Advances in Applied Microbiology, Volume 21

ADVANCES IN

Applied MicrobioIogy VOLUME 21

CONTRIBUTORS TO THIS VOLUME

L. T. Fan D. J. D. Hockenhull Philip H. Howard I. C. Kao Keido KO Lloyd E. McDaniel Juan F. Martin Hewitt W. Matthews Tomomasa Misato Prasanta K. Ray Jitendra Saxena

R. H. Shipman E . J. Vandamme

Barbara Fritche Wade Isamu Yamaguchi

ADVANCES IN

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

VOLUME 21

@

1977

ACADEMIC PRESS, New York San Francisco London A Subsidiary of Harcourt Brace Jovanovich, Publishers

COPYRIGHT 0 1977, BY ACADEMIC PRESS,INC. ALL RIGHTS RESERVED. NO PART OF THIS PUBLICATION MAY BE REPRODUCED OR TRANSMITTED IN ANY FORM OR BY ANY MEANS, ELECTRONIC OR MECHANICAL, INCLUDING PHOTOCOPY, RECORDING, OR ANY INFORMATION STORAGE AND RETRIEVAL SYSTEM, WITHOUT PERMISSION IN WRITING FROM THE PUBLISHER.

ACADEMIC PRESS, INC.

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United Kingdom Edition published by ACADEMIC PRESS, INC. (LONDON) LTD. 24/28 Oval Road. London N W l

LIBRARY OF CONGRESS CATALOG CARD NUMBER:59-13823 ISBN 0-12-002621-X PRINTED IN THE UNITED STATES OF AMERICA

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

ix

Production of Polyene Macrolide Antibiotics JUAN

F . MARTIN AND

LLOYD

E . MCDANIEL

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

11. Factors Affecting Polyene Macrolide Production ....................... I11. Interaction of Polyene Macrolide Antibiotics with the Producer Cells . . . . . IV . Genetics of Polyene-Producing Streptomyces ..........................

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

2 14 37 43 47

Use of Antibiotics in Agriculture

TOMOMASA MISATO. KEIDO KO. AND ISAMU YAMAGUCHI I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I1. Agricultural Antibiotics and the Pollution Problem . . ...... .... 111. Utilization of Medical Antibiotics as Agricultural Chemicals . . . . . . . . . . . . . . . . . IV . Antibiotics Developed as Agricultural Chemicals ........................... V . Future Prospects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References ...........................................................

53 54 55

60 82 83

Enzymes Involved in p-Lactam Antibiotic Biosynthesis

E . J . VANDAMME I. I1. 111. IV . V.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Structure of B-Lactam Antibiotics ........................................ Biosynthesis Mechanisms of B-Lactam Antibiotics and Their Enzymes . . . . . . . . Terminal-Stage Enzyme Reactions in a-Lactam Antibiotic Biosynthesis . . . . . . . Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

89 89 92 97 117 119

Information Control in Fermentation Development

D . J . D . HOCKENHULL I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I1 . Research and Development Budget ...................................... V

125 133

vi

CONTENTS

111. Project Initiation Request (Research or Development Program) . . . . . . . . . . . . . .

.......................... V. Periodical Report ................... VI. Laboratory and Plant Protocols . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VII. The Standard Operating Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VIII. Direct Experimental Records . . . ...................... M. Miscellaneous In ........... X. Minutes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . XI. Collection and Flow of Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . XII. Making the Best ....................... References . . . . .............................. ..... IV. Formal Program

133 135 138 142 143 146 153 153 134 156 159

Single-Cell Protein Production by Photosynthetic Bacteria

R. H. SHIPMAN,L. T. FAN,AND I. C. KAO I. Introduction . ...................................... 11. Process Consi ............... ................... ...................... 111. Conceptual Design . . . . . . . . . . . . . . . . . . _............. IV. Economic Analysis . . . . . , . , . , . . . . . . . . . . . . . . . References ........................................

161 166 172 176 181

Environmental Transformation of Alkylated and Inorganic Forms of Certain Metals JITENDRA

I. Introduction

SAXENAAND PHILIPH. HOWARD ..................................

185

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

186 194 198 205 210 212 215 217 219 220 222

Test Methods for Studying Transformation ..................... Analytical Procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Factors Affecting Transformation of Metals . . . . . . . . . . . . . . . . . . . . . . . Biochemical Pathways and Mechanisms for Transformation of Metals . . . . . . . . . General Discussion of Various Test Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Correlation between Laboratory and Field Results and M. Restoration of Metal-Contaminated Areas . . . . . . . . . . . ............ X. Categorization of Elements . . . . . . . . . . . XI. Summary and Conclusions,. , . . . . . . . . . . . . . . . . . . . . . . . References .....................................................

111. IV. V. VI. VII. VIII.

Bacterial Neuraminidase and Altered Immunological Behavior of Treated Mammalian Cells

PRASANTA K. RAY I. Introduction . . . . . . . . . . . . . , . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Receptor-Destroying Enzyme . . . . . . . . . . . . . . . . . . . . . . . . 111. Sialic Acid and Its Relationship to the Antigenicity of the Cell Surface . . . . . . . . 11. Neuraminidase-The

227 228 237

CONTENTS

....... Iv. Increased Immunogenicity of Neuraminidase-Treated Cells . . . . . . V. Regression of Established Solid-Tissue Tumor . . . . . . . . . . . . . . . . . . . . . . . . . . . . , VI. How Do Neuraminidase-Treated Tumor Cells React in the Host to Establish Specific Antitumor Immunity? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VII. A Probable Mechanism by Which Neuraminidase-Treated Tumor Cells Give Rise to Specific Antitumor Immunity ..................... VIII. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . .

vii 243 246

249 259 260 261

Pharmacologically Active Compounds from Microbial Origin

HEWITTw. MATTHEWSAND BARBARAFRITCHE WADE I. Introduction . . . . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Types of Pharmacological Activities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . , . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ,

SUBJECTINDEX . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . CONTENTS OF PREVIOUS VOLUMES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

269 269 286

287

289 293

This Page Intentionally Left Blank

LIST

OF CONTRIBUTORS

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

L. T. FAN, Department of Chemical Engineering, Kansas State University, Manhattan, Kansas (161)

D. J. D. HOCKENHULL, Glaxo Laboratories Ltd., Ulverston, Cumbria, England (125) PHILIPH. HOWARD,Lije Science Division, Syracuse University Research Corporation, Syracuse, New York (185) I . C. KAo, Biochemical Development Division, Eli Lilly and Company, Indianapolis, Indiana (161)

KEIDO KO, The Institute of Physical and Chemical Research, Wako-shi, Saitama, Japan (53) LLOYD E. MCDANIEL,WaksmunInstitute of Microbiology, Rutgers University, New Brunswick, New Jersey (1) MARTIN,*Departmnt of Nutrition and Food Science, Massachusetts Institute of Technology, Cambridge, Massachusetts (1)

JUAN F.

HEWITTW. MATTHEWS, Southern School of Pharmucy, Mercer University, Atlanta, Georgia (269) TOMOMASAMISATO,The Institute of Physical and Chemical Research, Wako-shi, Saitamu, Japan (53) PRASANTAK. RAY, Chittaranjan National Cancer Research Centre, Calcutta, India (227) JITENDRA SAXENA, Li$e

Science Division, Syracuse University Research Corporation, Syracuse, New Ym-k (185)

R. H. SHIPMAN, Department of Chemical Engineering, Kansas State University, Manhattan, Kunsas (161) *Present address: Departamento de Microbiologia, Facultad de Ciencias, Universidad de Salamanca, Salamanca, Spain.

ix

X

LIST OF CONTRIBUTORS

E. J. VANDAMME,Laboratory of General and Industrial Microbiology, University of Gent, Gent, Belgium (89) BARBARAFRITCHEWADE, Southern School of Pharmacy, Mercer University, Atlanta, Georgia (269) ISAMUYAMAGUCHI, The lnstitute of Physical and Chemical Research, Wakoshi, Saitama, Japan (53)

Production of Polyene Macrolide Antibiotics JUAN

F. MARTIN^

Department of Nutrition and Food Science, Massachusetts lnstitute of Technology, Cambridge, Massachusetts AND

LLOYDE. MCDANIEL Waksman Institute of Microbiology, Rutgers University, New Brunswick, New Jersey I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. History of the Discovery of New Polyene Macrolide Antibiotics .......................... . . . . . . . . . . . . . . . B. Chemical Characteristics of Polyene Macrolide Antibiotics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Microbial Species That Produce Polyene Macrolide

..................... A. B. C.

D. E. F. G. H. I. J.

K.

.......

ene Macrolide Production . . . . . . . . . . . . Nutritional Studies ................................. Carbohydrates as Carbon Sources . . . . . . . ........ Relation to the Utilization of Short-Chain Fatty Acids and Alcohols . . . . . . . . . . . . . . . Role of Oxaloacetate . . . . Role of Citrate . . . . . . . . . ...... Interrelation with Fatty Acid Biosynthesis . . . . . . . . . . . . . Nitrogen Sources ..................... ...... Regulation by Arcmatic Amino Acids . . . . . . . . . . . . . . . . . . Effect of Inorganic Orthophosphate . . . . . . . . . . . . . . . . . . . Regulation of the Biosynthesis of Polyene Macrolide Antibiotics by Adenine Nucleotides and/or the Energy Charge of the Cell .................................. Effect of Metal Ions. . . . . .

M. Effect of Redox Potential . . . . . . . . . . . 111. Interaction of Polyene Macrolide Antibioti Producer Cells . .

2 2 3 12 14 14 15 17 20 21 22 24 25 27

31 33 34 36 37 37

Production ........................... C. Polyene Macrolide Production in Mixed Culture with

......................................... D. Susceptibility of the Producer Strains to Polyene Macrolide Antibiotics ............................... E. Feedback Regulatory Effect of Polyene Macrolide Antibiotics on Their Own Production. . . . ...

39 40 41 42

'Present address: Departamento de Microbiologia, Facultad de Ciencias, Universidad de Salamanca, Salamanca, Spain. 1

2

JUAN F. MARTIN AND LLOYD E . MCDANIEL

IV. Genetics of Polyene-Producing Streptomyces ............... A. Natural Variants of Soil-Isolated Strains and Mutational Strain Improvement ................................ B. Effect of Actinophages on Antibiotic Production-Lysogenic Conversion of Antibiotic-Producing Strains ......................... References ............................................

43 43

45 47

1. Introduction

A. HISTORYOF THE DISCOVERY OF NEW POLYENE MACROLIDEANTIBIOTICS Since the early 195Os, when the first polyene macrolide antibiotics were reported, more than 90 different members of this group have been described, and more are being discovered each year. Sixteen polyene macrolides were known in 1957 (Dutcher, 1957), 41 in 1960 pining, 1960), 57 in 1963(Oroshnik and Mebane, 1963), and 84 in 1973 (Hamilton-Miller, 1973). Several more, which have been reported recently, are included in Table I. However, one must be cautious in considering many of these antibiotics to be new, since the question of the identity of many remains open. Most polyene macrolides have not been obtained in pure form, and the chemical compositions of most of them are unknown. Thus, tennecitin and pimaricin have been found to be the same; also lagosin and fungichromin. Polifungin A is identical with nystatin (Porowskaet al., 1972; Roszkowskiet al., 1972), but polhngin B is different from the latter. The pentaenes mycoticins A and B are identical, respectively, with the minor and major components of flavofungin and flavomycoin (Bognar et al., 1970; Uri and Bekesi, 1958). It appears also that the aromatic heptaene macrolides candicidin, trichomycin, levorin, and hamycin are in fact mixtures in different proportions of the same components rather than separate entities. It has been suggested that hamycin and trichomycin are identical (Divekar et al., 1966)and that candicidin is identical with trichomycin (Khokhlovaet al., 1963). Pyrolysis gas chromatography of these heptaene macrolides suggests that they are mixtures, with a single identical main component and varying proportions of similar minor components (Burrows and Calam, 1970; Calam, 1974). Countercurrent distribution studies indicated that of the four components of the levorin complex (&, Al, A,, A3), A,, the main component of the complex, is not distinguishable from the main component of candicidin (Bosshardt and Bickel, 1968). Khokhlova et al. (1963) indicated that strains of Streptomyces griseecs which produce candicidin, Streptomyces canescus (the producer of ascosin), and Streptomyces levoris (the producer of levorin) are the same species, but

PRODUCTION OF POLYENE MACROLIDE ANTIBIOTICS

3

that Streptomyces hachijoensis (the producer of trichomycin) is different. A new polyene antibiotic isolated from Streptomyces helvoloviolaceous is identical with component A3 of the levorin complex (Konev et al., 1973).

B. CHEMICAL CHARACTERISTICS OF POLYENE MACROLIDEANTIBIOTICS The polyene macrolides form a subdivision of the macrolide antibiotics containing hydroxylated macrocyclic lactone rings and usually one or more sugars. Biogenetically the macrolides are a homogeneous group, being synthesized from acetate and propionate via the polyketide pathway (Bu’Lock, 1967). The macrolide antibiotics are divided into two subgroups: (a) polyene antifingal antibiotics and (b) nonpolyene antibacterial antibiotics. The polyene subgroup has a system of conjugated double bonds, or chromophore, in the macrolactone ring. This results in an amphipathic molecule containing both a rigid planar lipophilic portion and a flexible hydrophilic polyhydroxylated region. The chromophore accounts for some of the characteristicphysical and chemical properties of the polyenes (strong light absorption, photolability, and poor solubility in water) and appears to be responsible for the differences in the biological modes of action of the polyene and the nonpolyene macrolide subgroups. The chromophorc gives a typical multipeak ultraviolet-visible light absorption spectrum which permits a rapid characterization and division of the polyene macrolides into dienes, trienes, tetraenes, pentaenes, hexaenes, and heptaenes according to the number of conjugated double bonds in the chromophore. A classification of the existing polyenes is given in Table I. The polyene macrolides have lactone rings of 26-38 atoms, which are much larger than those of the nonpolyene macrolides (e.g., a 14-membered lactone in erythromycin). The aminosugars and aromatic moieties found in polyene macrolide antibiotics attached to the macrolide rings are shown in Fig. 1. Recently, a new class of nonpolyene antifungal macrolides, the axenomycins, with large 34-member lactone rings has been reported. This group appears to be closely related in structure and biological activity to the polyene macrolides in spite of their nonpolyene character (Bianchi et al., 1974). Although purification of polyene macrolide antibiotics is difficult because of their low solubility and instability to heat and light, considerable progress has been achieved in recent years in the determination of the complex chemical structure of these compounds by utilizing sensitive analytical methods, such as electron impact and field desorption mass spectrometry (Rinehart et al., 1974), proton magnetic resonance, and X-ray structure analysis of single crystals for absolute configuration determination

P

TABLE I POLYENE MACROLIDE ANTIBIOTICS (CODENUMBER 22) Berdy's classification" Code number Name (alternative names) ~~~~~~~~~~~

