulfur, the remaining major nutrient element is plentiful in seawater in the form of sulfate. Whether the availability of this element might limit oil biodegradation in fresh water, especially in case of lowsulfur oils and nitrogen plus phosphorus enrichment, is not presently known, but seems to be a definite possibility. From the minor elements, Dibble and Bartha (1976) considered iron as potentially limiting for petroleum biodegradation in nitrogen- and phosphorus-supplemented seawater. Such limitation was in fact demonstrated in relatively clean but not in polluted coastal seawater samples. By projection, iron limitation should be even stronger in pelagic seawater. Hydrocarbon-degrading microorganisms typically grow at the oil-water interface, but Severance and LaRock (1973) recently described a Breuibacterium strain which, in agitated oil-water culture, leaves the water clear while reaching counts of 108 CFU/ml in the oil phase. It is not clear at this time whether the Breuibacterium in fact grows in the oil phase or is merely partitioned into it, since migration into the oil does not take place in quiescent culture. If indeed growth occurs within the oil phase, perhaps with the aid of metabolically formed water, the organism growing in this manner would be in especially poor competitive position to obtain mineral nutrients. In conclusion, biodegradation of oil in most aquatic environments is definitely limited by the scarcity of mineral nutrients. Since the hydrocarbon degrading microorganisms is generally compelled to maintain physical contact with the oil droplet, it is in relatively disadvantageous position to comPete for the scarce mineral nutrients with photoautotrophs and with degraders of dissolved organic matter. The situation is bound to worsen when water in oil emulsion (chocolate mousse) is formed or residual oils form tar aggregates. Along with other unfavorable conditions, the unavailability of

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mineral nutrients inside these emulsions or aggregates are certain to contribute to the longevity of these unpleasant residues.

C. INTERPLAY OF PHYSICOCHEMICAL AND BIOCHEMICAL FORCES IN SELF-PURIFICATION OF AQUATIC ENVIRONMENTS As a matter of organization, the environmental and biological factors that influence the persistence of polluting oil were discussed above one by one, but it should be understood that they interact with each other in a complex manner to determine the overall rate and extent of oil degradation. The spreading of an oil spill on the water surface provides an increased surface area for evaporation, dissolution, photooxidation, and biodegradation processes. Wind and wave action will promote evaporation, dissolution and emulsification and, in addition, will help to replenish oxygen used up by biodegradation. The initial evaporative and dissolution changes reduce the toxicity of the slick and create more favorable conditions for biodegradation. Autoxidative changes are likely to be negligible because of the discrepancy between the required activation energies and the moderate to low temperatures of the marine environment (Lefcourt, 1973). On the other hand, photooxidation may contribute to oil degradation significantly. Freegarde et al. (1970) irradiated crude oil in water in flasks swept by COz-free air. The produced CO, was trapped and polar degradation products were recovered from the water. Quantum efficiencies were calcuated and extrapolated to the marine environment. According to these extrapolations, at 8 hours of effective sunshine as much as 0.2 gm of oil/m2/daycan be eliminated by photodegradation. What adds additional importance to photodegradation is the fact that photooxidative attack occurs preferentially at tertiary carbon atoms (e.g., methyl branches) that often represent a hindrance to biodegradation (Pirnik et al., 1974). Extensive weathering and pickup of suspended sediment particles causes an increase in oil density and eventual sinking. Incorporation into generally anaerobic aquatic sediments leads to an increased persistence of the oil. The formation of water-in-oil emulsions and later the aggregation of weathered oil into sizable tar lumps are processes that reduce available surface and thus slow down further degradation.

IV. Microorganisms and Oil Pollution Abatement From the foregoing discussion, microbial degradation emerges as the most significant natural mechanism for the removal of nonvolatile hydrocarbon

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pollutants from the marine environment. Therefore, it is of importance to know how various oil spill cleanup techniques influence this self-purification process. In addition, the possibility of utilizing a properly managed biodegradation process for the cleanup of oil pollutants will be explored.

A. EFFECTSOF

CLEANUP TECHNIQUES OIL BIODEGRADATION

TRADITIONAL

ON

During the past decade the need to deal with oil spills of increasing frequency and magnitude created a whole new field of engineering and technology. The Proceedings of the Conferences on “Prevention and Control of Oil Spills” (1969, 1971, and 1973, American Petroleum Institute, Washington, D.C.) record many of these developments. A good summary of current procedures is presented by Nelson-Smith (1973). The mechanical collection of oil slicks is aided by floating booms. Containment is essential; only a relatively thick layer of oil can be efficiently collected by various skimmers, belt collectors, or vortex devices. Absorbents (straw, plastic foam) are often used to aid the collection process. Essential for such operations are fair weather conditions with low winds, waves, and current velocities. Unfortunately, this set of conditions is rather atypical in marine disasters. Currents in excess of 75 cmlsecond and a wind chop in excess of 15 cm height tends to render floating booms ineffectual (NelsonSmith, 1973). Consequently, the mechanical collection of oil is generally feasible only in protected bays and harbors. Alternative cleanup techniques are less dependent on favorable weather conditions, but they are generally inferior to mechanical collection in terms of environmental damage. In situ combustion is seldom feasible because of safety considerations and the high flash point of the spilled oil that results from even very short ( 2 3 hours) weathering periods. Even in presence of wicking agents, the combustion is incomplete and the products of such incomplete combustion (e.g., benzpyrenes) may increase the toxicity of the residue. Considering the long lag phases of biodegradation caused by the volatile products of some crude oils at cold water temperatures, Atlas and Bartha (1972b) speculated that ignition of such slicks may speed up their subsequent biodegradation. This proposition requires experimental evaluation and an assurance that an increased toxicity to higher marine organisms does not outweigh the benefit of more rapid biodegradation. Dispersion by detergents removes the slick from the water surface and prevents the heavy coating of rocks and beaches. Dispersion is actually aided by rough weather conditions. The emulsified oil can be expected to be subject to more rapid dissolution and microbial degradation due to the increased oil-water interface. Robihaux and Myrick (1972)tested the effect of 6

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dispersants on the oxygen uptake of oil-polluted water and reported positive effects. Atlas and Bartha (1973e) tested 8 detergent formulations recommended for dispersion or “herding” of oil pollutants and found that each one stimulated the microbial degradation of crude oil in laboratory experiments. These advantages are, unfortunately, more than balanced by detrimental effects. Early detergent formulations, especially the solvent-based types, proved to be highly toxic to marine organisms and, in some instances, caused more biological damage than the oil itself (Ludwigson, 1969). By the nature of their action, even the improved, less toxic formulations increase the exposure of organisms in the water column and in the benthic environment to the oil pollutant. The demand of the dispersed oil on dissolved oxygen may lower oxygen concentration to critical levels. Because of these drawbacks, dispersants have to be used selectively and only as a last resort. Sinking of oil with heavy hydrophobic agents (ground chalk, siliconized sand) is a purely cosmetic and ecologically thoroughly unsound practice which increases the exposure of benthic organisms and sequesters the oil in anaerobic sediments where they are largely immune to further biodegradation. If the sinking removes the oil to the deep sea floor rather than to shallow sediments, the combination of low temperature and high hydrostatic pressure (see Section III,B,3) assures the long persistence of the pollutant. The discussion of traditional oil cleanup techniques would not be complete without its economic aspects. The requirement to maintain a large preparedness in terms of labor force, equipment and materials, but use these only on intermittent emergency basis, renders such operations extremely costly. Cleanup expenses were estimated to average $6.50 per gallon of spilled oil (Anonymous, 1973). This translates to roughly $2000 per metric ton, leading in case of major oil spills to multimillion dollar expenses for cleanup alone.

B. STIMULATEDOIL BIODEGRADATION The limitations, side effects, and high expense of traditional cleanup techniques has stimulated interest in unconventional alternatives. The discrepancy between the potential and actual biodegradation rates of hydrocarbons (see Table IV) invites attempts to artificially increase the biodegradation rate of polluting oil in aquatic environments and, in this manner, to utilize microorganisms for the cleanup task. The previously discussed biological and environmental constraints of microbial hydrocarbon degradation (Sections III,A and B) are not equally amenable to manipulation. In practical terms, little can be done about the nature of the accidentally spilled oil, or the temperature of the ocean water. On the other hand, it appears possible to inoculate oil slicks with highly efficient hydrocarbon degraders, correct the

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nutritional deficiencies of the water and, perhaps, aerate stagnant waters to alleviate oxygen deficiency. The idea of stimulated oil biodegradation is not new, and early work in this area was previously reviewed by Atlas and Bartha (1973~).Furthermore, this topic receives more extensive treatment in a current review by Atlas (1977). The current wave of scientific and commercial interest in this subject was heralded by a feature article in Chemical and Engineering News (Anonymous, 1970). A literature study of the subject was commissioned by the U. S. Coast Guard (Friede et al., 1972), and a workshop on “The Microbial Degradation of Oil Pollutants” was held (December 4-6, 1972, Atlanta, Georgia). Several research groups proceeded to isolate and study highly effective strains or mixed enrichments of hydrocarbon degraders (Miget et al., 1969; Kator et al., 1971; Bridie and Bos, 1971; Atlas and Bartha, 1972a; Reisfeld et al., 1972). Various commercial inocula, such as “Petrodeg,” “Petrobac,” “Ekolo-Gest,” and “DBC-Bacteria”appeared on the market and were promoted as being effective for oil cleanup. We had opportunity to evaluate two commercial inocula (Ekolo-Gest and DBC-Bacteria), and we found that these were completely ineffective for promoting oil biodegradation in seawater when used as directed (Atlas and Bartha, 1973e). It is not clear whether these preparations were ineffective to begin with, or became so during storage. A general weakness of the inoculation approach was that a need for it was more often assumed than demonstrated. Oil spills are most likely to occur in coastal areas and along shipping lanes, and in these environments ZoBell (1946)and Mironov (1970) consistently found hydrocarbon degrading microorganisms. Dibble and Bartha (1976) found a pronounced difference in oil biodegradation rate and extent when they compared seawater samples from an oil-polluted and a nonpolluted coastal environment, but to date the best experimental support for the benefit of inoculation was provided by Kator et al. (1971). These authors sprayed mixed enrichment cultures on oil slicks floating on large seawater tanks. Ammonium sulfate was added to both the inoculated and the control tanks. The rate of oil biodegradation approximately doubled in response to the inoculum. A need for mineral nutrients in addition to inoculation is generally recognized, but the addition of nutrients in the form of water-soluble salts restricts the practical application of stimulated oil biodegradation to oily water contained in tanks, bilges, or holding ponds (Rosenberg et al., 1975). Treatment of free-floating oil slicks requires oil- rather than water-soluble nitrogen and phosphorus supplements. “Oleophilic fertilizers” (Atlas and Bartha, 1973d) are selectively available to oil degrading microorganisms only. This technique not only avoids the waste of the fertilizer, but prevents algal blooms that would inevitably follow the use of water-soluble formula-

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tions. Atlas and Bartha (1973d)selected parahized urea and octylphosphate as oleophilic fertilizers. Urea and n-paraffins form tightly attached intermolecular complexes (“adducts”)with overall oleophilic characteristics. Octylphosphate is itself an oily substance. Paranized urea and octylphosphate are both easily degradable, and they supply nitrogen and phosphorus for the breakdown of petroleum roughly 10- and 100-fold their own weight, respectively. Since in unpolluted seawater, in addition to nitrogen and phosphorus, iron may also become limiting (Dibble and Bartha, 1976), this element was also added in oleophilic form as ferric octoate. The required amounts of these materials per ton of oil and their respective costs are listed in Table V. Oil biodegradation rates in laboratory flasks were accelerated by these materials 30-50-fold as compared to untreated seawater (Atlas and Bartha, 1972c; Dibble and Bartha, 1976). Simulated field experiments with miniature floating oil slicks on flow-through seawater tanks gave a six-fold rate increase in response to oleophilic fertilizer if calculated over the entire experimental period. A 12-fold rate increase was calculated when the linear portions of the degradation curves were compared. Kator et al. (1972) proposed the use of paraffinized ammonium and phosphorus salts for fertilization of oil slicks. This formulation released over 90% of the nutrients to the water phase within the first hour of incubation, and is, consequently, not highly promising for practical application. Olivieri et al. (1976) used parathized MgNH4P04in field experiments of stimulated oil biodegradation. The experiment was moderately successful resulting in 63% oil disappearance in 3 weeks versus 40% in the untreated control. MgNH4P04 has low water solubility and is presumably not rapidly eluted from the oil slick. It takes care TABLE V AMOUNTS AND APPROXIMATE COSTOF OLEOPHILIC FOR THE STIMULATED BIODEGRADATION OF FERTILIZERS ONE METRICTONOF FLOATING OIL Amount Compound Urea-parain adduct (Sun Oil CRNF, comtrolledrelease nitrogen fertilizer)

(kd

Cost ($)

73.0

80.30

Pyrophosphoric acid dioctyl ester (octylphosphate, Stauffer Chemical Co.)

8.2

8.50

Ferric (2-ethylhexanoate) (ferric octoate, Shephard Chemical Co.)

0.15

0.33

258

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of both the nitrogen and phosphorus requirement, but contains phosphorus in an excess ratio. The reported stimulation of oil biodegradation is much lower than reported by Atlas and Bartha (1973d). This may be due either to the nature of the fertilizer or to the differences between the petroleum samples and experimental conditions. There is little doubt that oleophilic compounds and microencapsulation methods beyond those listed above will find future applications in the manufacture of oleophilic fertilizers. In environments without extensive oil pollution history, oleophilic fertilizers may be used for best advantage in combination with microbial inocula. While many organisms and enrichments were isolated and studied, an effective product with a reasonable shelf life is yet to be developed. For practical application, a single formulation would be desirable, and in presence of the oleophilic fertilizers the preservation of microorganisms in viable state may pose special problems. Again, microencapsulation techniques appear to offer the best solution. Jamison et al. (1975)found that gasoline could be removed from contaminated groundwater supplies by supplying forced aeration and water-soluble forms of nitrogen and phosphorus to stimulate microbial degradation. A patent has been issued to Raymond and Sun Research and Development Co. (Raymond, 1974)for reclamation of hydrocarbon contaminated ground water using stimulated microbial degradation. The use of such stimulated biodegradation would be restricted to enclosed systems. Genetic engineering may make important future contributions to the development of highly effective hydrocarbon-degrading inocula. Friello et al. (1976) succeeded in expanding the substrate range of a hydrocarbondegrading pseudomonad by plasmid transfer. For safety reasons, a welldefined microbial culture tailored for a broad substrate range and high metabolic activity may find a better acceptance than enrichments of unknown composition and possible side effects on marine life. Although there is certainly room for additional basic research, it appears that the concept of oil cleanup by stimulated biodegradation is ripe now for commercial development and could see practical application in the foreseeable future.