Amino sugar moiety

Producer strain ~

~

Nitrogen

Ionic character

~~~

221 TFUENES (A maxima 262, 272, 283 nm) 2211 Trienin type Streptomyces sp. Mycotrienin Streptomyces sp. Trienine Antibiotic MM-8 Sheptomyces sp. Triene Chainia minutisclerotina 2212 Other trienes Resistaphyllin S. antibioticus 222 TETRAENES (A maxima 291, 304, 308 f 2 nm) 2221 Pimuricin type S . tumemomucerans var. Aeromycin B (P-42-E) griseoarenicolor S . lucensis Etruscomycin (lucensomycin) (1163 FI) S . natalensis, Pimaricin (tennecetin) S . chattanoogensis, S . gilveosporus S . noursei var. Tetramycin jenensis Streptomyces sp. Tetrin A, B Antibiotic PA-I66 Sheptomyces sp.

-

NDb ND None ND

Yes Yes Yes ND

ND

Yes

-

Mycosaminec

Yes

-

Mycosamine

Yes

Amphoteric

M ycosamine

Yes

Amphoteric

Mycosamine

Yes

-

Mycosamine Mycosamine

Yes Yes

Amphoteric Amphoteric

Neutral

-

Aromatic moiety

2222 Rimocidin type Akitamycin S. akitaensis, (toyamycin) S. toyamaensis S. albus sterilis Albotetraen Rimocidin S. rimosus (PA-86) Antibiotic RP-9971 S. gascariensis 2223 Nystatin type Amphotericin A S. nodosus Nystatin A,, A*, A3 S. noursei, S. albulus (fungicidin) (polifungin) S. plumbeus Phmbomycin A Plumbomycin B S . plumbeus 2224 Other less-known tetraenes Antimycoin A S. aureus Chromin S. chromogenes Endomycin A S. endus (helixin A) Flavoviridomycin S. flavoviridus var. fungicidicus, S. hygroscopicus var. enhygrus Ornamycin (17731) S. erumpens, S. ornatus Protocidin Streptomyces sp. NO. 964-A Sistomycosin s. viridosporus Tetraenin A, B Chainia cinnamonea Tetramedyn S. mediocidicus Tetramycoin A, B Chainia grisea, Chainia grisea var. fusca

ND

Yes

Amphoteric

ND M ycosamine

Yes Yes

Basic

ND

Yes

Amphoteric

Mycosamine Mycosamine

Yes Yes

Amphoteric Amphoteric

ND ND

Yes Yes

ND ND ND

-

Yes Yes

Acid Amphoteric

ND

Yes

ND

Yes

Amphoteric

ND ND ND ND

Yes

-

Yes

-

-

(Continued)

VI

TABLE I (Continued) Berdy's classification" Code number Name (alternative names) Unamycin A Antibiotic A-5283 Antibiotic AC2-435 Antibiotic J4-B

Producer strain

S. fungicidicus Streptomyces sp. A5283 Streptomyces sp. ACz-435 Streptomyces sp. (S.fungicidicus) Streptomyces sp. Streptomyces sp. 0777

Antibiotic RP-7071 Antibiotic LIA 0777 223 PENTAENES 2231 Methylpentaenes (aldopentaenes) (A maxima 327, 340, 357 2 2nm) Aurenin S. aureorectus Cabicidin s. gougeroti Chainin Chainia sp. 3047 S. fdipensis Filipin complex (durhamycin) S . cinnamoneus var. Fungichromin cinnamoneus, (moldcidin B) (pentamycin) S. roseoluteus, (Gkxo-A246) S. cellulosae, (Lagosin) S. pentaticus Fungichromatin S. cellulosae Pentaneicin Streptooerticillium sp. S. sanguineus Pentaene Neopentaene Streptomyces sp. S. rubrochlm'nus Rubrochlorin Xantholicin B S. rantholiticus Antibiotic HA-106 Streptooerticillium cinnamoneum var. sparsum

cn

Amino sugar moiety

Nitrogen

Ionic character

Mycosamine ND ND ND

Yes Yes No No

Acid Amphoteric -

ND Mycosamine

Yes Yes

Basic Amphoteric

None None None None

No No No No

Neutral Neutral Neutrd

None

No

Neutrd

None None None None None None None

No No No No No No No

-

Aromatic moiety

-

Acidic

-

-

-

?

r

s0 P

Antibiotic HA-135

Streptoverticillium sporiferum Antibiotic HA-145 Streptooerticillium cinnamneum var. albospwum Antibiotic HA-176 Streptoverticillium cinnamoneum var. lanosum 2232 “Normal” pentaenes (amphoteric pentuenes with amino sugar)(h maxima 317, 331, 350 ? 2 nm) Aliomycin S. acidomyceticus Distamycin C S . distallicus Eurocidin A S . eurocidicus, S . albireticuli Eurocidin B S. albireticuli Fumanomycin S . laoendobrunneus Moldcidin A S . griseofuscus, Streptomyces sp. S. sp. J-4(S. fungicidicus) Onomycin I Pentafungin S. antimycoticus Pentaene G-8 S . anandensis Quinquamycin S . laoendulae E-20-27 Streptomyces sp. Antibiotic A-228 Antibiotic PA-153 Streptomyces sp. Sh-eptomyces sp. 17-41 Antibiotic 17-41 B Antibiotic 0371 Streptovetticillium jenensis krissii 2233 “Normal” pentaenes. (Capacidin type) Capacidin S. noursei (variant) 2234 Gangtokumycin type Gangtokumycin S . gangtokensis ( S . hygroscopicus)

None

No

None

No

None

No

ND ND M ycosamine

-

-

Yes

Amphoteric

Mycosamine ND Mycosamine

Yes Yes Yes

Amphoteric

ND ND ND ND ND ND ND

Yes Yes

Acidic Amphoteric

-

-

Yes Yes

Neutral Amphoteric

Yes

-

-

Yes

-

Yes

-

Yes

sl

Acidic

-

Basic

(Continued)

4

TABLE I (Continued) 00

Berdy's classification" Code number Name (alternative names)

Producer strain

Genimycin Actinosporangium sp. 2235 Carbonyl pentaenes ( h maxima 364 nm; broad peak) Flavohngin S. ruber, (mycoticin A) S. jlavofungini Flavomycoin S. roseoflavus var. jenensis Mycoticin B S. jlaoofungini Surgomycin S . surgutus 224 HEXAENES (A maxima 340, 358, 380 ? 2 nm) 2241 Probably macrocyclic heraenes S . oiridojlavus var. 18A2 Candihexin A, B S. viridoflavus var. 18A2 Candihexin E, F Cryptocidin Streptomyces sp. 963, S. bulgaricum Hexin Streptomyces sp. S. endus, Endomycin B (helixin B) S. hygroscopicus var. enhygrus Flavacid s. jlavus Mediocidin S . mediocidicus Tetrahexin Streptomyces sp. (tetraesin) ATCC 14972 2242 Fradicin type (unknown structures) Fradicin S . fiadiae Mycelin S . roseojlavus, S . diastatochromogenes, S. fiadiae

Amino sugar moiety

Nitrogen

Ionic character

-

Yes

-

None

No

Neutral

None

No

-

None ND

No

Neutral

Aromatic moiety

* W

Mycosamine None ND

Yes Yes Yes

ND ND

-

None ND ND

Yes Yes Yes

Acid Amphoteric Amphoteric

ND ND

Yes No

Basic

Acid

P

6

B

-

-

Mycelin IMO Antibiotic A-1404

S. diastatochromogenes S. fi-adiae A-1404, S. diastatochromogenes 207

2243 Carbonyl hexaenes (A maxima 385 nm; broad peak) S. uiridogsiseus Dermostatin (viridofulvin) 225 HEF'TAENES 2251 Aromatic heptaenes 22511 Containing p-aminoacetophenone S . paucisporogenes Antifungin 4915 Ascosin S. canescus Ayfactin, S. aureofaciens, S. uiridofaciens (AYF), (AE-56), (Aureofacin) S. cinnamoneus Aureofungin var. terricola Azacolutin S. cinnamoneus (F-17-C) var. azacoluta S. griseus Candicidin (G-252) (PA-150) S. griseus H-5592 Eurotin A S. jujuy ATCC 13670 Gerobriecin Hamycin S. primprina S. longisporolavendulae Heptafungin A S . leuoris Levorin Ao, A,, Az, A,, B (26/1)

ND ND

Yes

None

No

Neutral

ND Mycosamine Mycosamine

Yes Yes Yes

ND Amphoteric Acid

ND p-Aminoacetophenone p-Aminoacetophenone

Mycosamine

Yes

Amphoteric

p-Aminoacetophenone

ND

Yes

Amphoteric

p-Aminoacetophenone

Mycosamine

Yes

Amphoteric

p-Aminoacetophenone

ND ND Mycosamine Mycosamine Mycosamine

Yes Yes Yes Yes Yes

Basic ND Amphoteric Amphoteric Amphoteric

p -Aminoacetophenone ND p-Aminoacetophenone p-Aminoacetophenone p-Aminoacetophenone (Continued)

TABLE I (Continued) Berdy's classification" Code number Name (alternative names) Trichomycin A, B

Producer strain

S. hachijoensis, S . abikoends S . surinam Actinoplanes sp.

DJ-400Bz Sch 16656 22512 Containing N-mthylp-aminoacetop henone Candimycin S . echimensis DJ-400 B, Perim ycin (NC-968) (Fungimycin) (Aminomycin) 2252 Nonaromatic heptaenes Amphotericin B Candidin (Candidoin) (Candidinin) Mycoheptin (2814 H)

Amino sugar moiety

Nitrogen

Ionic character

Aromatic moiety

Mycosamine

Yes

Amphoteric

p- Aminoacetophenone

Mycosamine ND

Yes Yes

Amphoteric

p-Aminoacetophenone p-Aminoacetophenone

ND

.h

5 IJ

e %CI

ND

Yes

ND

N-Methyl?aminoacetophenone

S surinam

Mycosamine

Yes

Amphoteric

N-Methyl?aminoacetophenone

3

S. coelicolor var. aminophilus

Perosamined

Yes

Basic

N-Methyl?-

r

.

aminoacetophenone

r

ei z

S . nodosus S . oiridofious

Mycosamine Mycosamine

Yes Yes

Amphoteric Amphoteric

None None

Streptoowticillium mycoheptinicum, S . netropsis

Mycosamine

Yes

Amphoteric

None

S. chartreusis var. tbilisus Antibiotic X-63 Streptomyces sp. Antibiotic A-3 Streptomyces sp. 2253 Noncharacterized heptaenes Grubilin Streptomyces sp. BA-27 Heptamycin Streptomyces sp. Hepcin Actinosporangium griseoroseum Heptaene 757 Streptomyces sp. 757 Monicamycin Streptoverticillum annamoneum var. monicae Neoheptaene Streptomyces sp. S . noursei Nursimycin Takamycin S . takaensis, S . reticuli C-11 Antibiotic 26/1 S. globisporus Antibiotic 2814-H Streptomyces sp. IA-2814 Antibiotic 1645-P, Streptomyces sp. 1645-IAUR LIA 0331 S. chromogenes LIA 0179 Streptomyces sp. Tbilimycin

Mycosamine

Yes

ND

None

ND ND

Yes Yes

ND ND

None None

ND ND ND

ND ND ND

ND Acid ND

ND ND ND

ND ND

ND ND

Acid Acid

ND ND

ND ND ND

ND ND ND

ND ND ND

ND ND ND

ND ND ND

ND Yes Yes

Amphoteric Amphoteric ND

ND ND ND

ND ND

ND ND

ND ND

ND ND

“Berdy (1972). bNo data available. c3-Amino-3, 6-dideoxy-~-mannose. d4-Amino-4,6dideoxy-~-mannose. c c

12

JUAN F. MARTIN A N D LLOYD E . MCDANIEL


MOST POLYENES

OH

PERlMYClN (FUNGIMYCIN)

,N

CH, - H

o c 0 - c H 3

~

a

c

0

-

c

.

P-AMINOACETOPHENONE

ASCOSIN AUREOFUNGIN

AYFACTIN AZACOLUTIN CANDlCl DI N DJ -400.82 HAMYCIN HEPTAMYCIN LEVORIN TRICHOMYCIN SCH 16656 . P-1-METHYLACETOWNONE PERlMYClN CANDIMKIN

DJ- 40081

FIG. 1. Aminosugars and aromatic moieties existing in polyene macrolide antibiotics.

(Mechlinski et al., 1970). For detailed information on the chemistry of polyene macrolides, the reader is referred to the reviews by Oroshnik and Mebane (1963), Dutcher (1968), Hamilton-Miller (1973), and Mechlinski (1973).Only afew polyene macrolides have been fully characterized (Fig. 2). C. MICROBIAL SPECIESTHATPRODUCE POLYENE MACROLIDEANTIBIOTICS The biosynthesis of polyene macrolide antibiotics is a widespread property among the actinomycetes. From 34 to 88% of the Streptomyces species isolated from soil were found to produce polyene macrolides (Ball et al., 1957; Pledger and Lechevalier, 1956). Polyene macrolides are produced mainly by members of the genera Streptomyces, Streptoverticillium, Actinospwangium, and Chainia. In the Russian literature, species belonging to the genus Streptomyces are usually referred to as Actinomyces (e.g., Actinomyces levoris). In order to have a homogeneous description of the producing species, the name Streptomyces has been adopted here.

13

PRODUCTION OF WLYENE MACROLIDE ANTIBIOTICS

PENTAENES

TETRAENES OH

FILIPIN( (R=H)

R

no

no OH

FUNGICHROMIN (LAGOSIN. PENTAMVCIN HOLOClOlN El (R=OHI

MI OH on on OH OH

PlHARlClN(TENNECETIN)(R=CH3) ETRUSCONVCINiLUCENKlN. A R E N M N (R.Cl+i-CH$4rCk$)

MVCOTICIN A (R-H) (FLAVOFUNGIN) HVCOTlClN B (R=CH3)

no

EUROClDlN A (R=CHj) EUROClDlN E ( R = H )

RlHOClMN

TURIN A TETRIN E

(RA)

CHAININ

(R=OHI

HEPTAENES

HEX AENES

-a* AMPHOTERlClN

B

C A NDlOlN HVCOHEPTIN (FHVDRO, 5-DEHVDROCANMMN

FIG. 2. Polyene macrolides of known structure.

14

JUAN F. MARTIN AND LLOYD E . MCDANIEL

Recently a new polyene macrolide (Sch 16656)has been described from a species of the aquatic genus Actinoplanes (Wagman et al., 1975). Proticin, a phosphorus-containing triene antibiotic produced by Bacillus lichenijbrmis var. mesentericus (Vertesy, 1972), does not seem to be a typical polyene macrolide since it has gram-negative activity and no antifungal activity. By contrast, rhizopchin, a polyene antibiotic produced by Rhizopus chinensis, has antifungal activity (Arima and Odahara, 1971). A method of screening for new producers of polyene macrolide antibiotics based on the inactivation of these products by sterols was described recently (Bibikova et al., 1975). By testing for the inhibition of yeasts by Streptomyces isolates, in unsupplemented media and in media supplemented with sterols, it is possible to select those that produce polyene macrolides (active against yeast only in the absence of sterol) and those that produce nonpolyene antifungal agents (active both in the presence and in the absence of sterol).

II. Factors Affecting Polyene Macrolide Production The information available on the production of polyene macrolide antibiotics prior to 1967 was reviewed by Perlman (1967). The objective of this review is to summarize the significant new developments in this field. It is a well-established fact that in most species control mechanisms have evolved that allow only necessary enzymes to be made in the correct amounts. Thus, a normal cell is strictly regulated and does not produce waste metabolites in any environment. Recently, evidence has been found that microorganisms that overproduce metabolites of industrial importance are in fact subnormally regulated (Demain, 1972). In the last few years, considerable understanding of the microbial biosynthesis of secondary metabolites (idiolites) belonging to the macrolide class has been achieved. We are now in a better position than before to understand how nutritional and environmental factors affect the biosynthesis of polyene macrolide antibiotics.

A. NUTRITIONAL STUDIES Most nutritional studies of polyene-producing Streptomyces have been done with batch cultures, usually in shake flasks, but with occasional scaling up to pilot-plant or large-scale fermentors of those polyenes that are produced industrially (see Table 11). In most studies several nutritional factors or physical parameters are studied, singly or in combination, and the combinations giving the highest yields are selected for further use. McDaniel et al. (1976) used factorial and central composite experimental designs with analysis and response surface plotting by computer for optimizing nutrient combinations for polyene macrolide production.

PRODUCTION OF POLYENE MACROLIDE ANTIBIOTICS

15

TABLE I1 POLYENEMACROLIDE ANTIBIOTICS THAT ARE PRODUCED COMMERCIALLY Name

Type

Amphotericin B Candicidin Hamycin Levorin M ycoheptin Nystatin Pimaricin Trichomycin

Heptaene Aromatic heptaene Aromatic heptaene Aromatic heptaene Heptaene Tetraene Tetraene Aromatic heptaene

Empirical medium development studies have not contributed materially to an understanding of regulation and control of the metabolism of the producing strains. In batch cultures, any factors affecting primary metabolism (growth-related biosynthetic processes) of the producing cell are also likely to affect the biosynthesis of antibiotics. The effects of phosphate and divalent cations, for example, could not be attributed to their action on regulation of polyene macrolide synthesis or to unspecific roles at the transcriptional or translational levels. To avoid such uncertainty, a phosphate-limited washed mycelial system has been introduced (Martin and McDaniel, 1976), which has the advantage that it selectively eliminates the effect of growth. Results obtained by both growing and suspension cell systems will be reviewed. A significant core of evidence suggests that since polyene macrolide antibiotics are synthesized via the polyketide pathway, there is a close connection between antibiotic production, cell carbohydrate metabolism, and fatty acid synthesis. B. CARBOHYDRATES AS CARBON SOURCES Most polyene antibiotic fermentations have been carried out using glucose as carbon source. Studies in our laboratory (Ethiraj, 1969)have established that glucose is the carbon source of choice for the production of candicidin. Concentrations of glucose as high as 7% (Brewer and Frazier, 1962)to 9.5% (Liu et al., 1975)have been routinely used. However, in pilot fermentations of candidin it was observed that high initial concentrations of glucose retarded growth and resulted in abnormal fermentation patterns. Slow-feeding of glucose, initially described in the penicillin fermentation (Soltero and Johnson, 1954; Pirt, 1971) to bypass the negative effect of high glucose concentrations, resulted in increased synthesis of the polyenes candidin and candihexin (Martin and McDaniel, 1974). The same increase was obtained

16

JUAN F. MARTIN A N D LLOYD E. MCDANIEL

when the glucose level was maintained at 5 mg/ml or 15 mg/ml. There was a higher respiration rate during the antibiotic production phase (idiophase) when glucose was slow-fed with a very high proportion of the polyene remaining attached to the producing cell. Maximal growth rates and final cell mass accumulations were lower in slow-fed fermentations, but glucose utilization rates were higher than in control fermentations. These results suggest that there is a channeling of substrate to polyene formation under slowfeeding of glucose (Martin and McDaniel, 1974). A similar approach, namely carbohydrate dosing in relation to the physiological state of the producing organism, was proposed earlier (Hosler and Johnson, 1953). Proper choice of carbohydrate feeding at various ages of the producing organism makes it possible to increase the rate of nystatin production severalfold (Tereshin, 1976). This effect is due to an increase in the specific activity of the mycelium. Intermittent addition of glucose in the amphotericin fermentation results in a slight increase in the titer of amphotericin B (Brewer and Frazier, 1962). The addition of sugar during the fermentation has also been used in the production of the heptaene DJ-400 (Siewert and Kieslich, 1971). The higher antibiotic synthesis under slow-feeding of glucose suggests the regulation of the polyene macrolide synthases by the metabolites of rapid glucose utilization (effectors),which is bypassed when glucose is fed slowly. In this context it is interesting to note that several enzymes involved in the biosynthesis of fatty acids which are biosynthetically analogous to macrolides are subject to catabolite repression in Escherichia coli (Overath and Raufuss, 1967; Weeks et al., 1969). Catabolite regulation as defined by Magasanik (1961) appears to occur in the biosynthesis of the antibiotics penicillin (Johnson, 1952), actinomycin (Gallo and Katz, 1972), streptomycin (Demain and Inamine, 1970; Inamine et al., 1969), siomycin (Kimura, 1967), indolmycin (Hurley and Bialek, 1974), and bacitracin (Haavik, 1974). Both catabolite repression of antibiotic synthase formation and catabolite inhibition of the preformed enzyme occur in the actinomycin and streptomycin fermentations, but only enzyme inhibition appears to be involved in the control of siomycin formation. The inhibitory effect of glucose on bacitracin synthesis does not appear to be related to catabolite regulation, but rather is due to acid production and lowered pH. The effectors involved in catabolite regulation of antibiotic biosynthesis have not been identified. However, there are significant differences between catabolite regulation of the biosynthesis of these antibiotics, and the effect of glucose on polyene macrolide synthesis. Actinomycin formation occurred only after glucose was exhausted from the culture medium, and repeated additions of glucose resulted in a transient inhibition of phenoxazinone synthase and actinomycin production, whereas polyene macrolide synthesis requires glucose in the medium. Candicidin production

PRODUCTION OF POLYENE MACROLIDE ANTIBIOTICS

17

stops after glucose exhaustion. Glucose exhaustion is followed by characteristic autolytic changes: oxygen uptake decreases sharply, candicidin synthesis stops, and the antibiotic is released. The pH of the culture and the dissolved oxygen tension of the broth increase, and both dry weight and DNA experience large decreases (Martin and McDaniel, 1975a) (Fig. 3). Similar marked changes were also observed in the nystatin fermentation upon glucose exhaustion (Lopatnevet al., 1973). The evidence existing on catabolite regulation of polyene macrolide biosynthesis by glucose is, therefore, obscured by the fact that glucose is required for synthesis of the product. Early nutritional studies (Acker and Lechevalier, 1954), confirmed recently by Abou-Zeid (1973) and Tereshin (1976), indicated that glucose and mannose support identical growth and candicidin production in a synthetic medium. Less candicidin was produced when galactose, fiuctose, arabinose, or several polysaccharides were used. The disaccharides maltose, sucrose, and lactose supported small candicidin yields. Martin and McDaniel (1976), using a phosphate-limited resting cell system in a defined medium, obtained similar results. Lactose did not support candicidin synthesis under their conditions, but some antibiotic was produced with sucrose. Similarly, disaccharides were poor carbon sources for growth and mycoheptin production by Streptoverticillium mycoheptinicum (Tereshin, 1976). In the amphotericin fermentation glucose was substituted for by British gum (a dextrin) because it gives large amounts of amphotericin B relative to amphotericin A (Brewer and Frazier, 1962). Starch and glucose proved to be the best carbon sources for mycoheptin production. Specific antibiotic production was lower using starch as a single carbohydrate source, but biomass accumulation was greater with starch than with glucose (Tereshin, 1976).

C. RELATIONTO THE UTILIZATIONOF SHORT-CHAIN FATTYACIDS AND ALCOHOLS Acetate and propionate, although they are the immediate precursors for formation of polyene macrolide antibiotics, do not by themselves support antibiotic synthesis. The same is true of malic, lactic, succinic, and citric acids (Acker and Lechevalier, 1954; Abou-Zeid, 1973; Tereshin, 1976). The inability of acetate, propionate, and malonate to substitute for glucose as exogenous precursors of candicidin has been corroborated using a resting cell system (Martin and McDaniel, 1976). However, supplementation of a glucose basal medium with acetate, propionate, malate, or lactate produces a significant stimulation of candicidin synthesis in batch cultures (Acker and Lechevalier, 1954; Martin and McDaniel, 1976). Addition of acetate also stimulates fungimycin biosynthesis (Mohanet al., 1963). Radioactive acetate and propionate are effectively incorporated into the polyene macrolides nys-

18

JUAN F. MARTIN AND LLOYD E. MCDANIEL

FIG.3. Pattern of fermentation parameters in candicidin batch fermentations: (A) dry weight (A), cell DNA (O), residual glucose (M);(B) total candicidin (A), mycelium-associatedcandicidin (O), extracellular candicidin (0); (C) pH (---), dissolved oxygen tension (---), and oxygen uptake rate (-). From Martin and McDaniel (197.5). Reprinted with permission of John Wiley and Sons, Inc.

PRODUCTION OF FOLYENE MACROLIDE ANTIBIOTICS

19

tatin (Birchet al., l W ) , lucensomycin (Manwaringet al., 1969), amphotericin B (Perlman and Semar, 1965; Linke et al., 1974), candicidin (Liu et al., 1972a), and levorin (Belousova et al., 1971). Malonate and methyl malonate are also effectively incorporated into candicidin 0. F. Martin, unpublished). The inability of acetate, propionate, and malonate to substitute for glucose is explained by the bct that not only does glucose provide acetate and propionate, but through the pentose phosphate cycle it is also involved in the biosynthesis of the aromatic moiety (p-aminoacetophenone) and also provides NADPH for the successive reductive steps of the highly oxidized polyketide chain. Lower alcohols (methanol, ethanol, propanol) have been reported to stimulate the production of polyene macrolide antibiotics (Tereshin, 1976). In the case of candicidin, n-propanol, but not isopropanol, ethanol, or butanol, is effective (Martin and McDaniel, 1976). An interesting approach for the understanding of the intermediary metabolism of the producing strain has been used by Soviet investigators. They compared the formation and utilization of short organic acids in highproducing industrial strains and in low or nonproducers, and they studied the enzymes involved in the condensation of acetate and propionate in both types of strains. Toropova et al. (1972) observed that a strain which produces large amounts of polifungin (nystatin) excreted during the growth phase three to four times larger amounts of volatile organic acid (formic, acetic, and butyric) than did nonproducing strains. The acids were subsequently utilized during the antibiotic production phase. Similarly, accumulation of keto acids (acetoacetic and a-ketoglutaric) followed by their utilization, occurred in high-producing strains. There was no significant increase in the total amounts of keto acids in the culture liquids of the inactive strains. Pyruvic acid followed a similar accumulation and utilization pattern, although the differences between producing and nonproducing strains were smaller. Succinic acid was utilized during polifungin production while citric acid accumulated in the medium. Rafalski and Raczynska-Bojanowska(1972) found that intensive synthesis of polifungin is associated with a rapid decrease in the concentration of %carbon acids (pyruvic and lactic) and an increase of 4-carbon acids (oxaloacetic and malic). It appears that C3 acids must play a role in the biosynthesis of polyene antibiotics. Carboxylation of acetate and propionate has been effectively correlated with polyene macrolide biosynthesis (Roszkowskiet al., 1972). The total pool of acyl-CoA in a highly productive strain of Streptomyces noursei var. polijkngini was 4- to 5-fold greater than in a low-producing strain in cells grown either on carbohydrates or lipids (Raczynska-Bojanowska, 1974). Mutants that produce high yields of polifungin have acetyl-CoA and propionyl-CoA carboxylase activities double those of a nonproducer strain. A

20

JUAN F. MARTIN AND LLOYD E . MCDANIEL

correlation also exists between the carboxylase activity toward both acyl-CoA derivatives and antibiotic production with several low-producing strains. The acetyl-CoA and propionyl-CoA carboxylase activities in the wild-type strain reached a maximum at the onset of antibiotic production and decreased about 3-fold during the idiophase.

D. ROLE OF OXALOACETATE Further insight into the role of the C3 acids in polyene biosynthesis was gained by the work of Rafalski and Raczynska-Bojanowska (1972, 1973). In Streptomyces noursei, in addition to nonspecific ATP and magnesiumdependent acetyl-CoA carboxylase (acetyl-CoA:carbon dioxide ligase, EC 6.4.1.2) utilizing carbon dioxide for carboxylation, there is also a carboxyltransferase (methylmdonyl-CoA:pyruvate carboxyltransferase, EC 2.1.3.l), which uses oxaloacetate as a carboxyl group donor. Carboxylating Systems of S . noursei

+

+

+

+

+

Acetyl-CoA ATP C 0 2 H,O = malonyl-CoA ADP phosphate [acetyl-CoA carboxylase (acetyl-CoA ligase) EC 6.4.1.21

+

(1)

+

Propionyl-CoA oxaloacetate = methylmalonyl-CoA pyruvate (2) [carboxyltransferase (methy1malonybCoA:pyruvatecarboxyltransferase,) E C 2.1.3.1]

+

Phosphoenolpyruvate COz = oxaloacetate (phosphoenolpyruvate carboxylase, EC 4.1.1.31)

(3)

The second system is about 100 times more active than the first. AcetylCoA carboxylase and carboxyltransferase of S. noursei are both nonspecific and catalyze with practically equal efficiency carboxylation of acetyl-CoA and propionyl-CoA. Both the carboxylating and transcarboxylating activities are higher in the high-producing mutants. These high activities are associated in high-producing mutants with an equally high activity of the anaplerotic enzyme phosphoenolpyruvate carboxylase, which supplies oxaloacetate. The oxaloacetate pool is thus replenished by carboxylation of phosphoenolpyruvate, which in turn is synthesized from pyruvate and ATP (Fig. 4). These authors propose an active role of oxaloacetate in the biosynthesis of polyene antibiotics, the level of oxaloacetate being the limiting factor. In a series of UV mutants, the oxaloacetate pools were larger in high-producing strains in parallel with higher activities of phosphoenolpyruvate carboxylase and lower activities of malate dehydrogenase and thioesterase as compared to lowproducing strains. In summary, there is a positive correlation between formation of polyene macrolide antibiotics and the activities of the three carboxylating systems, while malate dehydrogenase (which decreases the pool of oxaloacetate) and

PRODUCTION OF FQLYENE MACROLIDE ANTIBIOTICS

21

Glucose

I

Glucosesphosphate Fructose

Erphosphate r.

Fructose I,6diphosphate o i ~ r o x y a c e t m i i 3phos~hafe ~ ~ e

/

1,3-Diphosphoolycemte

1

3Phosphoglycemte

t

2-Phosphoglycerate

i

cx q::;:;i Phosphoenoipyuvate

Oxaloacetate

Ppuvate

ACETYL-CoA U A L o N Y L - c o A ( 2, METHYLMALONYL-CoA PROPIONYL -Co A

w f

cot

FIG.4. Oxaloacetate cycle in polyene macrolide biosynthesis. (1) Acetyl-CoA and propionyl-CoA carboxylases; (2) transcarboxykse;(3) phosphoenolpyruvate carboxylase.

thioesterase (which deactivates acyl-CoA) are negatively correlated (Raczynska-Bojanowska, 1974).

E. ROLE OF CITRATE The effect of citrate on polyene antibiotic biosynthesis deserves special consideration because of the well established regulatory role of citrate on the acetyl-CoA carboxylase of different organisms (Volpe and Vagelos, 1973). Acetyl-CoA carboxylase is considered to be the rate-limiting enzyme in fatty acid synthesis (Ganguly, 1960; Vagelos et al., 1963). Citrate activation of acetyl-CoA carboxylase in mammals is accompanied by polymerization of inactive protomers to active polymeric filaments and is clearly associated with a conformational change of the enzyme. The biotinyl prosthetic group of the carboxylase has been considered to be the locus of the citrate-induced conformational changes. In contrast, there are conflicting reports about activation by citrate of microbial acetyl-CoA carboxylases. Activation occurs in cell-free extracts of Saccharornyces cerevisiae (Rasmussen and Klein, 1968) but not with purified acetyl-CoA carboxylase ofE. coli or S. cerevisiue (Matsuhashi et al., 1964; Alberts and Vagelos, 1968). In the biosynthesis of erythromycin, a nonpolyene macrolide produced by S. erythreus, the activity of propionyl-CoA carboxylase was increased in vivo

22

JUAN F. MARTIN AND LLOYD E . MCDANIEL

by citrate when the effectorwas added to the mycelium, but not when added to sonicates of S. erythreus. The increase in enzymic activity by citrate was abolished by actinomycin added simultaneously. It was concluded that citrate affects directly or indirectly the biosynthesis of propionyl-CoA carboxylase rather than its activity (Raczynska-Bojanowskaet d.,1970). The possible stimulation of biosynthesis of polyene antibiotics by citrate has not been studied in detail. Attempts to stimulate candicidin production by resting cells of S. griseus by the addition of citrate were unsuccessful (J. F. Martin and L. E. McDaniel, unpublished). Similarly, citric acid proved to be a very poor substrate for levorin production. However, when citrate was added at 24 or 48 hours after inoculation (at 5-9 mg/ml) to soybean or cornmeal cultures, there resulted a 30-40% increase in levorin production. Addition at the time of inoculation did not increase antibiotic production. Because of the possible industrial utility of this regulator phenomenon, the “citrate effect” deserves further study.

F. INTERRELATION WITH FATTY ACID BIOSYNTHESIS It had been speculated that biosynthesis of large lactone macrolides (up to

38 carbon atoms in the ring) takes place by condensation of two long-chain acyl-CoA derivatives or by the stepwise addition of malonyl-CoA units to a preformed fatty acyl-CoA starter unit. Evidence summarized below indicates that these hypotheses are incorrect and that macrolide synthases and fatty acid synthases are independent enzyme complexes: (a) Studies on the biosynthesis of the polyketide-derived 6-methylsalicylic acid (Light and Hager, 1968; Dimroth et al., 1970) have demonstrated that the polyketide synthase involved is a multienzyme complex similar to, but different from, the fatty acid synthase. (b) With Streptomyces erythreus which synthesizes the nonpolyene erythromycin by condensation of methylmalonyl-CoA units (Corcoran and Chick, 1966), no long-chain branched fatty acids occurred that could be direct precursors for the biosynthesis of the macrolide (Hofheinz and Grisebach, 1965). Martin and McDaniel (1975~)found that exogenous fatty acids were unable to support candicidin biosynthesis when endogenous condensation of acetyl-CoA units was prevented by cerulenin (a specific inhibitor of the P-ketoacyl-acyl carrier protein synthase, or condensing enzyme). This excludes the hypothesis that polyene macrolides are formed by the condensation of two long-chain fatty acids. Exogenous fatty acids do in fact support growth of E . coli when endogenous fatty acid synthesis is suppressed by cerulenin (Goldberg et al., 1973). Early studies (McCarthy et al., 1955; Brock, 1956) demonstrated that oils and fatty acids stimulate the production of the polyene macrolide antibiotics fungichromin and filipin but not am-

PRODUCTION OF POLYENE MACROLIDE ANTIBIOTICS

23

photericin B (Brewer and Frazier, 1962)and candicidin (Ethiraj, 1969). Several individual fatty acids were able to replace the oils in stimulating filipin synthesis-palmitic acid and its esters, oleic acid, methyl oleate, triolein, etc. (Brock, 1956)-but only oleic acid and the synthetic oleate esters Span 80 (monooleate), Span 85, and Tween 85 (trioleates) replaced oils for fungichromin production. Glycerol was entirely inactive in the stimulation of the production of both antibiotics. It appears, therefore, that it is the aliphatic chains of the natural oils, not glycerol, which stimulate polyene antibiotic biosynthesis. Direct correlation of polyene macrolide biosynthesis and lipogenesis has been found in a number of cases. Belousova et al. (1970a) reported that cultivation of S . levoris in spent broth from a previously grown batch of the same organism stimulated levorin biosynthesis and fatty acid synthesis as well. Cultures grown in spent broth for 1 day incorporated into fatty acids 1.5-3 times more labeled acetate than did a culture of the same age in fresh corn meal medium. It was also noted that different spectra of cellular fatty acids existed in cells grown in the two media. More anteisoacids were synthesized when S . levoris was grown in fresh corn medium, accompanied by lower levorin production. The same stimulatory effect of spent broth from S. noursei fermentations has been observed (Nugamanovet aZ., 1973). Rafalski and Raczynska-Bojanowska (1972) observed that increased activity of the carboxylating enzyme systems involved in polyene macrolide biosynthesis correlated well with increased lipogenesis. Although oils and fatty acids contain more energy per unit weight than glucose, the increased polyene macrolide production with oils cannot be attributed to the added energy available, since the yields are far greater than would be expected from the energy supplied. The stimulation of polyene antibiotic biosynthesis by oils might be a simple precursor effect. Catabolism of fatty acids will result in an increased pool of acetyl-CoA, which is subsequently used for polyene macrolide biosynthesis. However, regulation of the biosynthesis of polyene macrolides by mechanisms other than a direct precursor effect is possible. These include: (1)alterations of the permeability of the cell membrane, (2) feedback regulation of the biosynthetic pathway, and (3)oxygen transfer effects.