C. ENVIRONMENTAL IMPACT OF STIMULATED OIL BIODEGRADATION Before stimulated biodegradation can become a widely practiced oil cleanup technique, it must be assured that this practice is not detrimental to the aquatic environment, or at least less so than available cleanup alternatives. Concerns can be grouped around the following questions: (1)Are the hydrocarbon degrading microorganismsused as inoculum free of pathogenicity toward man, aquatic plants, and animals? (2) Are any intermediates of oil

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biodegradation unacceptably toxic? (3) Will oxygen depletion due to oil metabolism injure or kill aquatic organisms? (4) Does stimulated oil biodegradation increase hydrocarbon contamination of aquatic food chains? (5) Are any of the fertilizers toxic, or will they cause eutrophication problems? To see these concerns in perspective, it should be realized that stimulated biodegradation does not do anything that would not naturally occur over a longer time period. Of course, the time factor can become quite important in terms of oxygen depletion and local concentrations of hydrocarbon metabolites. Use of microorganisms for oil slick inoculation, especially if these organisms do not naturally occur in the respective aquatic environments, will require careful evaluation of their pathogenic potential. Safety criteria should be similar to those that apply to microbial insecticides (Burgess and Hussey, 1971). The impact of stimulated oil biodegradation on aquatic ecosystems is a poorly explored area and is likely to remain so until a demonstrable economic potential of this cleanup technique creates motivation and funding for the required research. Rosenberg et al. (1975), who demonstrated the usefulness of an Arthrobacter strain for cleaning out oil residues from tanker holds, tested the toxicity of the resulting emulsion on sea urchin embryos, and found that its toxicity was two orders of magnitude higher than that of the crude oil by itself. The toxicity could be reduced considerably by dialysis against seawater, indicating that most of the toxicity was due to polar metabolites derived from hydrocarbons. The dispersing agent produced by the Arthrobacter strain was not toxic, and the crude oil emulsified by it exhibited only moderate increase in toxicity. Berner et al. (1975) seeded intentionally oil-contaminated freshwater and estuarine environments with the hydrocarbon-utilizingyeasts Candida lipolytica and C . subtropicalis. C. lipolytica was not recoverable from the seeded sites after 3 and 5 months, but C. subtropicalis persisted at the two sites for over 12 and 7 months, respectively. Several other hydrocarbon-utilizing yeasts and filamentous fungi that were part of the seeding mixture were never recovered. Accordingly, the survival of seed organisms can be expected to vary widely. No harmful effects by the seed organisms or their metabolic products were evident on the algae, protozoa, and invertebrates inhabiting the experimental sites. Because of the limited scope of the above-discussed experimental work, the safety concerns raised earlier in this section have to be discussed at this time largely in a speculative manner. Petroleum hydrocarbons are rather specific substrates, not ordinarily present in living organisms. Hence, there is little reason to believe that a high petroleum degradation potential would be combined with pathogenic properties or vice versa. There is some evidence that oil biodegradation intermediates may be toxic (Rosenberg et al.,

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1975; Atlas and Bartha, 1973b), but this is likely to be a problem only in enclosed waters with a high oil to water ratio. In the marine environment, wave, tidal, and current mixing would ordinarily prevent the buildup of high local metabolite concentrations. The same consideration applies to oxygen depletion, which would affect primarily small and stagnant bodies of water. The overall proportion of polluting hydrocarbons that enter aquatic food chains would not be significantly increased by stimulated biodegradation, but higher concentrations in local populations could conceivably result from this practice. The toxicity of fertilizers would have to be scrutinized on compound by compound basis, but oil- rather than water-soluble compounds will have the advantage that they are needed in much lower amounts and that their contact will be primarily restricted to oil-degrading microorganisms. By necessity, they would have to be easily biodegradable, and thus would not be subject to biomagnification effects. In tests by Atlas and Bartha (1973d), oleophilic fertilizers failed to trigger algal blooms, while equal amounts of water-soluble fertilizers caused definite eutrophication. The bulk of the oilsoluble fertilizers is expected to become available to the respective aquatic ecosystem only gradually over a period of time as the biomass accumulated during the oil biodegradation returns to its normal level. Therefore, in large, circulatingbodies of water, the effect will most likely be negligible, but small enclosed bodies of water may be subject to delayed eutrophication.

D. ROLE OF STIMULATEDBIODEGRADATION IN INTEGRATED OIL POLLUTION CONTROLPROGRAMS

No two oil spill incidents are exactly alike in terms of the quality and quantity of spilled oil, the contaminated environment, season, weather conditions, etc. Consequently, control responses should be flexible and be tailored to the situation, rather than follow a rigid pattern. Stimulated biodegradation is not expected to replace all other control measures, but it should rather add further flexibility to integrated control programs. From the foregoing discussion stimulated biodegradation emerges as a highly cost-effective alternative, unaffected by storms or currents. Its environmental impact, as far as can be predicted from a clearly insufficient body of experimental work, is likely to be more severe than that of mechanical collection, but probably less severe than that of dispersion, sinking or burning. Water temperature is an important consideration in the application of the stimulated biodegradation technique. Other conditions being equal, moderate to high water temperatures favor the biodegradation approach while very cold water temperatures would generally counterindicate the use of stimulated biodegradation.

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26 1

In trying to visualize the role of stimulated biodegradation in case of a major spill, still every feasible effort should be made to surround the stricken tanker or offshore rig with a floating boom. Within the enclosure, the contained oil would be pumped or scooped up using mechanical devices. Downwind from the enclosure, the oil slick that has escaped containment would be treated by a fertilizer-inoculant formulation, using either workboats, rotary- or fixed-wing aircraft, as conditions dictate. The escaped oil thus treated will be at least partially mineralized before it reaches shore. The residue will have lost much of its “stickiness” due to microbial dispersion (Rosenberget al., 1975), and will not readily adhere to rocks or beach sand. Wherever the treated oil is carried by wind or current, within itself it carries the agents of its own destruction, the oleophilic fertilizer and the adhering hydrocarbon degrading microbes. Extreme proximity of an accident to recreationally valuable beaches and/or prevailing low water temperatures may dictate the use of chemical dispersants in preference to stimulated biodegradation. Conversely, warm water temperatures combined with severe weather conditions may force the abandonment of the mechanical cleanup approach altogether and may dictate a major reliance on stimulated biodegradation instead. In small or stagnant bodies of water the stimulated biodegradation approach may require the simultaneous use of aerating devices in order to prevent oxygen depletion. ACKNOWLEDGMENTS We wish to thank all investigators who permitted us to review preprints of their publications. The oil pollution work of both authors was supported by grants from the OfEce of Naval Research.

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Zajic, J. E . , and Suplisson, B. (1972). Biotechnol. Bioeng. 14, 331343. Zajic, J. E., Suplisson, B., and Volesky, B. (1974). Enuiron. Sci. Technol. 8, 66446% ZoBell, C. E. (1946). Bactaiol. Reo. 10, 1-49. ZoBell, C. E. (1950).Adu. Enzyrnol. 10, 443436. ZoBell, C. E. (1964).Ado. Water Pollut. Res. 3, 85-118. ZoBell, C . E. (1969).Proc.--Conf. Preo. Control Oil Spills, 1969 pp. 317-326. ZoBell, C. E. (1973).Microb. Degradation Oil Pollut., Workshop, 1972 La. State Univ. Pub1 No. LSU-SG-73-01, pp. 153-161. ZoBell, C. E., and Agosti, J. (1972). 72ndAnnu. Meet., Am. SOC. Microbiol. p . E l l . ZoBell, C. E., and Prokop, J, F. (1966). Z. Allg. Mikrobiol. 6, 143-162. ZoBell, C. E., Grant, C., and Haas, H. (1943). Bull. Am. Assoc. Pet. Geol. 27, 1175-1193.

Comparative Technical and Economic Aspects of Single-Cell Protein Processes JOHN

H. LITCHFIELD

Battelle Columbus Laboratories, Columbus, Ohio I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. End Uses for SCP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Food . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Feed . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. SCP Microorganisms, Processes, and Products A. Gaseous Hydrocarbons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Liquid and Solid Hydrocarbons. ...................... C. Alcohols-Methanol and Ethanol IV. Economic Aspects . . . . . . . . . . . . . A. Raw Materials Costs . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . B. Process Factors AEecting Costs C. Manufacturing Costs ................................ D. Capital Costs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Plant and Animal Protein Products Competing with SCP A. Food Uses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Feed U s e s . . . . . . . VII. Conclusions . References

267 268 268 269 269 270 274 284 291 291 293 294 296 296 296 299 299 301 301

1. Introduction The term “single-cellprotein” (SCP) was coined at Massachusetts Institute of Technology by Professor C. L. Wilson, in 1966, to represent the cells of algae, bacteria, yeasts, and fungi grown for their protein contents (Scrimshaw, 1968). We should note at the outset that cells of microorganisms contain carbohydrates, lipids, minerals, and vitamins in addition to proteins. Several symposia held in recent years (Davis, 1974; Mateles and Tannenbaum, 1968; Gounelle de Pontanel, 1973; Tannenbaum and Wang, 1975) and reviews (Gutcho, 1973; Kihlberg, 1972; Lipinsky and Litchfield, 1970, 1974; Litchfield, 1974, 1975, 1976, 1977; Rockwell, 1976) cover various aspects of SCP production and uses. This review covers SCP processes and products of current interest, particularly those produced from hydrocarbons or chemicals derived from them. During the course of this discussion, we should bear in mind certain basic questions concerning SCP. (1) Who will buy SCP and for what uses-food processors, food ingredient suppliers, feedstuff formulators, agricultural cooperatives, individual farmers?-Food or feed use? (2) What quality of SCP product does the buyer desire?-Human food grade as a protein sup261

268

JOHN H . LITCHFIELD

plement, or as a protein concentrate or isolate for functional use in foods; or feed grade for broiler chickens, laying hens, swine, or veal calves? (3)Where is the buyer located-in the United States, in advanced countries, such as Western Europe, Eastern Europe, or Japan, or in less developed countries in Asia, Africa, and Latin America? (4) What are the competing protein products in the buyer’s region-soybean, cottonseed, peanut, fish meal? (5) What price will the buyer be willing to pay, depending upon quality and nutritional value? The answers to these questions will to a large extent determine the microorganisms, substrate, and process to be used in producing the SCP product.

II. End U s e s for SCP The two end uses for SCP are food for humans and feed for animals. The desired end use determines composition, protein quality, and requirements for purification including removal of trace amounts of substrate and undesirable contaminants. A. FOOD The applications of SCP products in food include addition for nutritional value or for functional properties of the proteins or both. For human food use the SCP product must have satisfactory nitrogen, and protein contents, amino acid profiles, lipid contents, mineral, and vitamin contents. However, crude protein as determined by multiplying nitrogen contents of cells by a factor, e.g., 6.25, includes both protein nitrogen and nonprotein nitrogen sources such as nucleic acids. Also, some amino acids may be bound to the cell walls of microorganisms and may not be available biologically when SCP products are consumed (Young and Scrimshaw, 1975). Thus, nutrient contents as indicated by chemical analyses are not a s&cient measure of the nutritional value of a SCP product for human food use. In addition, the SCP must have a satisfactory biological value as demonstrated in actual animal feeding studies and in human clinical trials. Also the SCP product must be free from toxic substances in the cells themselves or introduced into the cells through residues in the substrate such as polyaromatic hydrocarbons and from contamination by pathogenic microorganisms or their toxins. From a sensory standpoint the product should have a satisfactory flavor, aroma, color, and texture. SCP products also have a potential applicability for their functional uses, including gel formation, water and fat absorption, and whipping, foaming, or dispersing properties. In general, to achieve desirable functional characteristics, a protein concentrate or isolate must be prepared from the microbial

SINGLE-CELL PROTEIN PROCESSES

269

cells by disrupting the cell wall and/or altering the cell wall structure to enable the subsequent separation of nucleic acids from the proteins. Functional SCP products must meet all the same safety requirements as do products designed for nutritional use, but need not meet the same protein nutritional quality requirements. SCP protein concentrates and isolates have been discussed in a recent review (Litchfield, 1977). B. FEED For feed applications, SCP products must meet many of the same criteria as those required for human food applications. Nitrogen and protein contents and amino acid profiles are indicators of potential usefulness of an SCP product in animal feed applications. Methionine supplementation is required to obtain satisfactory feeding performance in nonruminants with most SCP products. Also, 1ysine:arginineratios may have to be adjusted because of high lysine contents of most SCP products to achieve satisfactory results in broiler chicken rations (Shacklady and Gatumel, 1973). However, the true feeding value of a SCP product can be demonstrated only by studies in domestic livestock, such as broiler chickens, laying hens, turkeys, swine, and veal calves. In addition to desired levels of weight gained and efficiency of feed conversion when SCP is fed to a given animal species, the product must also meet safety requirements from the standpoint of freedom from toxic residues that might accumulate in the tissues of animals to be subsequently consumed by humans. Multigeneration feeding studies are required to determine any adverse affects upon reproductive performance in animals (Hoogerheide, 1974; De Groot, 1974; Taylor et al., 1974). Finally the SCP product must be readily accepted by livestock and acceptable to feed formulators and farmers from the standpoint of physical characteristics, including ease of formulation and blending, and storage stability.

Ill. SCP Microorganisms, Processes, and Products Now that we have discussed the criteria that SCP products would have to meet, we will consider microorganisms, substrates, and processes for producing SCP products. We will emphasize those processes that are being developed on a commercial scale. Hydrocarbons and chemicals derived from them that have been considered for use as substrates for SCP production on a commercial scale include methane, purified n-alkanes, crude oil, and fractions obtained from crude oil, such as gas oil and kerosine, methanol, and ethanol (Lipinsky and Litchfield, 1974; Litchfield, 1974, 1975). Both bacteria and yeasts have been investigated for their use in these processes. Ideally, in the aerobic growth of

270

JOHN H . LITCHFIELD

microorganisms on these substrates, cell mass, carbon dioxide, and water should be the only end products of microbial metabolism. However, inhibitory products may be produced during growth in some instances, as will be discussed subsequently.