1. Alterations of the Permeability of the Cell Membrane In a Streptomyces fradiae mutant that requires oleic acid for neomycin formation, the production of neomycin depends on the cellular fatty acid spectrum, even though neomycin is not a polyketide-derived antibiotic (Arima et al., 1973; Okazaki et al., 1973). Similarly, fatty acid addition is likely to alter the membrane composition and permeability of the polyene macrolide-producing strains, although the

24

JUAN F. MARTIN AND LLOYD E. MCDANIEL

possible influence of these alterations on polyene macrolide antibiotic biosynthesis remains an open question.

2. Feedback Regulation of the Biosynthetic Pathways Fatty acid and polyene macrolide biosynthesis is a typical example of a branched pathway leading to both a primary and a secondary metabolite (Martin, 1976). The regulation of the flow of precursors in branched pathways has been discussed in detail by Demain (1968). Stimulation of the production of the heptaene macrolide levorin by different oils has been shown, but biosynthesis of levorin under the same conditions was inhibited by the component fatty acids (oleic acid, linoleic acid, etc.) (Tereshin, 1976). Martin and McDaniel (1976) also showed that sodium oleate, sodium palmitate, or methyl oleate failed to support the production of candicidin by resting cells of S. griseus in the absence of another carbon source. When added to a glucose-based medium, fatty acids inhibited candicidin biosynthesis. This inhibition seems to involve feedback regulation of the common part of the pathway by high concentrations of long-chain fatty acids. Raczynska-Bojanowska (1974)also described feedback inhibition of the acetyl-CoA carboxylase of the nystatin-producer S. noursei by free fatty acids. It is therefore unclear why oils stimulate the biosynthesis of some polyene macrolides (e.g., fungichromin and filipin) in complex media while fatty acids have an inhibitory effect on the biosynthesis of others by resting cells (e.g., candicidin) or on the activity of biosynthetic enzyme systems (e.g., nystatin). Further studies on the nature of the regulatory mechanisms involved are required. Stimulation of polyene macrolide production may be due to an inhibitory effect of the fatty acids on the independent fatty acid synthase, therefore sparing acetyl-CoA for polyene macrolide biosynthesis. Feedback inhibition of fatty acid synthase by fatty acyl-CoA derivatives is well established (Lust and Lynen, 1968; Birnbaum, 1970; Izui et al., 1970; Satyanarayana and Klein, 1973; Flick and Bloch, 1975). 3. Effect of Antifoam Agents on Oxygen Transfer in the Culture Both oils and fatty acids have detergent properties that affect oxygen uptake and may interact positively or negatively on polyene biosynthesis. G. NITROGEN SOURCES Considerable differences in the ability of different amino acids to support candicidin biosynthesis were described by Acker and Lechevalier (1954). L-Asparagine was the best sole nitrogen source. L-Histidine, glycine,

PRODUCTION OF POLYENE MACROLIDE ANTlBIOTICS

25

L-glutamic acid, and L-aspartic acid were next in effectiveness as nitrogen sources. The D-isomers of the amino acids supported negligible growth and no candicidin production. Similar results were reported recently by AbouZeid (1973) for candicidin and for ayfactin (Abou-Zeid and Abou-el-Atta, 1971). It is interesting to note that p-alanine supported a 10 times higher yield than a-alanine and was the best amino acid source for the production of ayfactin. This result is interesting, as p-alanine is a component of the pantetheinyl moiety of coenzyme A and pantetheinyl-proteins of fatty acid and macrolide synthases. Radioactive p-alanine added to the growth medium of S. erythreus is incorporated into the fatty acid synthases of this organism (Corcoran, 1973). L-Asparagine has been successfully used as nitrogen source in a defined medium for candicidin production by resting cells of S. griseus (Martin and McDaniel, 1976). Inorganic salts are generally poor nitrogen sources for polyene production. However, ammonium salts were found to be acceptable in the case of mycoheptin (Tereshin, 1976). Complex nitrogen sources are the choice for large-scale antibiotic production, and a large variety of plant and animal proteins have been used. These include yeast extract, soya peptone, cottonseed meal, soybean meal, corn meal, casein and its hydrolyzates, corn steep liquor, and distillers’ solubles. Soybean meal is a good nitrogen source for producing many polyene macrolides (Brewer and Frazier, 1962; Mohan et al., 1963; Martin and McDaniel, 1974), probably because of its balance of nutrients (short fatty acid precursors, protein, cations, low phosphate, etc.) and its slow hydrolysis, which create physiological conditions during trophophase favoring antibiotic production in the idiophase (Nefelova and Pozmogova, 1960). Starch-fiee soya peptone supports high yields of candicidin (Liu et al., 1975; Martin and McDaniel, 1975a). In the levorin fermentation, soybean meal hydrolysis by proteases produces an initial increase in soluble nitrogen (Tereshin, 1976).An increase in ammonia that follows is due to deamination of amino acids. H. REGULATIONBY AROMATICAMINO ACIDS As can be seen in Table I, several polyene macrolides (ascosin, aureofungin, ayfactin, azacoultin, candicidin, DJ-400 B2 hamycin, heptamycin, levorin, trichomycin, and Sch 16656)belonging to the heptaene subgroup, have a p-aminoacetophenone moiety. A few others (candimycin, DJ-400 B1, and perimycin) have an aromatic N-methyl-p-aminoacetophenone moiety. The aromatic moiety of candicidin is synthesized from glucose via shikimate and the aromatic amino acid pathway to p-aminobenzoic acid (PABA), which is then incorporated into candicidin (Liu et al., 1972a) (Fig. 5), rather than through the alternative polyketide-derived route (Corcoran and Darby,

26

JUAN F. MARTIN AND LLOYD E . MCDANIEL

ERYMROSE 4- PHOY:

-

+,~OSPHOENOLPYRUVATE c-

1 \

3- DEOXY-D-ARABINO-HEPTULOSONIC ACID-. 7- PHOSPHATE (DHAP)

1

tI I

i

I I

SHlKlMlC ACID

/I\

PREPHENIC ACID

CHORiSMlC ACID

p-AMFIOBENA)H: ACID

I

TYROSINE

I

I

i

PHENYLALANINE CANDlClDlN

FIG.5. Proposed regulatory mechanism of the biosynthesis of aromatic polyene macrolides by aromatic amino acids.

1970). When either (ring UL)-['4C]-p-aminobenzoic acid or 7-[14C]-paminobenzoic acid was added to the candicidin-producingculture, between 33 and 40% of the radioactivity added was incorporated into candicidin. About 98% of the radioactivity in candicidin was recovered from the p aminoacetophenone moiety after hydrolysis. The high incorporation of PABA into candicidin compared with the lower degree of incorporation of glucose or shikimic acid indicates that PABA is a direct precursor of the aromatic moiety. Incorporation of PABA was linear for at least 10 hours and paralleled cellular uptake. The intracellular pool of PABA for antibiotic synthesis appears to be small and strictly regulated, as cellular uptake of PABA stopped soon after candicidin synthesis was blocked by the addition of cerulenin (Martin and McDaniel, 197%). Similar results were obtained in the case of the N-methyl+-aminoacetophenone moiety of perimycin (syn. fungimycin) (Liu et al., 1972b). The N-methyl group of the methyl-p-aminoacetophenone is derived from the methyl group of L-methionine. However, it is unclear whether methylation of PABA takes place prior to the attachment of the aromatic moiety or whether it is an a posteriori modification of the complete amethylfungimycin molecule.

PRODUCTION OF POLYENE MACROLIDE ANTIBIOTICS

27

The biosynthesis of candicidin was 50% inhibited by a mixture of 5 mM L-tryptophan, L-tyrosine, and L-phenylalanine (Liu et al., 1972a). Interestingly, the rate of incorporation of exogenous PABA was stimulated by 50%at inhibitory concentrations of the aromatic amino acids mixture. This was interpreted as indicating that PABA biosynthesis in the cell was retarded by the presence of an excess of aromatic amino acids, presumably by feedback inhibition, causing the exogenous supply of PABA to be more efficiently incorporated. Although no effect of the aromatic amino acids on mycelial growth was observed, an indirect effect could not be excluded with the batch system used. Further studies, using a resting cell system, indicated that the regulation of candicidin biosynthesis by aromatic amino acids is independent of growth and is exclusively produced by L-tryptophan, not by L-phenylalanine or L-tyrosine 0. F. Martin, unpublished). These results suggest that PABA biosynthesis is subject to end-product inhibition exerted by the presence of an excess of aromatic amino acids in the cell. This is a fine example of a regulatory system in which the biosynthesis of a secondary metabolite (candicidin) is regulated by the mechanisms controlling the biosynthesis of primary metabolites such as the aromatic amino acids, as represented in Fig. 5. Similar regulatory examples have been discussed by Demain (1974). A similar branched pathway leading to the biosynthesis of chloramphenicol and aromatip amino acids has been described in Streptomyces sp. 3022 (see review by Malik, 1972). However, the biosynthesis of chloramphenicol does not appear to be regulated by aromatic amino acids (Lowe and Westlake, 1971); i.e., the producing organism has a DHAP synthase insensitive to end-product inhibition or repression. I. EFFECTOF INORGANIC ORTHOPHOSPHATE Negative control of the biosynthesis of secondary metabolites by high inorganic phosphate concentrations has been observed in many Streptomyces fermentations (see reviews by Demain, 1972; Weinberg, 1974). Antibiotics, like most other secondary metabolites, are produced only at inorganic phosphate concentrations that are suboptimal for growth. Inorganic orthophosphate in the range of 0.3 to 300 mM permits excellent cell growth, but 10 mM concentration often suppresses biosynthesis of antibiotics. Several examples of suppression of polyene macrolide antibiotic production along with the range of permissive phosphate concentrations are listed in Table 111. Early reference to the phosphate effect on the biosynthesis of the polyene macrolide antibiotics nystatin and amphotericin B was made by Donovick

TABLE I11 SOME POLYENE MACROLIDEANTIBIOTICS WHOSE PRODUCTION IS CONTROLLED BY INORGANIC PHOSPHATE Range of phosphate permitting antibiotic production

(mM)

Antibiotics

Producer strain

Amphotericin B Ayfactin Candicidin Candidin Levorin Mycoheptin Nystatin

Streptomyces nodosus Streptomyces aureofaciens Streptomyces grkeus Streptomyces viridoflavus Streptomyces levwis Streptoverticillium mycoheptinicum Streptomyces noursei

~

~

~

P Referencesb

~

1.5-2.2 1-17 0.5-5

3, 6 1 4

0.5-5

5 2

0.3-4 3.55 1.62.2"

6

33

=Optimalconcentration of phosphate for antibiotic production. bKey to references: (1) Abou-Zeid and Abou-el-Atta (1971), (2) Belousova et al. (1970a), (3) Donovick and Brown (1965), (4) Liu et al. (1975), (5) J. F. Martin and L. E. McDaniel (unpublished), (6) Tereshin (1976).

t U

r 0 0

P

PRODUCTION OF POLYENE MACROLIDE ANTlBIOTlCS

29

and Brown (1965); however, no detailed study was published. The effect of phosphate on accelerating growth and increasing the rate of carbohydrate utilization by the nystatin producer was described by Popova et al. (1961). Belousova et al. (1970a) found that inorganic phosphate decreased the synthesis of levorin in a corn steep liquor medium. Glycerophosphate also inhibited antibiotic formation, although to a lesser extent. The same authors reported that a decrease in synthesis of levorin was accompanied by an increase of fatty acid formation without changing the percentage ratio of the different fatty acids in the cells (Belousova et al., 1970b). Abou-Zeid and Abou-el-Atta (1971) showed that high concentrations of phosphate inhibited ayfactin formation and increased the utilization of starch. Lopatnev et al. (1973) found that the extracellular phosphate concentration in the nystatin fermentation decreased during growth and remained at a very low level during the production phase. After exhaustion of glucose and cessation of growth, the extracellular phosphate concentration increased again. Liu et al. (1975) found that the addition of 10 mM phosphate to the candicidin producer resulted in a 2-fold increase in mycelial growth. Specific candicidin production was 96% inhibited under these conditions. After phosphate addition, the dissolved oxygen level in the broth decreased sharply, accompanied by increases in mycelial growth rate and sugar utilization. A shift from secondary metabolism to primary metabolism by the action of phosphate could be achieved at any time during idiophase (Fig. 6). Similar results were obtained in the biosynthesis of the nonaromatic heptaene candidin. In batch cultures, phosphate exerted its effect on both primary and secondary metabolism, and it was unclear whether its effect was primarily in stimulating growth or in inhibiting (or repressing) polyene macrolide formation. Further insight was gained during short-term incubation periods by following the incorporation of labeled precursors into candicidin by resting cells of S. griseus. Phosphate at'5 or 10 mM decreased the rate of incorporation of precursors into candicidin without affecting cellular uptake of precursors. Complete inhibition of precursor incorporation was achieved after 120 minutes using 10 mM phosphate. Addition of phosphate did not alter overall protein synthesis in the short-term experiments. The specific regulatory effect of phosphate on the formation or activity of some enzymes involved in candicidin synthesis was studied using chloramphenicol to inhibit protein synthesis. The halting of protein synthesis resulted in partial inhibition of antibiotic formation, suggesting that the formation of candicidin synthases occurs while the system is synthesizing candicidin. The activity remaining in the presence of chloramphenicol was subject to phosphate inhibition, showing that at least a part of the phosphate effect is on the activity of preformed enzymes (A. L. Demain and J. F. Martin, unpublished).

JUAN F. MARTIN AND

0 TIME (hr)

20

40

60 80 TIME (hr)

100

120

FIG. 6. Inhibition of candicidin formation by phosphate. The fermentation was run in a 30-liter fermentor. The medium contained 2.5% soya peptone, 6.5% edible grade glucose, and 5X A4 zinc sulfate. Sixteen hours after incubation the pH of the culture was brought up to 8.0 and maintained at that pH throughout the entire fermentation by adding 20% NaOH solution with an automatic pH control instrument. Two fermentations were run. In one fermentation, K2HP04 was added to the culture at the time indicated; the other fermentation served as control. From Liu et al. (1975). Reprinted with permission of American Society for Microbiology Publication Office.

Several mechanisms have been proposed to explain the effect of phosphate on the production of idiolites. These include the following: 1. Vegetative growth limitation due to a lack of phosphate shifts the metabolism to the production of idiolites. Bu'Lock (1974) suggested that different degrees of nutritional limitation are required to trigger the formation of different idiolites. 2. Herold and Hostdek (1965)attributed the phosphate inhibition of tetracycline biosynthesis to a shift of the carbohydrate catabolic pathways, glycolysis being favored at the expense of the pentose-phosphate cycle; hence NADPH becomes the limiting factor in antibiotic synthesis.

PRODUCTION OF POLYENE MACROLIDE ANTIBIOTICS

31

3. Robbers et al. (1972) observed that phosphate inhibition of the biosynthesis of ergot alkaloids is overcome by tryptophan, an inducer of ergot synthase. Phosphate seems to limit the synthesis of endogenous tryptophan. Tryptophan synthase activity increases 20- to 25-fold in low-phosphate cultures. The synthesis of PABA, a component of the aromatic polyene macrolides, may be the limiting step in polyene macrolide biosynthesis in the presence of excess phosphate. 4. In the biosynthesis of streptomycin (Miller and Walker, 1970),viomycin (Pass and Raczynska-Bojanowska,1968), and vancomycin (Mertz and Doolin, 1973), phosphate acts by preventing the formation of alkaline phosphatases required for antibiotic biosynthesis. Miller and Walker (1970) reported that inactive streptomycin phosphate was formed when inorganic phosphate was added to mycelia synthesizing the antibiotic. Walker (1971)has proposed that the dephosphorylation reaction is the final step in the biosynthesis of streptomycin. Dephosphorylation of streptomycin phosphate to active streptomycin is prevented by excess phosphate. Similar modification by phosphorylation takes place also in the antibiotics lincomycin and neomycin (Majumdar and Majumdar, 1970). Different mechanisms of phosphate effect probably exist in the biosynthesis of different antibiotics. But most likely all of them are different projections of a common parameter, the cellular energy charge, which determines the phosphorylating or energetic capability of the cell. J. REGULATIONOF THE BIOSYNTHESIS OF POLYENE MACROLIDEANTIBIOTICS BY ADENINE NUCLEOTIDES AND/OR

THE

ENERGY CHARGE OF

THE

CELL

The adenine nucleotides adenosine triphosphate (ATP), adenosine diphosphate (ADP), and adenosine monophosphate (AMP) stoichiometrically couple all the metabolic sequences of a living cell. The amount of metabolically available energy that is momentarily stored in the adenylate system is related to the energy charge of the cell, defined by Atkinson and Walton (1967) as follows: Energy charge (EC) = [(ATP)

+ 1/2 (ADP)]/[(ATP)+ (ADP) + (AMP)]

The energy charge is a regulatory parameter coordinating energy-utilizing and energy-regenerating metabolic sequences. Atkinson (1969)has reviewed response curves of activity for several enzymes as a function of the energy charge. Regulatory enzymes from sequences in which ATP is regenerated are highly active at low levels of energy charge and decrease sharply in activity as the energy charge increases above a value of about 0.75. Regulatory enzymes from biosynthetic sequences or others that consume ATP,

32

JUAN F. MARTIN AND LLOYD E. MCDANIEL

exhibit very little activity at low levels of energy charge, and their activities increase sharply at charge values above 0.75. Atkinson and co-workers (Chapman et al., 1971) predicted that the energy-charge values of a cell would be poised between 0.8 and 0.9 in order to maximize the regulatory effect of the energy charge. This is true for E. coli. In light of this theory, the effect of purine and pyrimidine nucleotides on the biosynthesis of candicidin has been studied. It was found that ATP, ADP, AMP, adenosine 3’,5’-cyclicmonophosphate (CAMP),and other purine and pyrimidine nucleotides, but not nucleosides or bases, inhibit candicidin biosynthesis in a resting cell system, similar to the effect of phosphate. It has been suggested that phosphate may be active in regulating polyene antibiotic biosynthesis through mediation of nucleotide levels in the cell. A large increase in the intracellular level of ATP has been found to follow phosphate addition. Steady-state values of energy charge were 0.7-0.8 in resting cells synthesizingcandicidin, and increase to 0.9 following phosphate or nucleotide addition. Since the energy charge acts as a fine control of enzyme reactions, particularly at the branch point, the effect of adenine nucleotides is probably due to a shift of the whole metabolic pattern of the cell. Possible regulatory target enzymes are some amphibolic enzymes of glycolysis, such as phosphofiuctokinase, or enzymes that regulate the entry of acetyl-CoA into the Krebs cycle (citrate synthase) or NAD(P)Hregenerating systems in the Krebs cycle, such as isocitrate dehydrogenase (Atkinson, 1969). Alternatively, phosphate and the adenine nucleotides may act at the transcriptional level by interacting, and therefore changing the substrate specificity of the RNA polymerase. DifEerent parts of the genome will be expressed (read) under low or high energy charge, resulting in growth or production of idiolites. The shift of the carbohydrate catabolic pathways upon phosphate addition proposed by Herold and Hostiilek (1965) is readily understood as an ATP-mediated regulatory effect. Additional evidence supporting the regulatory role of ATP on antibiotic production comes from studies indicating that the level of intracellular ATP drops sharply prior to antibiotic production (J. F. Martin and A. L. Demain, unpublished). The pattern of ATP changes observed during the fermentation might be explained according to Atkinson (1965) and Martin and McDaniel (1974) as follows: when phosphate is exhausted from the medium, the balanced assimilation of nutrients characteristic of trophophase is halted. Biosynthesis of macromolecules, NAD(P)H regeneration, and Krebs cycle reactions slow down since no intermediates are removed. ATP accumulates momentarily during the transition period since the cell is unable to carry out energy-requiring growth processes. This seems to be the case in the fermentation of chlortetracycline (Janglova et al., 1969). Owing to the de-

PRODUCTION OF POLYENE MACROLIDE ANTIBIOTICS

33

cline in growth rate and TCA-cycle activity, glucose catabolites accumulate, as has been found to happen also in the fermentation of nystatin (Toropovaet al., 1972). The entry of acetyl-CoA into the Krebs cycle is further made difEcult by the negative effector action of ATP on citrate synthase and phosphoenolpyruvate carboxylase, the enzymes that catalyze the entry of acetyl-CoA into the Krebs cycle (Hostiileket al., 1969). Acetyl-CoA is in this way shifted toward the production of polyene macrolides or fatty acids. After the transition stage, the intracellular level of ATP drops to very low steady levels probably reflecting utilization for polyene macrolide biosynthesis and turnover metabolism without replenishment of the intracellular pool (Janglova et al., 1969; J. F. Martin and A. L. Demain, unpublished). It appears that the antibiotic-producing cell is essentially unable to regenerate enough ATP because of phosphate limitation. The addition of phosphate or nucleotides reverses this situation, shifting secondary metabolism to that typical of growth.

K. EFFECTOF METAL IONS Early studies indicated that iron and zinc were essential for growth or antibiotic production (Acker and Lechevalier, 1954). Stimulation of the production of the tetraene macrolide, antimycoin, by calcium and magnesium ions was reported by Schaffner et al. (1958). Brewer and Frazier (1962) described stimulatory effects of manganous, nickel, zinc, and cobalt salts on the production of amphotericin B in soybean meal media. Manganous salts, especially in the presence of iron, were the most effective. The stimulatory effect of divalent cations on candicidin production was studied by Liu et al. (1975). Maximum effects were exerted by iron, zinc, and magnesium at 5 x M in a complex medium. However, in a to defined medium metal stimulation of candicidin production occurred only at M level, and higher concentrations were inhibitory. In all cases the 5 x an increase in candicidin yield was accompanied by a reduction of mycelial growth. Addition of zinc at any time during the fermentation resulted in increased candicidin synthesis, reduced dry weight, and an increased rate of glucose utilization. Zinc, iron, and magnesium appear to be common stimulators of polyene macrolide production. Differences among several polyenes may be due to the difFerent nutrients used and the content of metal ions in the complex media. Weinberg (1962, 1970) has reviewed the role of trace metals in secondary metabolism and has concluded that iron and zinc are “key” metals for actinomycetes and fungi. The concentrations of metals required for secondary metabolism are one or more log units higher than the minimum

34

JUAN F. MARTIN AND LLOYD E. MCDANIEL

required for growth (approximately lo-? M).Moreover, primary metabolism tolerates up to M of each metal, about 2 log units higher than the level that is inhibitory for secondary metabolism. Although a large number of examples of metals affecting production of idiolites is known, no unitary role appears to exist; rather, several possibilities in different systems have been suggested. The most likely role of metals is as activator of the synthases of secondary metabolism, although an effect at the transcriptional or translational level has been suggested in some cases. A probable role of metal ions on the biosynthesis of polyene macrolides is either the activation of polyene synthases or the stabilization of the intermediate substrates in polyketide biosynthesis, as suggested by Bu’Lock (1967). P-Polyketide intermediates have a great tendency to form coordination complexes with metal ions. Enzyme-bound growing chains of malonyl units may be maintained in a suitable configuration in this way to allow for the condensation of the malonyl-CoA derivatives and the decarboxylation and transacylation reactions. The stimulatory effect of magnesium ions on polyene macrolide biosynthesis is probably related to the activating effect on acetyl-CoA carboxylase (acetyl-CoA ligase, EC 6.4.1.2), a magnesium-requiring enzyme (Lynen, 1967), as has been found in in uitro studies of this enzyme (Rasmussen and Klein, 1968). Another possible role of metal ions in the production of candicidin, namely the removal of inhibitory concentrations of phosphate, has been suggested by Liu et al. (1975). However, attempts to demonstrate the sequestering effect of metal ions on high concentrations of inorganic phosphate by adding equivalent amounts of zinc and phosphate to soya peptone cultures of S. grlseus were not successful. L. EFFECTOF DISSOLVED OXYGEN

The general role of oxygen in the growth of aerobic microorganisms is well known (see reviews by Harrison, 1972, 1973). The rate of solution of oxygen in a medium has to be at least equal to the requirement of a microorganism for growth and metabolism in order for it not to be limited by oxygen supply. McDaniel and Bailey (1969), studying the effect of oxygen supply rates in shake flasks, found that maximum yields of candidin could not be obtained in conventional unbaffled Erlenmeyer flasks. It was necessary to use baffled flasks in which oxygen transfer rates (sulfite oxidation rates) of 1 mmole per liter per minute or higher could be obtained. The highest yield of fungimycin was obtained at oxygen transfer rates of0.5 mmole per liter per minute or higher (Mohan et al., 1963). In the case of candicidin the limiting rate was reported to be 0.8 mmole per liter per minute (Liu et al., 1975).

PRODUCTION OF POLYENE MACROLIDE ANTIBIOTICS

35

Dissolved oxygen levels are critical for the production of many antibiotics, including cephalosporin and capreomycin (Feren and Squires, 1969), penicillin and bacitracin (Brandl et al., 1966), oleandomycin (Surikova and Zavileiskaya, 1972), and erythromycin (Lobanova and Brinberg, 1971). Ethiraj (1969) studied the effect of dissolved oxygen tension on candicidin production. At 40% of saturation and above, the yields were constant. At 10 and 20% of saturation, candicidin production was reduced. Lopatnev et al. (1973) reported that a mixing speed of 300 rpm was required to supply enough dissolved oxygen to a culture of S. noursei producing nystatin. Lower mixing speeds resulted in depletion of dissolved oxygen and drastic reduction in the formation of nystatin. Under intensive agitation (oxygen transfer coefficient k# >6 min-'), a dependence of the specific productivity of nystatin on the specific rates of growth and carbohydrate utilization was found (Lopatnevet al., 1975);this was not observed with poor agitation (kIp c3.5 min-'). Maximum yields of nystatin were obtained only with the higher oxygen transfer conditions. Agitation speeds of 300 rpm or above were also required to keep the dissolved oxygen levels from falling below 20% of saturation in candidin and candihexin fermentations (Martin and McDaniel, 1974). Saturation levels of 60% or higher were maintained during the production of candicidin using an agitation speed of 420 rpm in a 30-liter fermentor (Martin and McDaniel, 1975a). Under these conditions, maximum oxygen uptake rates were 0.7 mmole per liter per minute in candidin and candihexin fermentations and 0.5 mmole per liter per minute with candicidin. If adequate agitation speeds are not provided, the dissolved oxygen levels will fall below the critical level (the level above which the rate of oxygen uptake by an organism is independent of oxygen concentration). According to Lopatnev et al. (1973), reduction of the dissolved oxygen to a concentration not lower than the critical level does not reduce nystatin synthesis. This is in contrast with the results of Ethiraj (1969), which indicated that the dissolved oxygen tension for maximum candicidin production is well above the critical level (3.5% of saturation). A correlation of the change of oxygen uptake rate with onset of polyene macrolide formation was found in the fermentations of three polyene macrolides, candidin, candihexin, and candicidin. The large increase in oxygen uptake which parallels cellular growth (DNA increase) during trophophase levels off or decreases sharply, coinciding with the onset of antibiotic formation (Martin and McDaniel, 1974, 1975a)(Fig. 3). It is obvious that a change in metabolism occurs at that time owing to exhaustion of some essential nutrient, typically phosphate. Under nutrient limitation, the balanced assimilation of nutrients with associated high oxygen demand is halted, producing a shift to secondary metabolism (Martin and McDaniel, 1974).

36

JUAN F. MARTIN AND LLOYD E . MCDANIEL

A factor directly affecting the interaction of dissolved oxygen in the broth and oxygen uptake by the cell is the addition of antifoam agents. Selection of antifoaming agents is generally done on an empirical basis. Polyether and polyglycol derivatives (e.g., Polyglycol P-2OO0, Dow Chemical Co.) at concentrations of 0.02% are routinely used in fermentations of polyene macrolides. Lvova et al. (1973) studied the comparative effect of natural oils and synthetic antifoam agents on the production of levorin. Most oils could be used only at low concentrations. Sperm oil could be used at a higher level. At 0.5-1% it exerted an antifoam effect and stimulated antibiotic biosynthesis. Polyether synthetic products (e.g., adecanol and propinol B-400) at concentrations of 0.02-0.M% had the significant advantage of suppressing foaming without affecting production, isolation, or purification of levorin.

M. EFFECTOF REDOXPOTENTIAL Another factor that is claimed to affect the production of polyene macrolide antibiotics is the redox potential (Eh)of the culture. The redox potential is easily defined in a simple chemical system by the equation of Nernst, but its exact meaning in complex microbial systems is not clear. In the presence of oxygen all redox systems tend toward the fully oxidized state, and in complex systems redox probes reflect the concentration of dissolved oxygen. In spite of the above, correlations have been made between the redox potential of the culture and different microbial processes (Harrison, 1972). Sukharevich et al. (1970a,b)stated that a change in the redox potential at constant aeration rates leads to substantial changes in the polyene macrolides synthesized by S. leuoris and S. mycoheptinicum, the producers of levorin and mycoheptin. Studies with S. mycoheptinicum revealed that low Eh values during the first 24-26 hours of the fermentation decreased both growth and total antibiotic production. This strain usually produces a heptaene macrolide (mycoheptin) together with a pentaene macrolide component. Specific yields of the pentaene decreased to one-third when low redox potential values were maintained, while that of the heptaene was at the same level as in control fermentations, thus providing a method for specific enrichment of the heptaene macrolide component (Sukharevich et d., 197Oa). In the production of levorin, high-producing strains of S. levoris were grown at reduced redox potentials adjusted with hyposulfite, hydrosulfite, and thioglycolic acid or at increased redox potentials achieved by changing the volume of the medium or by adding potassium ferricyanide. Experiments were done at constant high or low aeration levels and changing redox potentials. The levorin complex is a mixture of several components (Tsyganov and Yakovleva, 1968), the two main ones, levorin A and B, occurring in different proportions in several low- and high-producing strains. Increasing the redox potential of the medium with atmospheric oxygen or an oxidizing agent led