A. GASEOUSHYDROCARBONS Methane has been of interest as a substrate for SCP production because, in some regions of the world natural gas is still flared at wells that are remote from markets. Production of a feed-grade SCP from this natural gas that would be otherwise wasted would be a possible way of recovering this product. Methane is available in high purity as compared with other hydrocarbons and can be recovered easily from fermentation media without leaving any residues. A number of bacteria utilize methane as a carbon and energy source for growth. These include Methanomonas methanica (Hamer, 1968) Methanomonas methanooxidans (Hamer et al., 1967), Methylococcus capsulatus (Foster and Davis, 1966; Eroshin et al., 1968; Whittenbury et al., 1970; Harwood and Pirt, 1972) Pseudomnas methanica (Dworkin and Foster, 1956; Leadbetter and Foster, 1958), and a variety of mixed cultures of microorganisms (Vary and Johnson, 1967; Klass et al., 1969; Sheehan and Johnson, 1971; BewersdorfF and Dostalek, 1971; Sheehan, 1972; Wolnak et al., 1972; Wilkinson et al., 1974; Harrison, 1976). Also, a hngal culture, identified as Graphium, grows on natural gas (ethane and methane), ethane being the preferred substrate (Volesky and Zajic, 1971). However, as far as it can be determined, there are no reports in the literature on the growth of yeasts on methane as a carbon and energy source. Table I summarizes some of the studies reported in literature on the growth of methane utilizing microorganisms. For economic commercialscale production of SCP from methane, continuous-culture systems are more attractive than batch-culture systems. Higher productivities (weight of cells per unit volume of medium per unit of time) are attainable in continuous cultures of methane-utilizing bacteria then in batch cultures (BewersdorfF and Dostalek, 1971; Hamer et al., 1975). Also, in batch growth, a significant portion of the methane supplied may not be utilized, and continuous systems permit design for recycle of unutilized substrate. A bacterial SCP process has been developed to a pilot-plant scale at Shell Research Ltd. in England using M . capsulatus (Harrison et al., 1972; Hamer et al., 1975) and mixed cultures (Wilkinson et al., 1974; Harrison, 1976). Although high productivities and yield coefficients can be achieved in continuous production of bacterial SCP from methane, productivity is limited by transfer of oxygen and methane from the gas phase to the bacterial cells

TABLE I GROWTHOF SELECTEDMICROORGANISMS ON METHANEIN BATCHAND CONTINUOUS CULTURE

0rgan is in (s) Msthunomonus

Scale and conditions (aeration, agitation)

Temperature (“C)

Batch, 1-liter fermentor,

PH

Specific growth Cell density Yield per g d g m Productivity or dilution (d) rate (gm/liter, dry substrate utilized (gm/liter/hr, dry (hours-’) weight basis) (dry weight basis) weight basis)

28

6.1-6.7

0.112

1.025

6.9

0.140

0.4

-

-

Hamer et al. (1967)

1.00-1.03

-

Harwood and Pirt (1972)

0.67-0.68

-

Vary and Johnson (1967)

1.44

-

Klass et al. (1969)

800 ml medium 0 . 1 4 . 3 liter CH,/min, 640 rpm Methylococcus capsulutus

Continuous, 2.8 liter medium, 6-7% CHI, 17.1-19.4% 0, viv, 38.6-49.5 mYmin, 1450 rpm

37

HR, mixed culture

Batch, 1.26-1.44-liter shake flasks, 150 ml medium, 8% CH4 in air, 397 rpm

30

6.5

Batch 65% H,-35%

-

-

IGT-10, mixed culture

02,62.5 ml CH4/min

1.03

0.256-0.286 0.286

0.067

-

References

(CO t1 t h U

ed)

TABLE I (continued)

Organism(s)

N

21 h)

Scale and conditions (aeration, agitation)

Temperature (“C)

PH

Specific growth Cell density Yield per gm/gm Productivity or dilution (d) rate (gm/liter, dry substrate utilized (gm/liter/hr, dry (hours-’) weight basis) (dry weight basis) weight basis)

M -45 mixed culture

Continuous, 5-liter fermentor, 2.97 liter medium, 35-37% CHI, 60% air, 3-5% O2 recycled, 1510 rprn

45

6.8

0.303

TM-10 mixed culture

Batch, 2.5 liter medium, 7% CH,, 11.9%0 2 (v/v), 950 rpm

31

5.6-5.9

0.159

Continuous, 2 . 5 liter medium, 7% CHI, 11.9%0 2 (v/v), 950 rpm

31

5.6-5.9

Continuous, 10 liter fermentor, 8 . 9 liter medium, 1 atm, 470 rpm

32

5.7

Pseudomonas SP., Hyphomicrobium sp., Acinetobacter sp., and Flauobacterium sp., (mixed culture)

12.8

References

0.616

2.39

Sheehan and Johnson (1971)

1.10

0.90

0.08

BewersdorE and Dostdlek (1971)

0.08

1.87

0.90

0.15

BewersdorfY and Dostdek (1971)

0.06(d)

0.8

0.99

-

Wilkinson et al. (1974)

TABLE I1 GROWTHOF SELECTED MICROORGANISMS O N PROPANE AND BUTANE

Organism and substrate

w W 4

Scale and conditions (aeration, agitation)

Temperature (“C)

pH

Specific growth rate (hours-’)

Cell density (gm/liter,a dry weight)

Yield per gm/gni Productivity substrate utilized, (gm/liter/hr, dry (dry weight basis) weight basis)

References

Arthrobac ter simpler B-129 (n-propane)

Batch 10 liter medium 50% propane, 50% air (v/v), 5 litedhr, 400 rpm

29

6.5

-

0.603 (66 hr)

-

-

Orgel et al. (1971)

Candida rigida No. 113 (liquefied petroleum gas (LPG)

Batch 10 m3 medium 1020 kg butane supplied

28

5.8-6.0

-

25b (45 hr)

0.94 (0.253)”

-

Imada et al. (1972a)

Batch, 10 m3 medium, 1.8% LPG, v/v

30

6.0

-

27b (40 hr)

0.278‘

7.0

0.091

1.36

7.0

0.111

30b (112 hr) 22b (72 hr)

Nocardia paraffinica KY 4334 (a) n-Propane (b) n-Butane

Batch, 5-liter fermentor, 3 liter medium, gas flow 3 litershin 600 rpm

“Maximum values at time indicated. b“As is” basis.

“Based on substrate supplied.

0.95

Imada et al. (1972b) Sugimoto et al. (1972)

274

JOHN H . LITCHFIELD

(Hamer et al., 1975). Furthermore, either single or double gaseous substrate limitation may occur. Other problems in the production of bacterial SCP from methane include explosive hazards that require operation below 12.1% by volume of oxygen (Hamer et al., 1967) and high heat production at high productivities in the order of 10 kcal per gram of cells at a yield coefficient of 1.0 gm of cells per gram of methane (Hamer et al., 1975). Also inhibitory products may be produced during growth of bacteria on methane in continuous cultures as observed in the case of M . capsulatus (Eroshin et al., 1968; Hanvood and Pirt, 1972). Treatment of the growth medium with anion exchange resin apparently aided in removing an inhibitor from M . capsulatus (Harwood and Pirt, 1972). Because of the requirements for avoiding explosive hazards and for cooling the fermentor during operation, capital investments for production of bacterial SCP from methane may be significantly higher than from liquid hydrocarbon substrates. Several processes have been developed by Kyowa Hakko Kogyo Co. Ltd. in Japan for the production of bacterial SCP product from gaseous hydrocarbons. In particular, Brevibacterium ketoglutamicum ATCC No. 15,587 grows on gaseous hydrocarbons, such as methane, ethane, propane, n-butane, isobutane, propylene, butylene, or mixtures of them (Tanaka-et al., 1972). Also, liquefied petroleum gas (LPG) can be utilized by Candida rigida No. 113 (Imada et al., 197%,b) and n-butane by Nocardia paraffinica KY 4334 (Sugimoto et al., 1972). The LPG process using C. rigida 113, which utilizes n-butane, has been operated in fermentors containing 10 m3 (l0,OOO liters) of fermentation medium. Arthrobacter simplex B-129 utilizes n-propane and higher n-alkanes, and alkenes (Orgel et al., 1971). However, these studies were conducted only in laboratory-scale fermentors and no further commercial development of this process has occurred. Table 11 summarizes typical values obtained in these studies. Unfortunately only batch studies and no continuous culture studies on propane and butane utilization are reported in the literature. B. LIQUIDAND SOLIDHYDROCARBONS Many microorganisms utilize liquid hydrocarbons, aerobically, as carbon and energy sources for growth including bacteria, yeasts, molds, and actinomycetes. Tables 111and IV show typical data on growth characteristics of selected microorganisms on liquid hydrocarbons. Included are data on processes that have been practiced on a pilot-plant scale. In general, gas oil and purified n-paraffins are the liquid hydrocarbon products showing the greatest potential as substrates for commercial processes. Crude oil, fuel oil, and kerosine have been studied as substrates for

TABLE I11 GROWTHOF SELECTEDBACTERIA AND ACTINOMYCETES ON LIQUIDHYDROCARBONS

Organism and substrate Achroinobacter deloacwate No. 5301 (Diesel oil 5-10%)

Scdk and conditions: aeration, agitation

7.0-7.2

-

Temperature

Batch, 6000-liter

(“(3 35-36

fermentor, 3000 liter of medium, 1 (v/v)/ m in

Acinetobacter (Mierococcus) cerificuns (a) gas oil 20 gmlliter (a) Batch, 7.5 liter (b) n-hexadecane (b) Fermentor 4.5 liter (4 gmiliterj of medium, vlvi min, 900 rpm (c) 10.9 gmiliter (c) Batch, 7.5-liter fermentor, 3.5-7.0 mM OJliteri minutes

Mycobucterium phlei (Clo-Czon-alkanes)

pH

Specific growth rate or dilution (d) rate (hours-’)

Batch, 7.5-liter fermentor, 6-8 liter of medium, 1500 rpm

Cell density (gm/liter, dry weight)

Yield per gmigm Productivity substrate utilized (grniliterihr, dry (dry weight basis) weight basis) References

-

-

KO et al. (1964)

8-10 8-10

0.10-0.12 0.80-0.90

-

Ertola et al. (1969)

10-15

(48 hr)

30

7.0

(a) 0.4-1.0 (b) 1.0-2.0

30

7.0

1.33

-

1.20

-

Guenther and Perkins (1968)

30

6.8

0.43

25.8

1.05

-

Wagner et a1 (1969) -

(continued)

TABLE 111 (continued)

ec,

pH

Specific growth rate or dilution (d) rate (hours-’)

Nocardia sp. NBZ-23 (Clo-Czo n-alkanes), 1-5%

Batch, 7.5-liter 30 fermentor, 6-8 liters of medium 1500 rpm

6.8

1.25

14.7

0.98

-

Wagner et al. (1969)

Pseudomonus No. 5401 (fuel oil)

(a) Batch, 6000-liter 36-38 fermentor (b) Continuous, 36-38 6000-liter fermentor

7.0

0.16

1.00

-

0.12 (d) 0.25 (d)

-

KO and Yu (1968)

7.0

16 (24-26 hours) 10

1.2 2.0

0.598

-

Organism and substrate

N

2

Scale and conditions: aeration, agitation

Pseudomonus Batch, 5.1-liter aeruginosa fermentor, NCTC 5940 1 Iitedmin, 1000 rpm (n-octadecane 4 gmll)

Temperature

34

7.2

1.00

Cell density (gm/liter, dry weight)

Yield per gm/gm Productivity substrate utilized (gm/liter/hr, dry (dry weight basis) weight basis) References

8 55

Ertola et al (1965)

GROWTHOF SELECTEDYEASTS

Organism and substrate

Scale and conditions (aeration, agitation)

pH

35-36

30-40

-

35

30

4.0

0.25

15-20 (30-50 hr)

0.95-0.97

30-33

4.5-5.0

0.30

1.01-2.04

1.04-1.18

30

5.0

0.14

5.0

0.70

30

5.5

0.15

15-20

0.817

Candida sp., (C13-C11 n -alkanes) 1-4%

Batch, 500-liter fermentor, 220 liters of medium, 1 v/v/min 900 rpm

Candidu sp., (n-alkan es)

Continuous 100 m3 air-lift fermentor

Candida Batch, 15-liter guilliermondii, fermentor, 5 liters of medium, 0.4 (C12-C15 vlvlmin 770 rpm n-alkanes) Candida intermedia (n-hexadecane)

Batch, 1-liter fermentor, 1525 rpm

Specific Cell growth rate density or dilution (d) (gm/liter, Yield per gmlgm Productivity rate dry substrate utilized (gm/liter/hr, (hours-’) weight) (dry weight basis) (dry weight basis)

Temperature (“C)

Batch, 1 ton/day Candida sp. ATCC 20343, (C1a-C20 n -alkanes)

N 4 4

TABLE IV FUNGION LIQUIDAND SOLID HYDROCARBONS

AND

1.0

-

References Chinese Petroleum C o p . (undated)

Sonoda et al. (1973)

2.663.24

-

Kanazawa (1975), Kanazawa et al. (1975) Aiba et al. (1969)

Miller e t al. (1964)

(continued)

TABLE IV (continued) ~~

~

Organism and substrate

~~

Scale and conditions (aeration, agitation)

Temperature (“C)

pH

Specific Cell growth rate density Productivity or dilution (d) (gm/liter, Yield per gm/gm substrate utilized (gm/liter/hr, dry rate (hours-’) weight) (dry weight basis) (dry weight basis)

References

~

Candida

intermedia, Candida

lipolytica, mixed culture (paraffin wax 16 wt% in pristane) Candida

kofuensis (C,o-Cal n -alkanes) 3%, by vol Candida

lipolyticu (Cx4-Cia n-alkanes)

C. lipolytica (gas oil 19.87% n -alkanes)

Batch, 1-liter fermentor, 0.5 liter working vol, 1525 rpm

30

5.5

10-12

0.665-0.725

-

Batch, 20-liter fermentor, 10 literdmin, 800 rpm

37

3.5

0.25

23 (18 hr)

Batch, 14-liter fermentor, 6-8 liters of medium, 1500 rpm

25

5.5

0.22

13.5

(1) Batch, 3-liter fermentor, 1.3 liters working vol, 1.5 vivlmin, 1200 rpm

30

4.5

0.2

10.4

-

Miller and Johnson (1966b)

1.0

-

Ueno et al. (1974a, b,c)

0.90

-

Wagner et al. (1969)

0.65

Dostalek et a1 (1968a)

(2) Continuous 3-liter fermentor, 1.3 liters working V O ~ , 1-5 V/V/ min, 1200 rpm

30

4.5

0.2

10.4

C. lipolytica (n-alkanes)

Continuous

30

2.9

0.12 (d)

6.2

C. lipolytica (a) (C,o-Cls

Continuous, 1800-liter working vol fermentor, 1.5 vlvlmin

32

5.5

0.16 (d)

Continuous, C,,-C,, 12 m3 fermentor, alkanes) 2.400 liter medium, BP 3001 v/v/min 380°C 120 gmlliter

30

4.0

alkanes)

(b) Gas oil N 4 \o

Candida tropicalis (n-alkanes)

Continuous airlift, 50,000-liter fermentor

30

3.0

C . tropicalis

Continuous, 50-liter working vol fermentor, 3 mM 0,lYmin

30

4

(gas oil), 15 g/liter

0.65

Dostilek e t a / . (1968a, b)

0.90

0.14

Decerle e t al. (1969)

23.6

0.88

-

Evans and Shennan (1974)

-

25

0.18"

3-4

Champagnat and Filosa (1971), Filosa (1970b), Laine and du C h a l h t (1975)

0.15-0.24

10-30

1.0-1.1

1.7-3.0

Silver and Cooper (1972a,b), Cooper e t a/ (1975)

15

-

-

0.2 (d)

Laine (1970)

(continued)

TABLE IV (continued) Scale and conditions (aeration, agitation)

Organism and substrate

C.tropicalis (n-hexadecane), 10 gil

Specitic Cell growth rate density or dilution (d) (gdliter, rate dry (hours-’) weight)

Yield per gm/gm Productivity substrate utilized (gmlliterlhr, (dry weight basis) (dry weight basis)

Temperature (“C)

pH

(a) Batch, 1 4 - h ~ fermentor, &liter medium, 1 vlvlmin, 3500 rpm

-

-

0.29

10

1.0

-

(b) Continuous,

-

-

0.10

10

1.0

0.95

14-liter fermentor, 1 vlvlmin 3000 rpm aBased on gas oil.

References Blanch and Einsele (1973)

SINGLE-CELL PROTEIN PROCESSES

28 1

SCP production by bacteria and yeasts on a laboratory or a pilot-plant scale (Takahashi et al., 1963; KO and Yu, 1968). However, processes based on these hydrocarbons have not been sufficiently promising to justify further commercial development. During the early 196os, processes were developed by British Petroleum Company (BP) for producing SCP &om gas oil by yeasts. Early work demonstrated that Candidu lipolytica, Candida tropicalis, and related yeasts could utilize the n-alkanes in gas oil and thereby bring about dewaxing (Champagnat and Filosa, 1965, 1971;Champagnat and de Mayo, 1966).Approximately 60-75% of the n-alkanes in gas oil is utilized by Candida species. Gas oil-based processes can be operated nonaseptically but under commercially clean conditions using suitable plant sanitation measures without sterilization of the fermentor or nutrients. Contamination control can be maintained by continuously adjusting the pH of the medium in the range 2.9-5.0 (Champagnat and Filosa, 1965; Evans, 1968). Generally, gas oil is used. About containing 1045%CI5-C3,,n-alkanes boiling range 3OO0-38OoC 10% is utilized and 90% having reduced cloud and pour points is returned to an adjacent refinery (BP Proteins Ltd., 1974; Evans, 1968). BP has operated a 16,000 metric tons per year continuous process pilot plant at Lavera, France. Air-lift fermentors are used with continuous feeding of gas oil, ammonia, and mineral nutrients and continuous harvesting of the yeast produced. Removal of spent gas oil from the yeast product can be accomplished by solvent extraction, solvent leaching, and washing with surfactants (Champagnat, 1965; Champagnat and Laine, 1966; Filosa, 1970a,b; Laine, 1970; Laine et al., 1971). Productivity of this process increases with increasing n-alkane content of the gas oil and with increasing energy consumed up to 4.0 gmfliter per hour at 3kwkg (Laine and du ChafFaut, 1975). In a separate group of processes for producing SCP, yeasts utilize purified n-alkanes derived from either gas oil or kerosine. Molecular sieve adsorption methods are used to prepare n-alkanes in the molecular weight range CloCZ3in a purity of 97.5 to 99% (Evans, 1968; Reber and Symoniak, 1975). Yeasts that have been used for producing SCP from purified n-alkanes on at least a pilot-plant basis include Candida lipolytica (BP) (Evans and Shennan, 1974); Candida kofuensis (Mitsui Toatsu Chemicals, Inc.) (Ueno et al., 1974a,b,c)Candida novellus (Liquichemica Biosintesi, S.p.A.) Giacobbe et al., 1975)Candida tropicalis (Gulf Research & Development Co.) (Cooper et al., 1975), Candidu (Kanegafuchi Chemical Industry Co. Ltd.) (Takata, 1969; Kanazawa, 1975; Kanazawa et al., 1975), and an unspecified yeast (Dainippon Ink & Chemicals, Inc., undated). All the processes based on purified n-alkane substrates are designed to operate continuously as in the case of the gas oil processes. BP has operated a 4000-metric ton per year pilot plant at Grangemouth, Scotland for producing C. lipolytica from purified n-alkanes. This process is operated continuously

282

JOHN H . LITCHFIELD

under aseptic conditions in which the n-alkanes, mineral nutrients, and water are sterilized by passing through a heat exchanger and air and gaseous ammonia are sterilized by filtration. Initially the processes operate in a batch mode until the yeast cell dry weight in the medium reaches a sufficient level to permit continuous operation at a high productivity. The conventional b a e d , agitated fermentor vessel is used in the BP purified n-alkanes process. (Evans, 1968; Bennett et al., 1969; Watts, 1976). Other yeast processes based on purified n-alkanes, for example, those of Gulf (Cooper et al., 1975), Kanegafuchi (Kanazawa, 1975), and Liquichimica Biosintesi (Giacobbe et aZ., 1975), are operated nonaseptically using air lifttype fermentors. Contamination is controlled by maintaining the pH in the range 3 4 by adjusting the ammonia feed. In n-alkane processes the yeast product is separated by centrifugationsand washing. Since the n-alkanes are almost completely utilized by the yeast, separation of the product and removal of residual hydrocarbons are not a problem as in the case of gas oil-based processes (Bennett et al., 1969; Cooper et al., 1975; Kanazawa, 1975; Kanazawa et al., 1975). Other yeast-based processes for producing SCP from liquid hydrocarbons that have been operated on a pilot plant or production scale include the following: (1) Institute Franpis du Petrole, Soleige, France, 100 kg/day (Anonymous, 1974a, 1976a; Avrillon et al., 1972; Decerle et al., 1969; Gatellier and Glikmans, 1971, 1972); (2) Sovnaft, Kojetin, Czechoslovakia, 1000 metric tondyear (Anonymous, 197313);(3)Petrom, Jassyon, Romania, using a Dianippon Ink and Chemicals Co. process, 60,000 metric tondyear planned capacity (Anonymous, 1973a, 1974a); (4) All-Union Research Institute of Biosynthesis of Protein Substances, Bashkira and Gorki, U.S. S.R. (Anonymous, 197413). In 1976, British Petroleum discontinued operations of the gas oil-based pilot plant at Lavera, France (Anonymous, 1976d). Operation of a 100,000 metric tondyear plant constructed in Sardinia by British Petroleum and Ente Nazionale Idrocarburi (Italproteine) has been held up pending resolution of a dispute with the Italian government over approval for marketing the Toprina yeast product on questions of levels of hydrocarbon residues (Anonymous, 1976b,c).Also, the 100,OOO metric tonslyear plant constructed by Liquichimica Biosintesi, S.p.A. at Saline di Montebello, Italy has been blocked by this same dispute (Anonymous, 1976c,d). On the other hand, British Petroleum reached an agreement early in 1976 with Venezuelan government and private interests for planning a 100,000 metric tondyear SCP production plant at Puerto la Cruz. The yeast product would be used for animal feed as would be the case with the product from BP’s Sardinia plant (Anonymous, 1976b). Plans for constructing large-scale SCP production plants in Japan by Dainippon Ink and Chemicals Co., Kanegafuchi Chemical Industry Co.,

SINGLE-CELL PROTEIN PROCESSES

283

were suspended in 1973 because of public protests over questions on the safety of the products and subsequent Japanese government regulatory action banning the use of these products (Anonymous, 1973a, 1976d; Tung, 1975). However, pilot plant-scale studies and nutritional and safety evaluations of SCP products have continued in Japan (Anonymous, 1976d; Tung, 1975). Bacterial processes have also been investigated for the production of the SCP from purified n-alkanes. Acinetobacter (Micrococcus) cerificans, Achromobacter delvacvate, No. 5301, and Pseudomonas species (McKenna and Kallio, 1965; Ertola et al., 1965; KO et al., 1964; Yamada et al., 1968) and Actinomycetes including Nocardia sp. and Mycobacterium phlei (Wagner et al., 1969) use n-alkanes readily. Exxon Corporation has developed a pilot plant-scale process for producing Acinetobacter cerificans from purified n-alkanes (Perkins and Furlong, 1967; Guenther and Perkins, 1968).Also the Chinese Petroleum Corporation, Taiwan has investigated the production of SCP from n-alkanes using Achromobacter delvacuate, No. 5301 and Pseudomonas No. 5401 on a small pilot-plant scale (KO et al., 1964; KO and Yu, 1968).However, neither of these processes have yet been developed to a significant commercial scale of operation. Some of the factors that affect growth rate, yield, and productivity of microorganisms growing in liquid hydrocarbons are (1)oxygen transfer, (2) mass transfer of the substrate to the cell, and (3) heat production, (Einsele and Fiechter, 1971; Humphrey, 1974; Prokop and Sobotka, 1975). Oxygen transfer coefficients of 10-15 kg/m3/hr are required for growth of microbial cells on hydrocarbon substrates (Laine and du ChaRaut, 1975). Hydrocarbons having a chain length of C,,-C,, are soluble in water only to the extent of 6-16 mg/l. The uptake of hydrocarbon by growing cells from the medium is far greater than would be indicated by solubility alone, An exponential growth phase followed by a linear growth phase is often observed in the growth of microorganism on hydrocarbons at low inoculum sizes. Explanations for this observation include, for example, (1)direct uptake from the aqueous phase during exponential growth, and by direct contact of cells and droplets in the linear phase (Erickson and Humphrey, 1969), and (2) formation of biological flocs that limit substrate or oxygen transport (Blanch and Einsele, 1973). During the growth of C . lipolytica on n-dodecane, no appreciable linear growth phase was observed in batch cultures. Early washout occurred at less than maximum growth rate in continuous cultures. Growth rate was a function of hydrocarbon concentration and agitation rate (Moo Young and Shimizu, 1971). Hydrocarbons may be solubilized by surface-active compounds produced in growing cultures (Barnett et al., 1974). When Pseudomonas aeruginosa was grown on n-heptane, there was evidence for the incorporation of this

284

JOHN H . LITCHFIELD

substrate into micelles as a result of production of unidentified surface-active compounds in the culture which facilitated substrate transport into the cells (Velankar et al., 1975). Once the hydrocarbon reaches the cell surface, penetration appears to take place by passive diffusion. The exact mechanisms for hydrocarbon transport within the cell have not been completely resolved. A recent review gave a more detailed discussion of substrate and oxygen transport in the microbiological utilization of hydrocarbons (Prokop and Sobotka, 1975). The production of heat by microorganisms growing on n-alkanes is signscandy greater than with carbohydrate substrates. Theoretical values of heat evolution can be calculated from the heat of combustion of a given organism and yield. For example, calculated values range from 4400 kcalkg to 8000 kcaVkg for yields in the range of 1.2 to 0.9, gm cells (dry weight) per gm substrate utilized, respectively (Laine and du Chdaut, 1975; Kanazawa, 1975). Problems associated with cooling fermentors during SCP production from hydrocarbons will be discussed subsequently in connection with costs. Hydrocarbons having a chain length of C,, or longer are solids at temperatures in the usual ranges for microbial growth. Solid hydrocarbons are not soluble and are difficult to disperse in growth media. A mixed culture of C. lipolytica and C. intermedia has been grown in a mineral salt medium containing solid n-alkanes as a carbon source (Miller and Johnson, 1966b)(Table IV). Solid hydrocarbon is dissolved in a liquid hydrocarbon solvent that is not utilizable by the bacterial culture and thus the solvent act as an inert carrier (Miller and Johnson, 1966b; Wegner, 1967). As far as it can be determined, no processes in which solid n-alkanes are used as a carbon and energy source have been investigated in other than a laboratory scale. C. ALCOHOLS-METHANOL AND ETHANOL Methanol and ethanol are two chemicals derived from hydrocarbons that are of interest as substrates for SCP production. Methanol offers a means for recovering natural gas that would be flared at wells in producing regions that are remote from markets (Rosenzweigand Ushio, 1974). Use of methanol as a substrate for SCP production has a number of advantages as follows: (1) high solubility in water; (2) lack of explosive hazards of methanol-oxygen mixtures as compared with methane-oxygen mixtures; (3) freedom from traces of aromatic hydrocarbons; (4) ease of removal from the microbial cell product. Ethanol is produced from catalytic dehydration of ethylene. It has similar advantages for use in SCP production to those of methanol. Reviews have been published on the pathways of C1 compound metabolism by microorganisms (Ribbonset al., 1970)and on the utilization of methanol by bacteria and yeasts (Cooney and Levine, 1972; Kosaric and