PRODUCTION OF POLYENE MACROLIDE ANTIBIOTICS

37

to a decrease in the fraction of levorin B in the antibiotic complex. When the redox potential was lowered by adding a reducing agent, the fraction of levorin B increased. The changes in the redox potential of the culture did not affect the amount of biomass of S. levoris produced, but at lower redox potentials the total antibiotic yield was only 22% of that of the control. The large amounts of reducing agents (1-2%) required to lower the redox potential from the 450-mV level characteristic of normally aerated fermentations to the 1W150-mV level of “reduced broths may affect antibiotic formation by mechanisms other than the oxidoreduction potential. If the redox potential monitored with a platinum electrode reflects, as assumed, the redox potential of the intracellular components, it is interesting to correlate the NAD(P)/NAD(P)H ratio (the key parameter reflecting the redox potential for driving oxidation-reduction reactions) with changes in the total yield of polyene macrolide and the composition of the polyene macrolide complexes. At artificially low redox potentials, NAD(P)/NAD(P)H should be expected to be low and therefore favor the formation of reduced lipid substances (requiring large amounts of NADPH) at the expense of more oxidized compounds such as macrolides. In effect, Gatenbeck and Hermodsson (1965) have established that the concentration of reduced nucleotides determines the in vitro incorporation of acetate into polyketidederived compounds or fatty acids. Activities of NADP-dependent dehydrogenases responsible for regeneration of NADPH are not limiting for the biosynthesis of the polyene macrolide polifungin, but enzymic activities are nevertheless lower in the nonproducing mutants (Roszkowskiet al., 1971). A large response of intracellular NADH to changes in oxygen supply has been found in Klebsiella aerogenes (Harrison, 1972), but no correlation has been established so far between the redox potential of the culture and the intracellular level of NAD(P)H. The redox potential of the culture may affect the proportion of components in polyene macrolide complexes by favoring the specific reduction of some double bonds or keto groups of the polyketide chain. In addition to the effect on antibiotic production, Sukharevich et al. (1972)reported that decreased redox potentials affect the structure and form of the colonies on a solid medium of 14 species of Streptomyces. Low redox potentials also produce changes in the development of aerial mycelium.

111. Interaction of Polyene Macrolide Antibiotics with the Producer Cells A. MODIFICATION AND DEGRADATION OF POLYENE MACROLIDEANTIBIOTICSBY THE PRODUCER CELLS

In the production of antibiotics, the final amount of extractable product depends on the total amount secreted and the fraction that is degraded

38

JUAN F. MARTIN A N D LLOYD E . MCDANIEL

under the fermentation conditions. Reutilization of exogenous antibiotic by the producing cell does not seem to play a major role in decreasing the yield of antibiotic fermentation. Uptake of antibiotics (e.g., chloramphenicol) by producing cells can occur during the growth phase, but a decreased permeability of the producing cell to the excreted antibiotic develops during idiophase and is one of the mechanisms by which the producer cell avoids suicide (Demain, 1974). Antibiotics may be either modified or degraded under production conditions by mechanisms similar to those evolved by resistant target organisms (Benveniste and Davies, 1973). Phosphorylation of streptomycin by S. griseus (Nimi et al., 1971) and neomycin by S. j-adiae (Majumdar and Majumdar, 1970)or hydrolysis of chloramphenicol by Streptomyces sp. (Malik and Vining, 1971) and penicillin by penicillin acylases of Penicillium chrysogenum (Cole, 1966) are well-known modification mechanisms. There are several reports in the literature indicating that titers of some polyene macrolide antibiotics drop after termination of antibiotic formation (e.g., nystatin, candihexin) whereas those of others remain at the same level for several days (e.g., candidin, candicidin). Spizek et al. (1965b) reported that the addition to the culture medium of fungicidin in concentrations exceeding the amounts that the culture can produce induced enzymic systems that degraded (or transformed) the antibiotic approximately to the same level as that produced in control cultures. However, no proof of the involvement of enzymic activity was provided. In a similar study with candihexin and candidin, it was observed that when candihexin was added at the onset of the idiophase to candihexin-producingcultures there was degradation of the added candihexin (or lack of de n o w candihexin synthesis)and the antibiotic level remained at the same level during the fermentation (J. F. Martin and L. E. McDaniel, unpublished). However, when candidin was added at the same time to candidin-producingcultures there was no effect on the production of candidin as compared to the nonsupplemented control, and the final antibiotic yield was the total accumulation of exogenously added and de nooo synthesized antibiotic. Whether there is an enzymic conversion, a chemical degradation, or a feedback inhibitory effect on antibiotic production is unknown at present. Tereshin (1976) reported that amphotericin B may be used as an endogenous substrate by the producing cell, resulting in the formation of biologically inactive amphotericin B. It is unclear, however, whether formation of inactive components occurs by intracellular modification prior to antibiotic excretion or whether exogenously added amphotericin B is taken up by the cell and modified. The occurrence of nonactive components lacking the aminosugar moiety of polyene macrolides (mycosamine) has been described in the polifungin and candihexin fermentations (Roszkowski et al., 1972; Martin and McDaniel, 1975b), but

PRODUCTION OF POLYENE MACROLIDE ANTIBIOTICS

39

they are believed to be nonglycosylated intermediates rather than transformation products. Determination of the chemical structure of the inactive amphotericin B would help to clarify the inactivation mechanism in polyene macrolide-producerswhich may have some survival significance for the producing strain.

B. STABILIZATIONOF POLYENE MACROLIDE ANTIBIOTICS AFTER PRODUCTION The lability of polyenes to light or heat is well known (Oroshnik and Mebane, 1963; Hamilton-Miller, 1973). Thermal inactivation has been studied with filipin (Tingstad and Garrett, 1960), nystatin (Belenky et al., 1966), amphotericin B (Bonner et al., 1975), and the tetraene fumagillin (Garrett, 1954). Thermal inactivation of nystatin was associated with loss of the polyenic chromophore. In addition to thermal inactivation, two other mechanisms, (1) autoxidation and (2) UV light photolysis, are known to affect the stability of polyene macrolides. Autoxidation of rimocidin and pimaricin in solution has been reported (Dekker and Ark, 1959). It can be prevented by the addition to pimaricin of chlorophyllin or antioxidants, such as ascorbic acid, hydroquinone, or gallic acid. Rickards et al. (1970) studied the autoxidation of solutions of filipin and lagosin and concluded that the reaction products were the corresponding tetraene epoxides. This autoxidation was reduced by the addition of the antioxidant, 2-t-butyl-4-methoxyphenol.Antioxidants have also been reported to protect against the autoxidation of nystatin (Zhdanovich et al., 1967). The photolability of polyene macrolides is well documented. Polyene macrolides show a high specific absorption of long ultraviolet light due to the presence in their molecule of alternating double bond chromophores of different lengths. Dekker and Ark (1959) observed that photolysis of pimaricin can be prevented by chlorophyllin or dyes such as quinoneimine, triphenylmethane, and nitro and azo dyes that act by intercepting UV light. They suggested that UV photolysis brings about a trans to cis isomerization of the chromophore resulting in the loss of activity. Zondag et al. (1960) postulated a similar isomerization mechanism for photolysis of pimaricin. Cis-trans isomerization after UV irradiation has also been described in the heptaene macrolide DJ-400 (Siewert and Kieslich, 1971) and in levorin (Filippova et al., 1972). Thermal inactivation and autoxidation are likely to decrease the yield of polyene macrolides in long-lasting (4-7 days), highly aerated fermentations. Photolysis may also influence the yield of polyene macrolides in shake flasks or glass-jar fermentors although the glass and UV-absorbing substances in

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the broth will shield the excreted polyenes against radiations of less than 350 nm. Addition of antioxidants may stabilize the polyene macrolides in the broth but also will interfere with the redox potential of the culture. Complexing of polyene macrolides appears to be a mechanism that stabilizes them in the fermentation. The addition of cholesterol in equimolecular amounts to the candihexin or candidin fermentations increased the production of both products 0. F. Martin, unpublished). Cholesterol forms complexes with polyene macrolides and may act by protecting the extracellular polyene from thermal inactivation or autoxidation. Cholesterol-levorin and cholesterol-amphotericin B have been shown to have a stabilizing effect on polyene antibiotics (Klimov et al., 1971). Other complexing agents of polyene macrolides, such as sodium deoxycholate, which is known to solubilize water dispersions of polyene macrolides, completely inhibit polyene macrolide production. The inhibitory mechanism is unknown. Alternatively, the stimulatory effect of cholesterol on polyene macrolide biosynthesis may be due to the interaction of cholesterol with the polyene macrolides as they are secreted at the membrane level.

c. POLYENE MACROLIDE PRODUCTION IN MIXED CULTURE WITH YEASTS Different types of microbial interaction occurring in mixed cultures of bacteria have been reviewed by Meers (1973). Of these relationships, synergism is that type of interaction in which two species produce more of a given product when growing together than when growing alone. Several examples of this type of interaction have been described (Styczynska et al., 1965; Graveri, 1956). Yakovleva et a2. (1972) and Tsyganov et al. (1973) tested the effect of cocultivation of 56 species of microorganisms with S. levoris on the production of the heptaene macrolide levorin. The species studied included yeastlike fungi, filamentous fungi, and bacteria. Either no effect or an inhibtory effect on antibiotic production was observed when S. levoris was cultivated with bacteria, but considerable stimulation was obtained when it was grown with yeasts. The highest increase in titer (2-fold) was produced by Candida tropicalis. Synergistic stimulation of antibiotic production occurred only when the yeast was grown for 24 hours before inoculation of the Streptomyces, but not when both strains were inoculated simultaneously. The increase in antibiotic titers resulted from an increase in the mycelial dry weight as well as from a higher specific production. After this report an attempt was made to increase candidin and candihexin production by cocultivation of the producing strains of Streptomyces viridoflavus and S . viridoflavus mutant 18A2 with S. cerevisiae. Yeast was added after growth of

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the Streptomyces had occurred in order to avoid a nonspecific effect on growth. When logarithmically growing yeast cells were added, they overgrew the Streptomyces and no production of antibiotic was observed. However, when the same amount of autoclaved cells was added, there was a stimulation of production of both candidin and candihexin. Two hypotheses are offered to explain the stimulation of antibiotic production: (1)nutrients are excreted by the yeast or are provided by the autoclaved yeasts and utilized by the Streptomyces; (2) polyene macrolides bind to the target sterols of yeast membranes forming complexes that act as stabilizing agents. A reversible complete binding of polyene antibiotics to components of cell-&ee yeast extracts has been described (Klimov and Nikiforova, 1971). An exclusively nutritional role of yeast seems unlikely since yeast extract does not support synthesis of the two polyene macrolides. A polyene macrolide-stabilizing role of the yeast cells is in agreement with the similar role of cholesterol. It is interesting to note that species of Candida that give a high synergistic effect are usually more sensitive to the action of polyenes than filamentous fungi, which proved to have little synergistic action. Mixed cultures with yeasts or the addition of autoclaved yeasts may be important and economical tools for increasing industrial production of polyene macrolide antibiotics. D. SUSCEPTIBILITY OF THE PRODUCER STRAINS TO POLYENE MACROLIDEANTIBIOTICS Several antibiotics have growth-inhibitory effects on their producing strains (see review by Demain, 1974). The producer strains avoid suicide by different mechanisms; these include (1) production of the antibiotic in a phase subsequent to growth, (2) modification of the antibiotic to render it inactive, (3) alteration of the antibiotic target in the cell that becomes antibiotic resistant, and (4) decreased permeability to the excreted antibiotic. No such effect of polyene macrolide antibiotics would be expected a priori since cell membranes of Streptomyces, like other bacterial membranes, are believed to be lacking in sterols. However, there is increasing evidence that at least some polyene macrolides have an inhibitory effect on the growth of their producing strains and on the amount of antibiotic produced by that strain. Dolezilova et al. (1965)reported that a fungicidin-producingstrain of S. noursei was susceptible to the action of fungicidin. There was an inverse correlation between the producing ability and sensitivity to the produced antibiotic. A high-producing strain able to form 15,000 unitdm1 was resistant to concentrations of fungicidin up to 20,000 unitdml. A strain able to produce 6OOO unitdm1 was resistant only to concentrations below 2000 units of hngicididml, and a nonproducing strain of fungicidin was inhibited by 20

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JUAN F. MARTIN AND LLOYD E . MCDANIEL

unitdml. Similar results were reported with the levorin- and mycoheptinproducing strains (Tereshin, 1976). Levorin and mycoheptin exert a lethal effect on the growth of their producing strains proportional to the antibiotic concentration in the medium. Spores of high- and low-producing strains differ in their susceptibility to levorin. The number of survivor colonies of high levorin-producing strains were lo00 times greater than that of lowproducer strains when both strains were grown on a solid medium containing 4000 units of levorin per milliliter. The inhibitory effect of levorin and mycoheptin on their producing strains resulted in 4-5 days delay of growth. Deep colonies of substrate mycelium were formed in medium supplemented with antibiotic. The colonies returned to their original morphology if transferred to antibiotic-free medium. Preliminary studies on the effect of nystatin on some enzymes of the glycolytic pathway, pentose phosphate pathway, and tricarboxylic acid cycle indicated that glucose-6-phosphate dehydrogenase and transketolase of S t r e p t m y c e s rimosus are sensitive to the effect of nystatin (Egorov and Toropova, 1975). This result, together with the fact that the amount of enzymes of the pentose phosphate cycle in nonproducer mutants is three to four times greater than in the producer strain whereas those of the tricarboxylic acid are 2-4 times less, may indicate a regulatory role of the antibiotic on the intermediary metabolism of the producer stains. It is unclear, however, whether this effect may have an in vivo effect on regulation of the metabolism of the producer strains since the intracellular concentration of nystatin is unknown. E. FEEDBACK REGULATORYEFFECTOF POLYENE MACROLIDEANTIBIOTICS ON THEIROWNPRODUCTION The addition of fungicidin to a culture of S. noursei, which synthesizes cycloheximide and fungicidin, produced an increase in cycloheximide together with an apparent inhibition of synthesis of fungicidin. This was explained on the basis of the competition of the metabolic pathways synthesizing the two antibiotics for malonate, a common precursor in the biosynthesis of cycloheximide and fungicidin. Similarly the addition of candihexin to candihexin-producing cultures stopped further antibiotic production. The addition of candidin to cultures producing candidin did not produce inhibition of antibiotic synthesis. The existing evidence of feedback regulation of polyene macrolide production is only indirect since decreased antibiotic titers following the addition of antibiotic to the producer strain may result from inhibition of antibiotic synthesis or from an increased rate of degradation of the antibiotic. A study of the effect of exogenous antibiotic on the rate of incorporation of labeled precursors into the antibiotic would resolve this question.