SINGLE-CELL PROTEIN PROCESSES

285

Zajic, 1974). Bacteria that utilize methane as a carbon energy source for growth such as Pseudmonas methanica, Methanomonus methanooxiduns, Methanoococcus capsulutus, also utilize methanol (Whittenbury et al., 1970). However, there are other bacteria that do not utilize methane but do utilize methanol as a carbon and energy source for growth. Examples of these organisms include Pseudomonus extorquens (Harrison, 1973; Downs and Harrison, 1974), Hyphomicrobium sp. (Wilkinson and Hamer, 1972; Wilkinson and Harrison, 1973; Wilkinson et al., 1974), Methylomonus methanolica (Dostalek et al., 1972; Dostalek and Molin, 1975), and Pseudomonas utilis and P . inaudita (Yoshikawa et al., 1975). Certain actinomycetes and yeasts also utilize methanol readily, Examples include Streptomyces sp. (Kato et al., 1974) Torulopsis ghbrata (Asthana et al., 1971), T. methanosorba and T . methanodomercqii (Yokote et al., 1974); Kloeckera sp. No. 2201; Candidu boidinii (Sahm and Wagner, 1972), and Hansensula polymorpha (Levine and Cooney, 1973). Table V summarizes bacterial SCP processes based on methanol and Table VI yeast and actinomycete SCP processes based on methanol and ethanol. Bacterial processes utilizing methanol as the substrate have the advantages over yeast processes of higher growth rates, yield coefficients, and productivities. The oxidation of methanol by bacteria takes place by an enzyme system that is independent of nicotinamide adenine dinucleotide (NAD). On the other hand, oxidation of methanol by yeast apparently takes place through an alcohol oxidase that requires flavin adenine dinucleotide (FAD) (Ribbon et aZ., 1970; Sahm and Wagner, 1972, 1975). The lower yield coefficients obtained in yeast growth on methanol may result from a lower yield of adenosine triphosphate (ATP)from reduced FAD than from reduced NAD. Less heat is liberated during the growth of microorganisms on methanol than on n-alkanes. Depending upon yields of cells, typical values of heat liberated by bacterial growth on methanol range from 3300 to 5700 kcal/gm (MacLennan et al., 1973; Gow et al., 1975). Even at these levels of heat liberation, refrigeration would be required for cooling fermentors used in the production of SCP from methanol at growth temperatures in the range of 30" to 32°C. Emphasis has been placed on isolation of thermotolerant organisms that will grow on methanol in the temperature range 37" to 45°C. Both bacteria such as Pseudomonas sp. (Nagai, 1973) and Pseudomonas methylotropha (Methylophilus methylotrophus) (Young, 1973; Gow et al., 1975; Anonynous, 1976e) and yeasts, such as Hansenula polymorpha (Levine and Cooney, 1973) and an unspecified strain (Nagai, 1973), grow well on methanol in this temperature range. Imperial Chemical Industries Limited has developed the most advanced process for SCP production from methanol. Methylophilus methylotrophus is the organism used (Anonymous, 1976e). A novel type of air-lift fermentor,

GROWTHOF

TABLE V BACTERIAON METHANOL

SELECTED

Scale and conditions (aeration, agitation)

Temperature (“C)

pH

Hyphomicrobiurn sp., 0.44%

Batch, 10-liter fermentor

-

6.4

0.20

0.7 (12 hours)

-

-

Wilkinson and Hamer (1972)

Methanomonas methan ica , 3.5 ml%

Batch, 1.25-liter fermentor, 800 ml of medium 0.16 literdmin, 610 rpm

30

5.5-6.7

0.19

0.412 (44.3 hours)

-

-

Hamer (1968)

Methylomonas methanolica, 0.40 7%

(a) Batch, fermentor 4-liter working vol, 300 mM O i l liter/hr, 1.5 v/v/min, 1200 rpm

30

6.0

0.53

18.00

0.46

2.63

Dostalek et al. (1972)

(b) Continuous fermentor,

30

6.0

0.24

9.6

0.48

2.3

Dostilek et al. (1972), Dostilek and Molin (1975)

Organism and substrate concentration ~~

~

Specific Cell growth rate density or dilution (d) (@liter, rate (dry (hours-’) weight)

Yield per gm/gm substrate Productivity utilized (dry (gm/liter/hr, weight basis) (dry weight basis)

References

~

4-liter working vol, 300400 mM Odliterslhr, 1.5 vivimin, 1200-1500 rPm

Methylophilus (Pseudomonas) methylotrophus

3540

Continuous, pressurecycle air-lift fermentor, 1000 metric ton/year capacity

-

0.38-0.5

0.5

7

MacLennan et al. (1973), Young (1973), Gow et

ul. (1975), Anonymous (1976e)

Pseudomonas SP.

Pseudomonas

N

W

~

Continuous 1- to 50-liter fermentors

38

-

0.77

-

0.46

-

Nagai (1973)

Batch 2-liter fermentor,

30

6.4

0.22

1.2

-

-

Downs and Harrison (1974)

30

6.4

0.05

2.3-2.4

-

-

Downs and Harrison (1974)

31

6.3

0.31

4.5

0.41

-

ertorquens, 0.5%

0.5 literlmin, 1200 rpm Continuous, 2-1 fermentor, 0.5 literimin, 1200 rpm

TM-20, mixed culture, 0.1%

Batch, 10 liters working vol fermentor, 0.3 literimin, 690 rpm

VIV

Haggstrom (1969)

288

JOHN H . LITCHFIELD

TABLE GROWTHOF SELECTED ACTINOMYCETES

Organism and

Scale and conditions (aeration

Temperature

substrate

agitation)

(“C)

PH -

Batch, 500-ml shake flask, 100 ml medium

37

7.0

-

Semicontinuous, 60-liter fermentor (working volume), 1 v/v/min, 1500rpm

28

5.0

0.20 (maw) 0.14 (mean)

Batch, 30 1 fermentor, 17-liter medium, 17 literdmin, 500 rpm

40

3.5

0.45

Continuous (same conditions)

40

3.5

0.20 (d)

Continuous, Hansenula polymorpha DL-1, fermentor, 375 ml ATCC 26012 (methanol, 5 ml/ medium liter

37

4.5

0.22 (0.13 d)

Torulopsis glabrata (methanol)

Continuous, I-liter fermentor, 0.2 litedmin, 1700 rpm

30

4.5

0.19 (ma..) 0.065 (d)

Batch, 5-liter fermentor

30

5.5

0.092

Continuous, 1-50-1ite1 ferrnentors

30

-

0.263

Actinomycetes Sheptomyces sp. No. 239, (methanol) 1 gm/100 ml Yeasts Candida boidinii (methanol), 0.5%

Candidu ethanothennophilum, ATCC 20380 (ethanol), 20%

(1%)

Torubpsis methanoswba ATCC 20361 (methanol), I% Yeast (methanol)

Specific growth rate or dilution (d) rate (hours-’)

called the “pressure cycle” fermentor, has been developed for use in the process. This system is described by Gow et al. (1975). It is designed to maintain higher oxygen transfer rate without oxygen limitation, remove the heat liberated during growth at high productivity, maintain a homogeneous liquid phase, and avoid sterility problems encountered with conventional fermentors because of contamination through drive shafts and mechanical seals. ICI has developed a proprietary agglomeration process for the initial separation of bacterial cells from the growth medium, which permits the final centrifugation of a much higher solid slurry than is possible otherwise. A 1000-metric ton per year pilot plant has been operated for 3 years. A

289

SINGLE-CELL PROTEIN PROCESSES

VI AND

YEASTS

ON

Cell density (gm/liter, dry weight)

METHANOLOR ETHANOL

Yield per gm/gm substrate utilized (dry weight basis)

Productivity (gmllitedhr, dry weight basis)

0.35

-

Kato et a1 (1974)

30" (70 hours)

0.2gb

-

(a) Sahm and Wagner (1972,

-

0.84

-

8.0

0.95

1.6

1.2

0.36

0.2

Levine and Cooney (1973), Cooney and Levine (1975), Cooney et a/. (1975)

4.55

0.57

-

Asthana et al. (1971)

17 (47 hours)

-

-

Yokote et al. (1974)

-

0.39

-

Nagai (1973)

3.2 (6 days)

References

1975) (b) Reuss e t a / . (1975) Masuda et al. (1975)

50,000-75,OOO-metric ton manual capacity plant is now under construction for start-up in late 1979 (Anonymous, 1976e). Mitsubishi Gas Chemical Company, Inc., Toyko, Japan has developed pilot plant processes from the growth of yeast that will tolerate temperatures in the range of 40°C when grown on methanol as a substrate and bacteria of the genus Pseudomonas that will grow on methanol at 38°C (Nagai, 1973). However, these processes have not yet been scaled up to the extent of the ICI process. Mitsubishi Petrochemical Co., Ltd., Tokyo, Japan has investigated both bacterial and yeast SCP processes based on methanol in both batch and

290

JOHN H . LITCHFIELD

continuous culture systems (Masuda and Yoshikawa, 1973; Tung, 1975). A bacterial SCP has been produced from methanol in a 500-metric ton per year pilot plant. Ethanol can be used by certain bacteria and yeasts as a substrate for SCP production. It has the advantage over methanol of acceptability to regulatory agencies as a raw material for producing a food grade end product. Organisms that utilize ethanol of interest in SCP production include Acinetobacter calcoaceticus (Laskin, 1975), Candida acidothermophilum and Candidu ethanothermophilum (Masuda et al., 1975), and Candidu utilis (Amoco Foods Co., 1974; Ridgeway et al., 1975). The yeast-based SCP processes have been developed to a greater extent than bacterial SCP processes using ethanol as a substrate. Table VI summarizes the growth characteristics of selected yeasts on ethanol as the substrate. At the present time, Amoco Foods Co. is producing C. utilis from ethanol in a 5000-ton per year plant at Hutchinson, Minnesota (Amoco Foods Co., 1974). In this aseptic, continuous process, the ethanol concentration is maintained at approximately 200 ppm. The nitrogen source, either aqueous or anhydrous ammonia, is supplied continuously to maintain pH in the desired range. Other macro and micro elements are also supplied continuously. All liquid streams except ammonia are sterilized at 149°C (300°F). Ammonia is sterilized by filtration and air by compression and filtration. Typical operating conditions are 30"C, pH 4.6, and aeration and agitation to give oxygen absorption rates in the range 100 to 140 d f l i t e r per hour. Cell concentrations of 6-7 gmfliter (dry weight basis) are obtained at a space velocity of 0.3 hr-' (Ridgeway et al., 1975). In addition, a 1000-metricton per year pilot plant-scale plant for growing C. UtiZis on ethanol has been operated at Kojetin, Czechoslovakia, with planned scale-up to 60,OOO metric tons per year (Wells, 1975). However, information on the operating conditions used in this process has not yet been published. Mitsubishi Petrochemical Co., Ltd. is developing a pilot plant-scale process for producing C. acidothermaphilum and C. ethanothermophilum from ethanol, but it has not reached a commercial-scale production level (Anonymous, 1974e; Masuda et al., 1975). The advantages claimed for this process are operation in the pH range 2.5 to 4.0, and at temperatures up to 40°C. Exxon Corporation in a joint venture with Nestle Alimentana, S.A. has conducted small pilot plant-scale studies on the production of Acinetobacter calcoaceticus from ethanol (Laskin, 1975). Again, detailed information on this process has not yet been released.

SINGLE-CELL PROTEIN PROCESSES

29 1

IV. Economic Aspects A number of factors affect the cost of producing SCP products from hydrocarbons, methanol, or ethanol. These include the organism, feedstock and nutrients, process conditions, product recovery requirements, and cooling requirements. From the standpoint of organisms used, whether bacteria, yeasts, actinomycetes, or fungi, growth rate, yield from a given substrate, stability in continuous culture, and size of cell are important factors. Growth rate and yield influence productivity and in turn the size and capital investment of facilities required for a given production level. Genetic stability of the organism is important since it is desirable to practice SCP processes in a continuous mode over extended periods of time to obtain higher productivities than possible in batch cultures. Large cells such as those of yeasts are more readily separated from aqueous growth media at a lower cost by centrifugation than are small size cells such as those of bacteria. A.

RAW

MATERIALS COSTS

The costs of feedstocks such as hydrocarbons, methanol, and ethanol, and nutrients such as ammonia and phosphoric acid are important components of direct manufacturing costs of SCP Products. Table VII presents some recent prices of these raw materials and quantities required for selected processes based on representative published data. Gas oil, although a relatively low cost source of n-alkanes, has fallen into disfavor because of the complex and costly purification processes that must be used to prepare a SCP product that will meet the requirements of regulatory agencies in various countries. n-Alkanes can be prepared in 98-99% purity by molecular sieve separation processes (Masuda, 1972; Reber and Symoniak, 1975). Purified n-alkanes prepared in this manner should meet U. S . Food and Drug Administration standards for purity for white mineral oil (Code of Federal Regulations, 1976). This degree of purification increases the cost of substrate considerably over that of gas oil. However, product purification costs are less. It is most important to note that the prices given in Table VII are those of commercial suppliers. Large petroleum companies may use a different basis for determining the cost of hydrocarbons, methanol, or ethanol produced internally. Rapid increases in the price of crude oil over the past several years has also led to corresponding increases in cost of hydrocarbon substrates such as gas oil or n-alkanes to SCP producers. The prices of methanol and ethanol have also risen during this same period.

h

W

MATERIAL PRICE

RANGES AND

TABLE VII APPROXIMATE QUANTITIES REQUIRED FOR SELECTED SINGLE-CELL PROTEINPROCESSES Approximate quantity (kg/kg of SCP product, dry basis)

Raw material Energy source Gas oil Purified n-alkanes Ethanol Methanol Nitrogen and phosphorus sources Ammonia, anhydrous Phosphoric acid, feed grade, 85%; 100% basis Other mineral saltsb FeSO,. 7H,O MgS04.7H,0 Mn S 0 4 . H 2 0 KCI ZnSO,. H,O

Recent price 1976O (U.S. dollars/kg)

Yeast processes

Gas oil

Purified n-alkanes

Ethanol

Methanol

2.0

0.11 0.25 0.39 0.14

1.1

-

-

1.05 -

0.21 0.44

0.16 0.086

0.14 0.052

0.09 0.05

0.13 0.095

0.032

0.022

0.03

0.03

0.07 0.58

~

Typical prices from U.S. supplier sources. 'Prices for feed-grade chemicals.

-

-

Bacterial process

0.9

-

SINGLE-CELL PROTEIN PROCESSES

293

Ammonia is the preferred nitrogen source for use in SCP production on the basis of its use in pH control, ease of handling, and cost. The price of ammonia is directly related to the cost of energy in the form of natural gas used for its production. For a source of phosphorus, feed grade phosphoric acid is preferred over fertilizer grade because of the presence of undesirable impurities, such as iron, arsenic, and fluoride in the latter. Other mineral nutrients required as sources of K, Mg, Mn, Zn, and Fe, should be supplied as hydroxides or sulfates rather than chlorides to minimize corrosion of stainless steel equipment.

B.

PROCESS

FACTORS AFFECTING COSTS

The importance of process conditions in various SCP Processes has been discussed previously. Factors such as pH, temperature, aeration and agitation systems and recycle of nutrients also affect cost of operation. A low pH in the range of 2.5 to 4.0 minimizes contamination problems in nonsterile systems and high temperatures in the range of 3545°C minimize cooling requirements. The aeration systems used (bagled and agitated vessels or air lift fermentors) also influences productivities and yields, and energy consumption and therefore, direct manufacturing costs. It is important to note that it may not be economical to operate at maximum productivity. In the case of Candida sp. grown on gas oil, increases in productivity above 3 gmlliter per hour decrease rapidly with increasing energy consumption although maximum productivity of 4 gmfliter per hour can be obtained (Laine and du Chaffaut, 1975). Recycle of nutrients in continuous processes is an important means for maximum utilization of raw materials. The cost of recovery of the SCP product is dected by the concentration of cell solids in the growth medium, extent of solubility of the substrate in the cells, cell size, density, viscosity, requirements for removing residues of unused substrates and toxic substances from the cells and susceptibility of the flavor, aroma, color, and nutrient contents of cells to damage by heat during drying. Also, the cell recovery process should yield a final product having low level of microbial contaminants including organisms that are pathogenic to either domestic livestock or man. The costs of initial separation of cells from the growth medium depend largely on the physical properties of the cells themselves (Wang, 1968a,b, 1969; Labuza, 1975). Removal of water from the cells by drying is a costly procedure. It is important to remove as much water as possible in initial procedures prior to drying. Processes other than the centrifugation that have been used for initial dewatering of cells include filtration, flocculation, and

294

JOHN H . LITCHFIELD

evaporation. The energy cost of cell separation by evaporation using a multiple effect system may still exceed those of centrifugation (Labuza, 1975). The use of a proprietary agglomeration process for initial cell separation followed by centrifugation for final dewatering of higher solids cell slurries developed by ICI has been discussed previously (Young, 1973; Gow et al., 1975; Anonymous, 1976e). Heat is released during the growth of microorganisms on hydrocarbons, methanol, and ethanol. With growth temperatures in the range of 3Oo-32"C and cooling water temperature in the range of 20°C, refrigeration will be required in SCP processes. However, the requirements for refrigeration can be lowered by using organisms that grow in the range 35"-45"C. For example, the estimated cooling requirement for the British Petroleum purified n-alkanes or gas oil processes for a 100,W-metric ton per year plant is approximately 110 x 10' kcalhr (BP Proteins Ltd., 1974). Substantial quantities of water are used in all SCP process to make up the growth medium. Estimated water requirements for a 100,000-metricton per year SCP plant for various processes are as follows: yeasts, n-alkanes, 18.2 million liters; bacteria, methanol, 45.5 million liters; and bacteria, methane 18.2 million liters (Ratledge, 1975). It is obvious that the spent growth medium must be recycled to minimize fresh water requirements and costs and waste water treatment requirements and costs.

C . MANUFACTURINGCOSTS Table VIII presents published comparisons of manufacturing costs for selected SCP processes based on hydrocarbons and methanol as substrates. One must be careful in comparing such cost breakdowns which have been computed in different years, and even if in the same year on Mering raw material, utilities, labor, maintenance and depreciation cost bases. For example, the British Petroleum gas oil process data take into account a dewaxing premium and the absence of n-alkane extraction unit. It is apparent that manufacturing costs are highly dependent upon the cost of the carbon and energy source to the SCP producer. Current prices given in Table VII indicate that percentage of total manufacturing costs attributable to the carbon and energy source may even be greater in the future for all products derived from petroleum, depending upon the relative increases in the costs of nitrogen, phosphorus, energy, and labor in the geographic regions where major SCP plants are located. In any event, the values given in Table VIII are based on estimates and would have to be confirmed by actual plant operating experience.

TABLE VIII COMPARISON OF PUBLISHEDMANUFACTURING COSTSFOR SELECTEDSINGLECELL PROTEINPROCESSES Percent of total manufacturing costs as calculated in year indicated Yeast processes Purified n-alkanes

Bacterial process

Gas oil, British petroleuma (1969)

British petroleumb ( 1969)

(1974)

Carbon and energy source

13

40

4546

Mineral nutrients and other chemicals

30

18

12-14

57'5 17.2

Utilities

25

18

12-24

18.3

11

16-44

3.8

11

-

-

-

-

3.2

-

17-25

-

15

Cost component

Labor Maintenance Insurance and taxes

32

Materials and supplies Depreciation "Bennett et a / . (1969). bBennett et a/.(1969). CAnonymous(1974a). dAnonymous (1974a), Giacobbe, et a / . (1975). ?Young (1973).

Gulp

Liquichimicad (1974, 1975)

1

Methanol ICI' (1973)

63

24

296

JOHN H. LITCHF‘IELD

D. CAPITALCOSTS SCP processes are both capital and energy intensive. In addition to recent increases in energy costs in most countries where SCP production facilities are located, plant construction, and equipment costs have also increased substantially. Capital cost estimates in the early 1970s for the British Petroleum n-alkanes process at a 100,OOO metric tonslyear scale were $25 to $30 million and $30 to $40 million for plant and built-up costs, respectively. For the Gulf n-alkanes process at a 45,000 metric tonslyear scale, estimated capital costs were $20 to $26 million, and for the Liquichimica (Kanegafuchi)process at a 100,OOO metric tonslyear scale, $39 million excluding working capital (Anonymous, 1974a). By 1975, typical estimates of fixed capital costs for a 100,OOO metric tons/ year SCP plant (U. S. Gulf Coast) were $106.7 million, $90.5 million, $72.2 million, and $66.0 million for gas oil, purified n-alkanes methane, and methanol-based processes, respectively (Brownstein and Constantinides, 1975). In 1976, the estimated cost of the ICI plant for producing bacterial SCP from methanol at a 50,OOO to 75,000 metric tondyear scale was $70 million (Anonymous, 1976e).

V. Plant and Animal Protein Products Competing with SCP Table IX summarizes recent selling prices for various food and feed grade microbial cell products including yeast SCP products grown on hydrocarbons. These prices are expressed on both a product basis and on crude and “true” protein bases. Crude protein (N x 6.25) is the usual basis for selling animal feedstuffs. However, since microbial cells contain signhcant quantities of nonprotein nitrogen constituents such as nucleic acids, the 6.25 factor may overstate the actual protein contents of SCP products. “True” protein calculated from amino acid analysis may be a more reliable measure of the protein contents of SCP products. Table X presents recent selling prices of selected plant and animal protein products used for feed or feed. Since nonprotein nitrogen contents of these products are relatively lower than those of SCP products, prices are expressed on product and crude protein (N x 6.25) bases. A. FOODUSES

For food applications, soybean protein is the major plant food protein product competition that SCP products would have to meet at the present

TABLE IX COMPARISON OF SELLINGPRICE RANGEOF SELECTEDSINGLE-CELLPROTEIN PRODUCTS Typical protein content (%, dry basis) Product, substrate, and quality

Price range, 1975-1976 (U.S. dollarsikg")

Crude protein (N X 6.25)

True protein'

Product basis

Crude protein basis

True protein basis

52

46

1.07-1.10

2.06-2.12

2.33-2.39

52

44

0.99-1.01

1.80-1.84

2.25-2.30

52

41

0.55-0.59

1.06-1.13

1.34-1.44

52

45

0.99-1.01

1 .90-1.94

2.20-2.24

48

40

0.40-0.55

0.83-1.15

1.00-1.38

60

52

0.456

0.75

0.87

Candida lipoliytica

66

58

0.566

0.85

0.97

(gas oil) feed grade Kluyw o r ny ces fragilis (whey) food grade

54

48

0.99-1.05

1.83-1.94

2.06-2.19

Candida utilis (ethanol) food grade Candidu utilis (NF) (sulfite waste liquor) food grade Candida utilis (sulfite waste liquor) food grade Saccharom yces cerevisiae (molasses) food grade Brewers' dried yeast, feed grade Candida lipolytica (n -alkanes) feed grade

aProtein content based on amino acid contents. bCalculated on basis of U.S. dollars equivalent of United Kingdom prices in 1975. 'Prices from industry sources.

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TABLE X COMPARISON OF TYPICAL SELLING PRICESOF SELECTED PLANTAND ANIMALFOODAND FEEDPROTEINPRODUCTS

Product Food proteins Soy protein concentrate Soy protein isolate Casein (edible) Feed proteins Soybean meal (defatted) Fish meal Menhaden Anchovy (Peru) Cottonseed meal Peanut meal Meat and bone meal

Price range, 1976" (U.S.dollars/kg)

Crude protein content (N x 6.25, dry basis)

Product basis

Crude protein basis

70-72 90-92 89

0.70-0.84 1.54-1.67 1.17

0.97-1.20 1.67-1.86 1.31

44 49

0.18-0.20 0.20-0.22

0.41-0.45 0.40-0.45

60 65 41 51 50

0.40-0.44 0.40-0.44 0.17-0.22 0.21-0.22 0.20-0.24

0.67-0.73 0.62-0.68 0.41-0.54 0.41-0.43 0.40-0.48

Typical prices from U . S . supplier sources.

time. This conclusion applies to either nutritional or functional uses of SCP products in food. Soybean protein concentrates containing 70-72% protein and soybean protein isolate containing 9045% protein are available as food ingredients commercially. Soybean products in general, have lower methionine contents than is desirable for human nutrition. These products would require methionine supplementation as would most SCP products to be acceptable as a balanced source of protein for human nutritional use as a sole source of protein in the diet. However, soybean protein isolates and concentrates are sold to the food industry primarily for their functional value in food rather than for their nutritional value. Any SCP product designed to be sold as a functional food ingredient would have to compete on functional and cost equivalent bases with these soybean products to enjoy acceptance in the marketplace. In the future, other plant protein products, such as glandless cottonseed protein and peanut protein concentrates and isolates, may also be potential competitors of SCP products. However, market acceptability of these products has not yet been established since only experimental quantities have been produced. Furthermore, one cannot discount potential competition from proteins of animal origin, such as functional fish protein isolates, milk

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299

proteins, or meat proteins. All these agricultural commodities fluctuate widely in price depending upon supply and demand and often may sell as prices below levels that can be met by a SCP product produced in a capitalintensive production facility.

For feed stuff applications, major competitors for SCP products at the present time are soybean meal (44%crude protein), and fish meal (6045% crude protein). Price comparisons of SCP products with these established feed ingredients must take into account not only protein contents, but also the need for supplementary methionine to bring their feeding value to comparable levels. In the United States, the animal feed industry uses least-cost feed formulation methods that trade off one feed stuff against another on the basis of nutritional value and price. This is also increasingly the case in Japan and Western Europe. At one time, it appeared that SCP products would be attractive in Western European countries as a substitute for soybean meal or for fish meal. However, wide price fluctuations in these commodities may result in SCP products being noncompetitive over substantial periods of time taking into account the high capital and operating costs of SCP production facilities. Under such circumstances, consumers of SCP products could benefit to the detriment of suppliers.

VI. Nutritional Value and Safety Extensive information is now available on the composition of various SCP products, including proximate analyses and contents of amino acids, nucleic acids, vitamins, and minerals (Waslien, 1975). Also the nutritional value of bacterial and yeast SCP products grown on hydrocarbons and methanol have been evaluated in animal species ranging from rodents to domestic livestock including broiler chickens, laying hens, swine, and calves (BP Proteins, Ltd, 1974; Gow et al., 1975; Litchfield, 1975; Shacklady and Gatumel, 1973). Typical Biological Values (BV) for Candida lipolyticu strains grown on gas oil or n-alkanes are 54 and 61, respectively. After supplementation of the yeast SCP products with 0.3% DL-methionine, the corresponding Biological Values are 96 and 91, respectively, as compared with 65 and 97, respectively, for soybean protein and dried whole egg supplemented with 0.3% DL-methionine (Shacklady and Gatumel, 1973). For animal feed applications, SCP products give the best performance in the range 5 to 15%of the ration. At levels above 15%,significant decreases in

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performance occur in broiler chickens, and this level appears to be the practical limit for use in swine rations (Litchfield, 1975). As a milk replacer for calves, a 7.5% level of C . lipolytica grown on either gas oil or purified n-alkanes appears to be the practical limit of use (Shacklady and Gatumel, 1973). The Protein Advisory Group (PAG) of the United Nations has issued guidelines for SCP products and for evaluation of novel sources of protein including preclinical and human testing (Protein Advisory Group, 1970a,b,c, 1971, 1972, 1974). In addition to these guidelines, regulatory agencies in most countries have requirements for efficacy and safety of food additives that must be met by a SCP product destined for sale in a given country. For example, in the United States the Food and Drug Administration has promulgated regulations for food yeasts and products derived from them (Food and Drug Administration, 1963; Code of Federal Regulations, 1976). If a SCP product is to be added to food as a major or even supplementary source of protein, then the nucleic acid contents must be reduced to acceptable levels to prevent adverse reactions such as the development of kidney stones and gout in humans consuming these products as a significant portion of the diet (Scrimshaw, 1975). Processes for reducing the nucleic acid contents of SCP products include acid precipitation, acid or alkaline hydrolysis, heat shock and incubation for endogenous nuclease action, and use of exogenous nucleases (Litchfield, 1977; Sinskey and Tannenbaum, 1975). The PAG has accepted a 2 gm per day limit of ribonucleic acid (RNA) from an SCP product (Protein Advisory Group, 1972). Considerable concern has been expressed on the safety of SCP products grown on hydrocarbon substrates (Anonymous, 1973a, 1976c,d). The major questions relate to the possible presence of traces of polycyclic aromatic compounds or residual n-alkanes in the SCP product. Extensive studies have been conducted in rats and mice to determine carcinogenicity, mutagenicity, and teratogenicity of SCP products grown on hydrocarbon substrates, without adverse effects being noted (Engel, 1973). The PAG Ad hoc Working Group on Single Cell Proteins has reviewed the status of information on n-alkane and aromatic hydrocarbon residues and the presence of odd-carbon fatty acids in SCP products and the safety of hydrocarbon-grown SCP products for use in animal feeding (Protein Advisory Group, 1976a,b). It was concluded that the low levels of residual n-alkanes present and contents of odd-carbon fatty acids do not present a hazard, and that safe and nutritionally acceptable SCP products for use in animal feeding applications can be produced. Also, conditions used in continuous cultivation of strains of microorganisms used in SCP production would minimize the possibility of mutants arising during growth that might produce toxic substances.