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Levorin seems to have little effect as a selective agent on the range of variability in the production of antibiotic by single colonies of a given population. On the contrary, when the original nonmutated S. mycoheptinicum was grown in agar supplemented with mycoheptin, variants resistant to mycoheptin were isolated which exceeded by 15-40% the production of antibiotic by the parent strain (Tereshin, 1976). Based on the effect of polyene macrolide antibiotics on their producer strains, a procedure for selection of high-producing strains has been proposed by Romankova et al. (1971). It includes repeated passages of the strains in submerged culture, followed by plating of vegetative mycelium on solid medium and selection of highly active variants. It is claimed that strains producing an increased amount of levorin, trichomycin, and amphotericin B were obtained by this procedure. Repeated submerged transfers of S. hachijoensis resulted in a variant whose activity was 90% higher than that of the original strain (Fursenko et al., 1972).

IV. Genetics of Polyene-Producing Streptomyces A. NATURALVARIANTS OF SOIL-ISOLATED STRAINS AND MUTATIONALSTRAINIMPROVEMENT Selection of high-producing mutants after empirical mutagenesis treatment is one of the first steps prior to and during industrial production of an antibiotic. Such work has been carried out with the producers of amphotericin B and nystatin (Thoma, 1971) and most probably with other polyene producers, but only recently have detailed studies on the subject been published. A few recent publications on mutational studies and actinophage conversion of the producing strains are indicative of increasing interest in the genetics of polyene-producing Streptomyces following the pioneering studies on Streptomyces genetics by Hopwood and his co-workers (Hopwood et al., 1973). The original soil isolate of S. levoris was found to consist of as many as nine morphological types of colonies. The isolate produced 6000-8000 units of levorin per milliliter. Several mutagens were used in a strain improvement program. Mutagenesis with ethyleneimine, ultraviolet light, or N-nitrosomethylurea followed by repeated passage in deep culture, resulted in the isolation of a high-producing strain (S. levoris 28) which produced 35,000-40,000 units/ml (Tereshin, 1976). According to Zhukova et al. (1975), S. levoris 28 consists of three morphological colony types; the main one accounts for 99.7% of the total. Different morphological variants ranged from 20 to 180% of the antibiotic production of the original strain. Streptomyces levoris 28 also produces a nonpolyene antifungal antibiotic, levoristatin.

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JUAN F. MARTIN A N D LLOYD E. MCDANlEL

Population variants differ in the ratio of levorin to levoristatin which they produce. Soil isolates of S. mycoheptinicum also showed a large population variance. In deep culture this species produces a nonaromatic heptaene macrolide, mycoheptin, and a pentaene macrolide antibiotic. The pentaene macrolide content is usually two to three times higher than that of mycoheptin (Tsyganov et al., 1965). Soil isolates always have a ratio of pentaene to heptaene macrolide higher than two. To increase the yield of the desired heptaene, the ratio of components was modified either by lowering the redox potential of the medium (see Section I1,M) or by mutation. Using N-nitrosomethylurea as mutagenic agent, a large degree (17%) of morphological variance was obtained with high variability in antibiotic production. All mutants produced both the pentaene and the heptaene although their ratios varied from 1.9 to 5.0. However, an increased heptaene to pentaene ratio was found to occur always at the expense of high antibiotic titers. High spontaneous variability in the form of spores and sporophores and in antibiotic titers was also found in the amphotericin B-producing organism Streptomyces nodosus. Zhukova et al. (1970) proposed that the high variability is due to the heterogeneous nature of the strain, consisting probably of several genotypes. Mutants with abundant sporulation and sporophores in the form of short tight spirals were highly active in amphotericin B biosynthesis whereas those with poor sporulation and a markedly changed form of sporophore were low or nonproducers. This correlation is frequently observed in Streptomyces which produce antibiotics. It possibly indicates that both phenomena, antibiotic production and sporulation, are regulated by similar metabolic parameters or that antibiotics play a role in the sporogenesis of bacteria (see reviews by Sadoff, 1972; Sarkar and Paulus, 1972). Natural variants of S. hachijoensis, the producer of trichomycin, differ in cultural and physiological properties from the parent strain as well as in the proportion of the components, trichomycins A and B, in the antibiotic complex (Fursenko et al., 1972). The candidin producer, S. viridoflauus, consists of two clearly different morphological types of colonies: yellowish and nonpigmented variants. Nonpigmented variants arise in the normally yellow population with a frequency of 1 3 % (McDaniel, 1976) and are normally nonproducers. Nonpigmented mutants having antifungal activity were found with very low frequency; they excrete a different antibiotic, the hexaene candihexin, instead of the parental heptaene candidin (Martin and McDaniel, 1974). Studies have also been carried out on the development of nystatinproducing strains. Nonproducer strains of S. noursei were selected from

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nystatin-producing strains by mutation with nitrosomethylurea (Toropova et

al., 1971). Producers and nonproducer strains of S. noursei were similar in morphological, cultural, and biochemical properties. The original soil-isolate of S. noursei produced, in addition to the polyene macrolide nystatin, cycloheximide, actinophenol and compounds of the dioxopiperazine type. After mutagenesis with X-rays, UV, or nitrogen mustard, strains were obtained that did not produce either cycloheximide and actinophenol or nystatin (Spizek et al., 1965a). Strains that do not produce any compounds of the cycloheximide series synthesize about double the amount of nystatin (Dolezilova et al., 1965). Another strain, S. noursei var. polifungini, produces in addition to cycloheximide a complex tetraene macrolide named polifungin consisting of polifungins Al, A,, A3, and B (Kotiuszkoet al., 1972), identical with nystatin. Five UV mutants which lost the ability to synthesize cycloheximide still produced the nystatin components, but differences were observed in the ratio of individual components. Two additional mutants were able to produce biologically inactive tetraene macrolides which lack the aminosugar moiety (mycosamine) (Roszkowski et al., 1972), a structureactivity relationship also found in candihexin complex (Martin and McDaniel, 1975b). As indicated before, production by high-producing polifungin mutants was correlated with increased specific activities of the carboxylation systems: acetyl-CoA carboxylase, methylmalonyl-CoA carboxyl transferase, and PEP carboxylase (Walski and Raczynska-Bojanowska, 1972). B. EFFECTOF ACTINOPHAGESON ANTIBIOTIC PRODUCTION-LYSOGENIC CONVERSION OF ANTIBIOTIC-PRODUCING STRAINS The role of phage infections in antibiotic-producing strains is unclear, but they pose a problem for many industrial fermentations. Lysogenicity in Streptomyces species by temperate phages was first reported by Welsch (1956, 1959). Phages and phage mutants of Streptomyces coelicolw (Hopwood et al., 1973) and other streptomycetes have been studied in detail (Welsch, 1969). Early studies showed that many strains of S. leuoris were lysogenic and polylysogenic (Rautenshtein and Muradov, 1965). Streptomyces levoris strain 2638 is reported to contain at least four different temperate phages designated 2638/1, 2638/II, 2638/III, and 2638/IV. Treatment with acridine orange resulted in variants devoid of three prophages. These variants were still able to produce levorin. When the fourth prophage was removed with increased concentrations of acridine orange, the strain was unable to produce the antibiotic (Rautenshtein and Muradov, 1968). Lysogenization of a strain deprived of all the four prophages with phages

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2638/1, 2638/II, and 2638/III, produced strains without the ability to synthesize antibiotic. Lysogenization with phage 2638/IV resulted in variants capable of producing antibiotic. Thus, it appears that prophage 2638/IV is involved in antibiotic production. It must be taken into consideration that acridine orange, which acts primarily by eliminating (curing) plasmids also acts as a mutagenic agent, especially at high concentrations, and therefore the loss of antibioticproducing ability in some of the strains might be due to mutation of chromosomal genes rather than the curing of extrachromosomal DNA. There are a number of recent reports indicating that extrachromosomal DNA elements (plasmids or defective phages) are possibly involved in the biosynthesis of the antibiotics kasugamycin and aureothricin by Streptomyces kasugaensis (Okanishi et al., 1970), oxytetracycline by S . rimosus (Boronin et al., 1975), and turimycin by Streptomyces hygroscopicus (Kahler and Noack, 1974). In S. coelicolor, Kirby et al. (1975)reported that antibiotic synthesis and resistance are plasmid-determined. Structures resembling incomplete phage particles were found in electron micrographs of antibioticproducing strains (Kurylowicz et al., 1974), and covalently linked circular DNA of small size has been found in S. coelicolor (Schrempf et al., 1975). The levorin complex is a mixture of levorin A and levorin B. Levorin A contains four components, A,,, Al, A,, and A3, which differ in antifungal activity as well as in toxicity (Tsyganov and Yakovleva, 1%9a,b). Infection of a high-producing strain of S. levoris with a specific virulent mutant of a temperate phage produced increased variation in cultural characteristics and antibiotic levels. Variants isolated from secondary growth of S. leuoris after phage infection were phage-resistant because of lysogenization. The ability of phage-resistant variants to produce levorin ranged &om 0 to 280% relative to the original strain. However, they lost phage resistance during subsequent subculture as the result of the loss of prophages. Phage-resistant variants and variants that have lost their phage resistance differ from the parent strain in the relative content of the components of the levorin complex (Rautenshtein et al., 1972a). A similar phenomenon has been described in Streptomyces netropsis. Four cultures of S. netropsis that produce a polyene macrolide antibiotic active against Verticillium duhliae, the causative agent of cotton wilt (Askarova et al., 1972), were also found to be lysogenic. These strains gave rise spontaneously to variants that lost their prophage along with the ability to produce the antibiotic that the parent cultures produced. The variants regained the ability to produce the antibiotic by lysogenization with temperate phages isolated &om the original strains (Muradov and Rautenshtein, 1973). The relationship between lysogenic conversion and ability to produce antibiotic is by no means clear. However, it is interesting to speculate on the

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possibility that some of the genes (more likely regulatory genes) coding for antibiotic synthesis are coded by phages or are turned on by phage-coded genes. Expression of genes coding for antibiotic would occur only after lysogenic conversion. Loss of the prophage could possibly be the reason for the well known strain degeneration phenomenon (loss of the ability to produce antibiotic). Lysis of antibiotic-producing strains is a rather common phenomenon. It is generally due to virulent mutants of temperate phages existing in lysogenic cultures (Rautenshtein, 1967). Virulent mutants of the temperate phages of S. netropsis were usually found on the fourth day of growth when high titers of antibiotic were reached. However, if the cultures were treated on the first day with concentrations of antibiotic ranging from 500 to 4000 units/ml, virulent mutants arose during the first 24 hours (Muradov and Rautenshtein, 1972). Similarly, the antibiotic vancomycin produced by Streptomyces orientalis induced the formation of virulent mutants of its phages at concentrations below 1OOO units/ml (Rautenshtein and Deshchits, 1970). Streptomyces rimosus, the producer of oxytetracycline, was also found to be lysogenic under conditions of industrial production. Lysis, however, was caused not by a virulent mutant of the temperate phage, but by a lytic factor that appears to be induced by the temperate phage in the course of transformation of the prophage into mature particles (Rautenshtein et al., 197213). Thus there appears to be a close relationship between actinophage infection and the production of polyene macrolide antibiotics, which deserves further research.

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JUAN F. MARTIN AND LLOYD E . MCDANIEL

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Use of Antibiotics in Agriculture TOMOMASA MISATO, KEIDO KO,'

AND ISAMU YAMACUCHI

The Znstitute of Physical and Chemical Research, Wako-shi, Saitama, Japan I. Introduction ........ 11. Agricultural Antibiotics an em .......... A. Advantages ........................................ B. Limitations ...................... 111. Utilization of M A. Streptomycin B. Chloramphenicol ................................... C. Cycloheximid D. Griseofulvin E. Novobiocin ......................... . . . . . . . . . . . . . . . IV. Antibiotics Developed as Agricultural Chemicals ............ A. Brief History of Research on Agricultural Antibiotics in Japan ............................................. B. Blasticidin S ....................................... C. Kasugamycin .................... D. Polyoxins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Validamycin ....................................... F. Tetranactin .......................... G . Cellocidin ......................................... H. Ezomycin .............................. I. Other Pro otics .......................... V. Future Prospects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . .

53 54 54 55 55 58 59 59 60

60 60 61 62 66 69 73 76 79 79 80 82 83

1. Introduction

Attempts to control plant diseases by the use of antibiotics have been made by plant pathologists all over the world since the discovery of penicillin. In western countries, however, only a few of these antibiotics have been developed for practical use. These are streptomycin, tetracycline, cycloheximide, and griseofulvin. Streptomycin, the first antibiotic introduced in agriculture, was used in the United States for the control of pear fire blight. This antibiotic and a mixture of streptomycin and tetracycline have been used for the control of bacterial plant diseases, and cycloheximide and griseofulvin have been used for the control of fungal plant diseases. Cycloheximide is a very powerful fungicide, but, unfortunately, highly toxic to plants, which restricts its use against plant diseases. Griseofulvin is a much less phytotoxic systemic fungicide, but its use is also restricted, because the relation of its manufacturing cost to its performance under field conditions is not satisfactory. 'Formerly K. T. Huang. 53

54

TOMOMASA MISATO, K E I W KO, AND ISAMU YAMAGUCHI

In Japan, these four antibiotics had been used only on a very limited scale for practical control of plant diseases until the curative effect of blasticidin S on rice blast was discovered by the author’s research group in 1958. The successful application of blasticidin S against rice blast has stimulated the development of agricultural antibiotics and led to the discovery of several excellent antibiotics, such as kasugamycin, polyoxins, and validamycin. Blasticidin S and kasugamycin are now in practical use for rice blast control instead of mercuric fungicides, and polyoxins and validamycin have been used to control the sheath blight of rice plant instead of arsenic fungicides (Misato, 1969a,b, 1975a,b; Huang and Misato, 1970). The amount of antibiotics used in Japan is shown in Table I. Reviews on many antibiotics tested for agricultural use in western countries have already been published (Dekker, 1963, 1969, 1971, 1975; Thirumalachar, 1968; Woodbine, 1962; Zaumeyer, 1956). It is the purpose of this paper to discuss the present status of antibiotics as plant disease control agents. The discussion will mainly be limited to antibiotics that show promise for practical application or are already so used, especially in Japan. For the older literature, the reader may refer the reviews mentioned above.