SINGLE-CELL PROTEIN PROCESSES

30 1

VII. Conclusions Single-cell protein products can be produced by growing bacteria or yeasts on a variety of hydrocarbon substrates and on methanol or ethanol. At the present time, the following factors are retarding the construction and operation of commercial-scale plants for producing feed-grade SCP products from hydrocarbons: (1)high costs of hydrocarbon feed stocks; (2)high construction and equipment costs; (3) wide price fluctuations in competing protein feedstuffs, such as soybean and fish meal, to levels below those that can be met by SCP products from capital and energy intensive processes; (4)delays in approval of SCP products grown on hydrocarbons by regulatory agencies in counties where major production facilities are planned or have been constructed. A food grade yeast SCP product is being produced from ethanol in the United States for use as a functional food ingredient. Also, the construction of a facility for producing a bacterial SCP product from methanol for feed use is under way in the United Kingdom. The long-term economic viability of these products will depend upon their ability to compete on price, availability, and utility in foods or feeds with conventional animal and plant protein sources. REFERENCES Aiba, S., Haung, K. L., Moritz, V., and Someya, J. (1969). J . Ferment. Technol. 47, 203 and 211. Amoco Foods Co. (1974). “Torutein Product Bulletin.” Chicago, Illinois. Anonymous. (1973a).Eur. Chem. News 23(573), 14. Anonymous. (197313). Eur. Chem. News 24(591), 12. Anonymous. (1974a). Ear. Chem. News 25(622), 18. Anonymous. (1974b). Chem. Eng. News. 52 (33), 30. Anonymous. (1976a). Eur. Chem. News 28(720), 6 . Anonymous. (1976b). Wall Street]. 56 (115),p. 6. Anonymous. (1976~).Eur. Chem. News 28(730), 10. Anonymous. (1976d). Chem. Week 118(17), 79. Anonymous. (1976e). Chem. 2nd. (London) No. 20, 859. Asthana, H., Humphrey, A. E., and Moritz, V. (1971). Biotechnol. Bioeng. 13, 923-929. Avrillon, R., Franckowiak, S., Gatellier, C., and Glikmans, G. (1972). U.S. Patent 3,698,998. Barnett, S. M., Velankar, S. K., and Houston, C. W. (1974). Biotechnol. Bioeng. 16, 863-865. Bennett, I. C . , Hondermarck, J. C., and Todd, J. R. (1969).Hydrocarbon Process. 48(3), 104-108. Bewersdod, M., and Dostalek, M. (1971). BiotechnoL Bioeng. 13, 49-62. Blanch, H. W., and Einsele, A. (1973). Biotechnol. Bioeng. 15, 861477. BP Proteins, Ltd. (1974). “The Toprina Cycle.” London, England. Brownstein, A. M., and Constantinides, A. (1975). 169th Natl. Meet., Am. Chem. S O C . , Philadelphia, Paper No. 86. Champagnat, A. (1965). U.S. Patent 3,186,922. Champagnat, A,, and de Mayo, C. (1966). U.S. Patent 3,257,289. Champagnat, A,, and Filosa, J. (1965). U.S. Patent 3,193,390.

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Subject Index A

C

Alamethicin activity of, 183 structure of, 181 Alanine production by immobilized cells, 4 Alborixin microbial source of, 191 structure of, 185 6-Aminopenicillinic acid production by immobilized cells, 3, 15, 22 Aspartic acid production economics of, 16-17 by immobilized aminoacylase, 9 by immobilized aspartase, 9 by immobilized bacteria, 14, 15 by immobilized cells, 5 by immobilized E. coli, 10-13 stability of, 13 reactor design and, 16

Cephalosporin synthesis by immobilized cells, 4 Citrulline production by immobilized cells, 5 by immobilized Pseudomonas putida, 15, 19-29

D Dethiobiotin microbial synthesis of, 161-163 Dianemycin microbial source of, 191 structure of, 186 L-Dopa production by immobilized cells, 4 E

Enniatin, structure of, 181-182

B Biotin biosynthetic pathways to, 146-148 biotin synthesizing reaction, 147, 153155 dethiobiotin synthetase, 147, 151-153 7,8-diaminopelargonic acid, 147, 149151 7-keto-8-aminopelargonicacid, 147, 149 pimilyl-CoA synthetase, 148 regulation of biotin synthesis, 155-157 chemical synthesis of, 158-159 microbial degradation of, 170-172 microbial synthesis of, 160-161 uses of, 146 Biotin antimetabolites, 163 actithiazic acid, 164-166 adenine, 169-170 amiclenomycin, 168-169 a-dehydrobiotin, 166-167 a-methylbiotin, 167-168 a-methyldethiobiotin, 167-168 stravidin, 168-169 307

F

Fructose production by immobilized cells, 5, 6

G Glucose-6-PO4 and glucose-l-P04 production by immobilized cells Gramicidins, structure of, 181 Grisorixin microbial source of, 190 structure of, 184 H

5-Hydroxytryptophan production mobilized cells, 5 I

Immobilized cells, agar and, 2 3 , 8

by

im-

308

SUBJECT INDEX

Immobilized cells, continued alginate and, 2, 8 bifunctional reagents for, 108 cellulose triacetate and, 2 3 , 8 collagen and, 2, 8 cross-linking cells and, 2 entrapment of in matrix, 2 general uses of, 2 historical, 1 ionic binding and, 2 liquid membrane and, 8 methods of immobilization and, 2 polyacrylamide and, 2 3 , 8 polystyrene and, 2 for transformations, prospects for future of,

24

effects of detergents on, 18 stability of, 19 by immobilized cells, 4 Menthol synthesis by immobilized cells, 4 Methionine synthesis by immobilized cells, 4 Monensin activity of an promoting feed utilization in ruminants, 215-216 biosynthetic pathways to, 202-203 microbial source of, 190 microbial transformation of, 203 structure of, 184 Mycotoxins acute and chronic toxicity of, 85 biological effects of, 85 chemical structures of, 88, 89, 90, 91, 92,

94 L Lactose hydrolysis by immobilized cells, 3 Laidlomycin microbial source of, 190 structure of, 185 Lasalocids antibacterial activity and, 213-214 synthetic derivatives of, 213-214 antiCoccidia activity and, 211-213 synthetic derivatives of, 213 biosynthetic pathways to, 19-202 microbial source of, 191 structures of, 187 Lenorem ycin microbial source of, 191 structure of, 186 Lonom ycin microbial source of, 190 structure of, 185 Lysine production by immobilized cells, 4 Lysocellin microbial source of, 191 structure of, 187

M Macrotetralides, structure of, 181-183 Malic acid production by immobilized Brevibacterium moniagenes, 15, 17 economics and, 19

am-

carcinogenic effects and, 95-96 dermatoxic effects and, 91-93 mutagenic effects and, 96-98 nephrotoxicity and, 90-91 neurotoxic effects and, 93 teratogenic effects and, 98 effects of on carbohydrate metabolism, 100-102 on lipid metabolism, 105-106 on mitochondria1 respiration, 102-105 in nonmammalican systems, 98-100 on nucleic acid and protein synthesis,

107-121 hepatotoxicity of, 85, 86 historical, 83, 84 interaction of with macromolecules, 122-

133 nonhepatoxic lesions and, 84

N NADP production by immobilized cells, 3 Narasin microbial source of, 191 structure of, 185 Nigericin microbial source of, 190 structure of, 184 0 Oil pollutants behavior of, 228-229

309

SUBJECT INDEX

microbial attack on, 253-261 sources of, 226-227

P Penicillin synthesis by immobilized cells, 4 Petroleum hydrocarbons enrichment of microbial populations with, 231-232 environmental constraints on microbial attack on, 246-253 microbial emulsification of, 236-246 sublethal effects of, 234-235 suppression of microbial populations with, 232-233 Polyether antibiotics antimicrobial activity of, 203, 208-209, 211 cardiovascular activities and, 216-218 classification of, 178, 183 by chemical structure, 178-179 ionophore antibiotics, 181 by mechanism of action, 178 coccidiostat activity of, 210-211 historical, 177 pharmacology of, 216 physical constants of, 204-207 proposed numbering system for, 188-189 toxicity of, 209, 211

nutritional value and, 299-300 use of product as feed, 269 use of product as food, 268-269 L-Sorbosone production by immobilized cells, 3 Steroid hydroxylation by immobilized cells, 3 Sterol metabolism by microorganisms conversions by mutants, 50-52 conversions in presence of enzyme inhibitors, 4 - 4 9 conversion of sterols with modified structure, 3 7 4 3 mechanisms of degradation of steroid nucleus, 33-34 degradation of sterol side chain, 35 Sterol side-chain cleavage chemical, 2 9 3 0 historical, 29 microbial, 29-30 as part of complete metabolism, 30-33 Sucrose hydrolysis by immobilized cells, 3

T Tryptophan production by immobilized cells,

> Tyrosine production by immobilized cells, 4

S

U

Salinomycin microbial source of, 190 structure of, 184 Septamycin microbial source of, 191 structure of, 186 Single-cell protein processes economics of, 291 capital costs, 296 competing animal and plant products, 296-298 manufacturing costs 294-295 raw material costs 291-294 historical, 267-268 microbial processes and, 269, 271-272 on gaseous hydrocarbons, 270-274 on liquid and solid hydrocarbons, 274283 on alcohols, 284-290

Urocanic acid production by immobilized Achromohacter liquidurn, 15, 21 by immobilized cells, 5

V Valinomycin, structure of, 181-182

Z Zearalanol, structure of, 61 Zearalenone acute toxcity of, 78 chronic toxicity studies, 79-81 assay of, 62-63 endocrine activities and, 75-77 fermentation process and for cultures, 63-64

310 Zearalenone fermentation process and, continued for surface culture, 6 4 4 7 for submerged culture, 68-73 general drug action of, 74-75 historical, 59-60 mechanism of biosynthesis of, 73

SUBJECT INDEX

metabolism of in animals, 77-78 Occurrence of in molded grain, 60 pharmacology of, 74 recovery of from solutions, 63 safety of, 78 structure of, 61 chemical name, 62

CONTENTS OF PREVIOUS VOLUMES Volume 1

Aerosol Samplers

Harold W. Batchelor Protected Fermentation

A Commentary on Microbiological Assaying F . Kauanagh

Milo3 Herold and Jan NeEasek The Mechanism of Penicillin Biosynthesis

Application of Membrane Filters

Arnold L. Demain

Richard Ehrlich Preservation of Foods and Drugs by Ionizing Radiations W . Dexter Bellainy

Microbial Control Methods in the Brewery Gerhard J . Hass

The State of Antibiotics in Plant Disease Control

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

Daoid Pra mer

brandt Microbial Synthesis of Cobamides

The Microbiological Transformation of Steroids

D. Perhan

T. H . Stoudt

Factors Affecting the Antimicrobial Activity of Phenols 6 . 0. Bennett

Biological Transformation of Solar Energy

Willium J . Golueke

Germfree Animal Techniques and Their Applications

Arthur W. Phillips and James E . Smith

B

Oswald and

Clarence

G.

SYMPOSIUM ON ENGINEERING ADVANCESI N FERMENTATION PRACTICE

Insect Microbiology

S . R. Dutky

Rheological Broths

The Production of Amino Acids by Fermentation Processes

Properties

of

Fermentation

Fred H . Deindoeffer and john M . West Fluid Mixing in Fermentation Process

Shukuo Kinoshita

J . Y. Oldshue

Continuous Industrial Fermentations Philip Cerhardt and J 4 . C . Bartlett

Scale-Up of Submerged Fermentations

W .H . Burtholemew The Large-Scale Growth of Higher Fungi Radeliffe F . Robinson and R. S . Dutiidson

Air Sterilization

Arthur E . Humphrey AUTHOR INDEX-SUBJECT

INDEX

Sterilization of Media for Biochemical Processes

Volume 2 Newer Aspects of Waste Treatment

Lloyd L. Kempe Fermentation Kinetics and Model Processes

Nanrlor Purges

Fred H . Deindoerfer 311

312

CONTENTS OF PREVIOUS VOLUMES

Volume 4

Continuous Fermentation

W.V .Maron Control Applications in Fermentation George J . Fuld AUTHOR INDEX-SUBJECT

INDEX

Induced Mutagenesis in the Selection of Microorganisms S . 1. Alikhanian

Volume 3

The Importance of Bacterial Viruses in Industrial Processes, Especially in the Dairy Industry F . J . Babel

Preservation of Bacteria by Lyophilization Robert J . Heckly

Applied Microbiology in Animal Nutrition Harlow H . Hall

Sphaerotilus, Its Nature and Economic Significance Norman C . Vondero

Biological Aspects of Continuous Cultivation of Microorganisms T . Holme

Large-Scale Use of Animal Cell Cultures Donald]. Merchant and C . Richard Eidain

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

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

Submerged Growth of Plant Cells L. 6. Nickell AUTHOR INDEX-SUBJECT

INDEX

Oxidation of Aromatic Compounds by Bacteria Martin H . Rogoff Screening for and Biological Characterizations of Antitumor Agents Using Microorganisms Frank M. Schabel, J r . , and Robert F . Pittill0

Volume 5

The Classification of Actinomycetes in Relation to Their Antibiotic Activity E l i 0 Baldacci

Generation of Electricity by Microbial Action J . B . Dacis

The Metabolism of Cardiac Lactones by Microorganisms Elwood Titus Intermediary Metabolism and Antibiotic Synthesis J . D . Bu'Lock Methods for the Determination of Organic Acids A . C . Hulrne AUTHOR INDEX-SUBJECT

INDEX

Correlations between Microbiological Morphology and the Chemistry of Biocides Adrian Albert

Microorganisms and the Molecular Biology of Cancer 6. F . Cause Rapid Microbiological Determinations with Radioisotopes Gilbert V. Lecin The Present Status of the 2,3-Butylene Glycol Fermentation Sterling K . Long and Roger Patrick Aeration in the Laboratory W. R . Lockhart and R. W. Squires

3 13

CONTENTS OF PREVIOUS VOLUMES

Stability and Degeneration of Microbial Cultures on Repeated Transfer

Fritz Reusser Microbiology of Paint Films

Richard T . Ross The Actinomycetes and Their Antibiotics Selman A. Waksman

Biodegradation: Problems of Molecular Recalcitrance and Microbial Fallibility M. Alexander Cold Sterilization Techniques john B . Opfell and Curtis E . Miller Microbial Production of Metal-Organic Compounds and Complexes

D. Perlman Fuse1 Oil

A. Dinsinoor Webb and John L. zngraham AUTHOR INDEX-SUBJECT

INDEX

Development of Coding Schemes for Microbial Taxonomy

S. T . Cowan

Volume 6

Effects of Microbes on Germfree Animals

Global Impacts of Applied Microbiology: An Appraisal Carl-Gbran Heden and Mortima P. Starr

Uses and Products of Yeasts and Yeast-Like Fungi

Thomas D. Luckey

Walter J . Wickerson and Robert 6. Brown Microbial Processes for Preparation of Radioactive Compounds

D. Pearlinan, Aris P. Bayan, and Nancy A . Guijjire Secondary Factors in Fermentation Processes

Microbial Amylases Walter W . Windish and Nagesh S. sfhatre The Microbiology of Freeze-Dried Foods Gerald J. Silzjerman and Samuel A. Gold-

blith

P. Margalith Nonmedical Uses of Antibiotics

Herbert S. Goldberg Microbial Aspects of Water Pollution Control

Low-Temperature Microbiology Judith Farrell and A. H . Rose AUTHOR INDEX-SUBJECT

INDEX

K . Wuhrinann Volume 8 Microbial Formation and Degradation of Minerals

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

Zrwin W . Sizer

Arnold L. Deinain Genetics in Applied Microbiology

A Discussion of the Training of Applied Microbiologists B . W. Koft and Wayne W . Uinbreit AUTHOR INDEX-SUBJECT

Industrial Fermentations and Their Relations to Regulatory Mechanisms

INDEX

S . 6. Brudby Microbial Ecology and Applied Microbiology

Thomas D. Brock The Ecological Approach to the Study of Activated Sludge Wesley 0 . Pipes

Volume 7 Microbial Carotenogenesis

Alex Ciegler

Control of Bacteria in Nondomestic Water Supplies Cecil W . Chambers and Riorman A. Clarke

3 14

CONTENTS OF PREVIOUS VOLUMES

The Presence of Human Enteric Viruses in Sewage and Their Removal by Conventional Sewage Treatment Methods

Malo-Lactic Fermentation

Ralph E . Kunkee AUTHOR INDEX-SUBJECT

Stephen Alan Kollins Oral Microbiology

INDEX

Volume 10

Heiner Hoffinan Media and Methods for Isolation and Enumeration of the Enterococci Paul A. Hartman, George W . Reinbold, and

Detection of Life in Soil on Earth and Other Planets. Introductory Remarks

Robert L. Starkey For What Shall We Search?

Devi S. Sarasujat

Allan H . Brown Crystal-Forming Bacteria as Insect Pathogens Relevance of Soil Microbiology to Search for Life on Other Planets

Martin H . Rogoff Mycotoxins in Feeds and Foods

Emanuel Borker, Nino F . Insalata, Colette P. Leui, and john S . Witzeman AUTHOR INDEX-SUBJECT

INDEX

G . Stotzky Experiments and Instrumentation for Extraterrestrial Life Detection Gilbert V . Leoin Halophilic Bacteria

D. J . Kushner

Volume 9 The Inclusion of Antimicrobial Agents in Pharmaceutical Products A. D. Russell, june Jenkins, and I . H. Har-

rison Antiserum Production in Experimental Animals

Applied Significance of Polyvalent Bacteriophages

S. G. Bradley Proteins and Enzymes as Taxonomic Tools

Edward D. Garber andJohn W . Rippon Mycotoxins

Alex Cieglw and Eioind B . Lillehoj

Richard M . Hyde Microbial Models of Tumor Metabolism G. F . Guuse Cellulose and Cellulolysis

Brigitta Norkrans Microbiological Aspects of the Formation and Degradation of Cellulose Fibers

L. Juruiek, J. Ross Coloin, and D. R. Whitaker The Biotransformation of Lignin to HumusFacts and Postulates

R. T. Oglesby, R. F . Christman, and C . H . Driver Bulking of Activated Sludge Wesley 0. Pipes

Transformation of Organic Compounds by Fungal Spores Claude Vezina, S. N . Sehgal, and Kartar

Singh Microbial Interactions in Continuous Culture Henry R. Bungay, I l l and Mary Lou

Bungay Chemical Sterilizers (Chemosterilizers)

Paul M . Borick Antibiotics in the Control of Plant Pathogens M . j . Thiruinalachar AUTHOR INDEX-SUBJECT

INDEX

CUMULATIVE AUTHOR INDEX-CUMULATIVE TITLE INDEX

315

CONTENTS OF PREVIOUS VOLUMES

Collection of Microbial Cells Daniel 1. C. Wang and Anthony J . Sinskey

Volume 11 Successes and Failures in the Search for Antibiotics Selman A. Waksman

Fermentor Design

Structure-Activity Relationships of Semisynthetic Penicillins

The Occurrence, Chemistry, and Toxicology of the Microbial Peptide-Lactones A. Taylor

K . E . Price

R. Steel and T . L. Miller

Resistance to Antimicrobial Agents J . S . Kiser, G . 0. Gale, and G . A. Keinp

Microbial Metabolites as Potentially Useful Pharmacologically Active Agents D. Perlman and G. P. Peruzzotti

Microinonospora Taxonomy George Luedemann

AUTHOR INDEX-SUBJECT

Dental Caries and Periodontal Disease Considered as Infectious Diseases

Volume 13

INDEX

Williain Gold The Recovery and Purification of Biochemicals

Chemotaxonomic Relationships Among the Basidiomycetes

Robert 6. Benedict

Victor H . Edwards Ergot Alkaloid Fermentations William ). Kelleher T h e Microbiology of the Hen's Egg R. G . Board Training for the Biochemical Industries 1. L. Hepner

Proton Magnetic Resonance SpectroscopyAn Aid in Identification and Chemotaxonomy of Yeasts P. A . J . Gorin a n d ) . F . T. Spencer Large-Scale Cultivation of Mammalian Cells R. C . Telling and P. J . Radlett Large-Scale Bacteriophage Production

K . Sargent AUTHOR INDEX-SUBJECT

INDEX

Microorganisms as Potential Sources of Food

Jnanendru K . Bhattacharjee

Volume 12 History of the Development of a School of Biochemistry in the Faculty of Technology, University of Manchester

Thoinas Kennedy Walker Fermentation Processes Employed in Vitamin C Synthesis

Milo5 Kulhanek Flavor and Microorganisms P Margalith and Y. Schwartz Mechanisms of Thermal Injury in Nonsporulating Bacteria M . C . Allwood and A. D. Russell

Structure-Activity Relationships among Semisynthetic Cephalosporins M. L. Sassiver und Arthur Lewis

Structure-Activity Relationships in the Tetracycline Series

Robert K . Blackwood and Arthur R. English Microbial Production of Phenazines ). M. Ingrum and A . C . Blackwood The Gibberellin Fermentation E . G . Jeffrey. Metabolism of Acylanilide Herbicides

Richard Bartha and Dacid Prainer

3 16

CONTENTS OF PREVIOUS VOLUMES

Fermentation Equipment

Therapeutic Dentrifrices

G . L. Solonions

j . K . Peterson Some Contributions of the U.S. Department of Agriculture to the Fermentation Industry George E . Ward

The Extracellular Accumulation of Metabolic Products by Hydrocarbon-Degrading Microorganisms Bernard j . Abbott and Wi//iaiii 15. Gledhill

Microbiological Patents in International Litigation

AUTHOR INDEX-SUBIECT

INDEX

john V . Whittenburg Industrial Applications of Continuous Culture: Pharmaceutical Products and Other Products and Processes

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

H . M . Tsuchija AUTHOR INDEX-SUBJECT

INDEX

Volume 15 Medical Applications of Microbial Enzymes

Irwin W . Sizer Immobilized Enzymes

K . L. Smiley and 6. W . Strandberg Microbial Rennets Joseph L. Sardinas Volatile Aroma Components of Wines and Other Fermented Beverages A. Dinsinoor W'ebb and Carlos j . .\.luller Correlative Microbiological Assays

Volume 14 Development of the Fermentation Industries in Great Britain

john j . H . Hustings Chemical Composition as a Criterion in the Classification of Actinomycetes H . A. Lecheualier, Mary P. Lechecalier, and Yancy N . Gerber Prevalence and Distribution of AntibioticProducing Actinomycetes john N . Porter

Ladislac J . Harika Insect Tissue Culture W. F. Hink Metabolites from Animal and Plant Cell Culture

Irving S. Johnson and George B . Boder Structure-Activity Relationships in Coumermycins John C . Godfrey and Kenneth E . Price Chloramphenicol

Vedpal S. Malik Biochemical Activities of Nocardia R. L. Raymond and V . W . jamison

Microbial Utilization of Methanol Charles L. Cooney and Dacid W . Lecine

Microbial Transformations of Antibiotics

Oldrich K . Sebek and D. Perlman In Vioo Evaluation of' Antibacterial Chemotherapeutic Substances A. Kgthrine Miller Modification of Lincomycin

Barney). Magerlein

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. E .

Erickson Microbiology and Fermentations in the Prairie Regional Laboratory of the National

CONTENTS OF PREVIOUS VOLUMES

Research Council of Canada 1946-1971

R. H . Haskins AUTHOR INDEX-SUBJECT

Fungal Sterols and the Mode of Action of the Polyene Antibiotics ). M. T . Hainilton-Miller

INDEX

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

Volume 16 Public Health Significance of Feeding Low Levels of Antibiotics to Animals Thornas N .Jukes Intestinal Microbial Flora of the Pig

R . Kenworthy Antimycin A, a Piscicidal Antibiotic Robert E . Lennon and Claude Vezina Ochratoxins

Kenneth L. Applegate and)ohn R . Chipley Cultivation of Animal Cells in Chemically Defined Media, A Review Kiyoshi Higuchi Genetic and Phenetic Classification of Bacteria R . R . (,'olt~ell Mutation and the Production of Secondary Metabolites Arnold L. Deinain Structure-Activity Relationships in the Actinom ycins Johannes Meienhofer and Eric Atherton Development o f Applied Microbiology at the University of Wisconsin William B . Sarles AUTHOR INDEX-SUBJECT

3 17

Microbiology and Biochemistry of Soy Sauce Fermentation F . M. Young and B . ). B . Wood Contemporary Thoughts on Aspects Applied Microbiology P . S. S. DazLson and K . L . Phillips

of

Some Thoughts on the Microbiological Aspects of Brewing and Other Industries Utilizing Yeast G . 6 . Stetcart Linear Alkylbenzene Sulfonate: Biodegradation and Aquatic Interactions CC'illiarn E . Gledhill

The Story of the American Type Culture Collection-Its History and Development (1899-1973) William A . Clark and Dorothy H . Geary Microbial Penicillin Acylases E . ). Vandamme a n d ) . P . Voets SUBJECT INDEX

Volume 18 Microbial Formation of Environmental Pollutants Martin Alexander

INDEX

Volume 17 Education and Training in Applied MicrobiolOgY

Wayne W. Uinbreit Antimetabolites from Microorganisms Dacid L . Pruess and ) a i m s P . Scannell Lipid Composition as a Guide to the Classification of Bacteria Norman Shaw

Microbial Transformation of Pesticides lean-Murc Bollag Taxonomic Criteria for Mycobacteria and Nocardiae S. G . Bradley a n d ) . S. Bond Effect of Structural Modifications on the Biological Properties of Aminoglycoside Antibiotics Containing 2-Deoxystreptamine Kenneth E . Price, John C. Godfrey, and Hiroshi Kawaguchi

3 18

CONTENTS OF PREVIOUS VOLUMES

Recent Developments of Antibiotic Research and Classification of Antibiotics According to Chemical Structure J d nos Berd y

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

SUBJECT INDEX

Volume 20 Volume 19 Culture Collections and Patent DepositioAs T . 6. Pridhain and C . W . Hesseltine Production of the Same Antibiotics by Members of Different Genera of Microorganisms

Hubert A . Lechecalier Antibiotic-Producing Fungi: Current Status of Nomenclature C . W. Hesseltine and J . J . Ellis Significance of Nucleic Acid Hybridization to Systematics of Actinomycetes S. G . Bradley

The Current Status of Pertussis Vaccine: An Overview

Charles R . Manclark Biologically Active Components and Properties of Bordetella pertussis Stephen 1. Morse Role of the Genetics and Physiology of Bordetella pertussis in the Production of Vaccine and the Study of Host-Parasite Relationships in Pertussis

Charlotte Parker Problems Associated with the Development and Clinical Testing of an Improved Pertussis Vaccine

George R . Anderson Current Status of Nomenclature of AntibioticProducing Bacteria

Erwin F . Lessel

Problems Associated with the Control Testing of Pertussis Vaccine

Jack Cameron Microorganisms in Patent Disclosures

Iming Marcus Microbiological Control of Plant Pathogens Y . Henis and I . Chet

Vinegar: Its History and Development Hubert A. Conner and Rudolph J . Allgeier Microbial Rennets

M . Sternberg Microbiology of Municipal Solid Waste Composting Meluin S . Finstpin and Merry L. Morris

Biosynthesis of Cephalosporins

Nitrification and Denitrification Processes Related to Waste Water Treatment

Preparation of Pharmaceutical Compounds by Immobilized Enzymes and Cells Bernard J . Abbott

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

Toshihiko Kanzaki and Yukio Fujisawa

Cytotoxic and Antitumor Antibiotics Produced by Microorganisms 1. Fuska and B . Proksa SUBlECT INDEX

Volume 21

Synthesis of L-Tyrosine-Related Amino Acids by P-Tyrosinase

Production of Polyene Macrolide Antibiotics

Hideaki Yamada and Hidehiko Kumagai

Juan F . Martin and Lloyd E . McDaniel

CONTENTS OF PREVIOUS VOLUMES

Use of Antibiotics in Agriculture

Tomomasa Misato, Keido K O , and lsamu Yainaguchi Enzymes Involved in P-Lactam Antibiotic Biosynthesis

E . J. Vandamme Information Control in Fermentation Development

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

R . H . Shipmun, L. T . Fan, and 1. 0'.Kao

A 7 8

8

c 9 D O

E

l

F 2

G 3 H 4 1 5 J 6

319

Environmental Transformation of Alkylated and Inorganic Forms of Certain Metals

Jitendra Saxenu and Philip H . Howard Bacterial Neuraminidase and Altered Immunological Behavior of Treated Mammalian Cells Prasunta K . Ray Pharmacologically Active Compounds from Microbial Origin Hewitt W. Mutthews und Barbara Fritche

Wade SUBJECT INDEX

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