II. Agricultural Antibiotics and the Pollution Problem One of the greatest needs in the world is the production of food for billions of people. At present, such production requires the use of pesticides, but, in turn, their use brings the possibility of environmental pollution. Environmental hazards caused by conventional agricultural chemicals are classified into two categories: (1) a nonselective toxicity (parathion); (2) concentration and accumulation of toxic compounds in the environment (DDT and BHC). Pollution-free pesticides, therefore, should have selective toxicity to target organisms and be susceptible to photolysis and degradation by soil microorganisms. In these respects, antibiotics are supposed to be useful biodegradable pesticides. As is true for every scientific technique, the use of agricultural antibiotics too has advantages and limitations. A. ADVANTAGES 1. Since agricultural antibiotics are sprayed at very low concentrations (10-50 ppm), the amount of compounds sprayed in a unit area is fir less (UlO-l/lOO) than of other conventional pesticide chemicals. Also antibiotics should be rapidly degraded by soil microorganisms. Therefore it is expected that the use of agricultural antibiotics will not bring environmental pollution.

USE OF ANTIBIOTICS IN AGRICULTURE

55

2. Antibiotics are produced by utilizing agricultural products obtained from biological photosynthetic conversion of solar energy. The production of antibiotics does not consume much stored energy, such as oil and coal. 3. Novel bioactive compounds with very complex chemical structures that are outside the domain of organic synthesis can be isolated and manufactured on a commercial basis. 4. Various antibiotics can be produced by using a single set of equipment and facilities. This advantage brings a low initial cost of antibiotics. B . LIMITATIONS 1. Antibiotics are generally mixtures of various structurally related components, like polyoxins. This complexity makes difficult the analysis on a microscale and the safety evaluation of compounds. 2. Tolerance or resistance of pathogenic microorganisms to antibiotics has occurred shortly after application of antibiotics for the control of plant diseases. In order to reduce or avoid the emergence of tolerant fungi and bacteria in the field, the alternate or combined application of chemicals with different mechanisms of action is recommended.

111. Utilization of Medical Antibiotics as Agricultural Chemicals The successful use of antibiotics against bacterial diseases of human beings has led to large-scale screening of antibiotics effective for plant disease control in the world. Many antibiotics developed for medical purposes were investigated for activity against plant pathogens. Furthermore, screening of soil organisms for production of antibiotic substances was started with the prime purpose of plant disease control. However, the results obtained with antibiotics and antibiotic-containing culture broth did not fulfill the high expectations. Many were too unstable under field conditions or showed toxic side effects on plants. Most antibiotics were rather expensive, even when used as a crude product. The number of antibiotics that were promising for practical use appeared to be rather restricted (Dekker, 1963). The medical antibiotics streptomycin, oxytetracycline (terramycin), and chloramphenicol are now used in various countries against several bacterial plant diseases. The antibiotics cycloheximide and griseofulvin, developed for agricultural purposes, are also used against several fungal plant diseases. Numerous other antibiotics, among them a number of polyenes, macrolides, polypeptides, purine- and pyrimidine derivatives, have not reached the stage of practical application against plant diseases.

TABLE I AGRICULTURAL ANTIBIOTICSUUSED I N JAPAN

Registration

Antibiotics

Diseases

Amounts used in Japan (1974) Weight cost (ton) (103 yen)

Antifungal antibiotics

1959 1959 1961

1965

1967

Cycloheximide Wettable powder Griseofulvin Paste Blasticidin S Dust Wettable powder Soh tion Kasugamycin Dust Wettable powder Solution Polyoxins Dust Wettable powder Solution

Onion downy mildew, shoot blight of Japanese larch Fusarium wilt of melon

"8 17

35,020

2

4,700

1250 3 152

75,000 2,547 102,426

7930 265 10

507,762 221,805 8,820

387 418 34

32,121 960,982 38,216

Rice blast

Rice blast

Rice sheath blight, fungal diseases of fruits and vegetables

t

z

1970 1972

1957 1964 1964

1968

1974

Ezomycin Wettable powder Validamycin Dust Wettable powder Antibacterial antibiotics Streptomycin Wettable powder Cellocidin Wettable powder Chloramphenicol basic copper Wettable powder Novobiocin Solution

+

Insecticidal antibiotics Tetranactin

Stem rot of kidney bean 0

0

3893 94

513,876 143,256

349

692,086

0

0

10

33,130

0

0

-

-

Rice sheath blight

Bacterial diseases of fruits and vegetables Rice bacterial leaf blight Rice bacterial leaf blight

Bacterial canker of tomatoes

Insects Carmine mite of fruits and tea

58

TOMOMASA MISATO, KEIDO KO, A N D ISAMU YAMAGUCHI

In the following sections, the antibiotics streptomycin, oxytetracycline, chloramphenicol, cycloheximide, griseofulvin, and novobiocin will be discussed. A vast number of antifungal antibiotics developed for medical purposes, which show no promise for practical application against plant diseases, do not fall within the scope of this review.

A. STREPTOMYCIN Streptomycin was the first antibiotic applied against plant diseases. It has been used to control fire blight of apple and pear by foliar application at the concentration of 200 ppm in the United States since 1954. By this work, economic control of plant diseases by the use of antibiotics has been recognized. In Japan it was found that foliar application of 200 ppm of streptomycin was effective to control wild fire of tobacco (Hidaka and Murano, 1955, 1956, 1959), and since then this antibiotic has been in practical use. A mixture of streptomycin and oxytetracycline was also applied against bacterial canker of peach, citrus canker, soft rot of vegetables, and various other bacterial diseases. It is interesting that streptomycin shows an inhibitory effect on plant fungal diseases. Sakurai et aE. (1958) observed its curative effect against downy mildew of rice by dipping rice seedlings infected with Phytophthoru mcrospora in 100-400 pprn of streptomycin solution for 3 hours. In experiments on the translocation of streptomycin in host plants, Mukoo et al. (1954) dipped potato seedlings in 100 ppm of streptomycin solution and found that the absorbed compound reached to the top of the seedlings after 3 hours. Using inhibition zone technique, Hidaka and Murano (1956) noted that streptomycin which was absorbed from tobacco stems translocated to upper leaves and then reached lower leaves. When the antibiotic was absorbed from leaves, it did not translocate. These findings were confirmed by experiments using 35S-labeledstreptomycin sulfate. Streptomycin is effective against bacterial leaf blight of the rice plant at a concentration of 200-500 ppm, but at 500 pprn or more it is toxic to rice leaves not only in expressing chlorotic spots, but also in reducing rice yield, especially when sprayed at near-heading stage. Arada et al. (1961) noted the almost complete losses of assimilated starch near chlorotic spot and its reduced metabolic activity. Kamimura and Takahi (1960)pointed out that mixtures of streptomycin sulfate and iron chloride or citrate were effective to reduce the phytotoxicity of the antibiotic. Tabei and Mukoo (1955) found the occurrence of streptomycin-resistant plant pathogenic bacteria of potato ring rot and soft rot, and they devised a culture medium for the isolation and quantitative estimation of a

USE OF ANTIBIOTICS IN AGRICULTURE

59

streptomycin-resistant strain of rice leaf blight pathogen, Xanthomonas oryzae, from infected plants. Wakimoto and Mukoo (1963) isolated sixteen streptomycin-resistantX . oryzae strains that could grow in medium containing 100 pprn of streptomycin. These streptomycin-resistant strains showed cross-resistance to chloramphenicol and cellocidin. B. CHLORAMPHENICOL By spraying it at 100-200 ppm concentration, chloramphenicol has been used to control bacterial leaf blight of rice in Japan since 1964. The antibiotic is industrially provided by chemical synthesis. The active L base, which is separated from the mixture of L and D forms produced by chemical synthesis, is used for chemotherapy. For purposes of plant protection, the mixture of both forms before separation is used. Recently, the antibiotic was mixed with basic copper chloride and used for controlling some bacterial diseases of fruit plants and vegetables. (ACTIDIONE) C. CYCLOHEXIMIDE

Cycloheximide was found as a by-product of streptomycin in the culture medium of Streptomyces griseus. Naramycin, a stereoisomer of cycloheximide, is produced from Streptomyces naraensis in high amounts without the by-production of streptomycin. Cycloheximide inhibits the growth of various plant pathogenic microorganisms at very low dose levels, but the application is limited because of its high phytotoxicity. Sprays containing 2 ppm of the antibiotic have been used for controlling downy mildew of onions in Japan since 1959, and a 3-ppm solution was found to be effective against shoot blight of Japanese larch (Takaoka, 1961, 1968). This antibiotic is used as a fungicide in forests. When a 500-ppm cycloheximide oil formulation was applied three times to trunks of 4-year-old pines at the height of one-third from the soil level, the antibiotic was absorbed and translocated to the upper part of free branches. By this method, D6ke et al. (1964)succeeded in reducing the damage rate of Japanese larch due to shoot blight by one-third. Akai and co-workers(1962a,b, 1963, 1964) found that cycloheximide-injured leaves contained a reduced amount of chlorophyll and a higher level of RNA. Chromosome aberrations and abnormal cell divisions in root-tip cells of wheat were also observed. Idemizu. (1960) noted that semicarbazone derivatives of cycloheximide showed reduced phytotoxicity while its potential activity against fungi was constant. However, oxime and acetate derivatives showed weak phytotoxicity and antifungal activity.

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TOMOMASA MISATO, KEIDO KO, A N D ISAMU YAMAGUCHI

D. GRISEOFULVIN Utilization of griseofulvin for plant protection was first introduced by Brian et al. (1951). They succeeded in controlling early blight of tomato and Botrytis disease of lettuce. In Japan, Hoshino and Sawamura (1957) and Terui and Kagawa (1959) observed its inhibitory effect against conidial germination of pathogenic fungus of apple blossom blight. By dusting a mixture of apple pollen grains and lycopodium containing 25% griseofulvin dust at the time of artificial pollination, blossom blight of apple plants could be controlled. By painting a diluted paste of 50%wettable powder of griseofulvin on the diseased parts of melons, fusarium wilt of melons could be controlled. This antibiotic has been used in Japan since 1959, but its cost seems to be too high for agricultural use.

E. NOVOBIOCIN Novobiocin, also called cathomycin, is produced by Streptomyces spheroides and other actinomycetes. Wakimoto et al. (1967) succeeded in reducing the occurrence of bacterial canker of tomato by dipping tomato seedlings in 100 ppm of novobiocin aqueous solution overnight before transplanting. It has been used since 1968.

IV. Antibiotics Developed as Agricultural Chemicals The development of agricultural antibiotics has not been limited to controlling plant diseases, but has extended more widely and more actively over various areas including utilization of insecticides, herbicides, and plant regulators in Japan. As shown in Table 11, many compounds of microbiological TABLE I1 PESTICIDAL COMPOUNDS

OF

MICROBIOLOGICALORIGIN

Fungicides

Antifungal antibiotics" Antibacterial antibiotics" Antiviral antibiotics"

Blasticidin S, etc. Streptomycin, etc. Aabomycin, etc.

Insecticides

Miticidal antibiotics" Bacterial toxins

Tetranactin Bacillus thuringensis

Herbicides

Herbicidal antibiotics

Anisomycin

Growth regulators

Fungal products'

Gibberellins

"In practical use as pesticides.

USE OF ANTIBIOTICS IN AGRICULTURE

61

origin are already used as pesticides or show promise for practical application. Blasticidin S, etc., as antifungal antibiotics, streptomycin, etc., as antibacterial antibiotics, tetranactin as a miticide, and gibberellins as plant growth regulators are used. Aabomycin as an antiviral antibiotic, a product of Bacillus thuringensis as a insecticidal antibiotic, and anisomycin derivatives as herbicides have been tested for practical use in the field. In the following sections, after a brief history of studies on agricultural antibiotics research in Japan, the agricultural antibiotics blasticidin S, kasugamycin, polyoxins, validamycin, cellocidin, tetranactin, and ezomycin will be discussed separately. A few other antibiotics will be mentioned only briefly.

A. BRIEF HISTORYOF RESEARCHON AGRICULTURAL ANTIBIOTICS IN JAPAN Practical plant disease control using antagonistic microorganisms was studied by Nishikado and his co-workers (1950).They discovered some isolates of actinomycetes that were antagonistic to pathogenic microorganisms of bacterial wilt of tomato, black spot of pear, scabs of wheat and barley, southern blight of cowpea, and other diseases. They noted the remarkable antagonistic effect of Trichodemna lignorum against tobacco sclerotium blight pathogen, and devised a mass culture system for this antagonistic organism. Using ' this method, they formulated spore dust of this fungus with over 15 x 1OO spores per gram and showed that sclerotium rots of tobacco and of devil's tongue were practically controlled by using the dust diluted 20-30 times with rice bran or dried soil and sprayed near the roots of these crops. Research on control of rice blast by the use of antibiotics was conducted during World War I1 by Yoshii et al. They found that cephalothecin, an antibiotic produced by Cephalothecium sp., increased the resistance of rice plants to rice blast fungus, Pyricularia w yz ae (Yoshii, 1949, 1950a,b, 1953, 1954). In 1956 Suzuki discovered antiblastin, an effective antibiotic against rice blast under laboratory conditions, and tested its large-scale practical application at agricultural experiment stations in Japan. The experiments failed because of instability of the agent (Suzuki et al., 1956). Nationwide application tests of this antibiotic stimulated interest in the development of new agricultural antibiotics, and extensive research along this line has been started since that time. For example, application tests of many antibiotics, such as antipiriculin (Nakayama et al., 1956), blastmycin (Watanabe et al., 1957), blasticidin A (Fukunaga et al., 1955), trichomycin (Hosoya et al., 1952), hygroscopin (Nakazawa et al., 1954), eurocidin (Nakazawa, 1955), pentamycin (Umezawa et al., 1958), and aureothricin (Umezawa et al., 1949) have been carried out. Although these antibiotics

62

TOMOMASA MISATO, KElDO KO, A N D ISAMU YAMAGUCHI

were effective under laboratory conditions, most were found to be impractical in field tests because of their instability. Misato et al. (1959)found a curative effect of blasticidin S (Takeuchiet aZ., 1958) against rice blast in a foliar spray at concentrations of 10-20 ppm. This antibiotic was found to be effective in wide-scale application tests in 1959and 1960, and it has been in practical use since 1961. After the success of blasticidin S for practical use, research on agricultural antibiotics has become active. Cellmidin against bacterial leaf blight of rice plant (Okimoto and Misato, 1963a), kasugamycin against rice blast (Umezawa et al., 1965), polyoxins against rice-sheath blight (Suzukiet al., 1965), validamycin against rice-sheath blight (Iwasa et al., 1971a), and tetranactin against the adults of carmine mite (Ando et aZ., 1971a), all were registered for practical use.

B. BLASTICIDINS Blasticidin S is the first successful agricultural antibiotic developed in Japan. It was isolated from the culture filtrates of Streptomyces griseoc h r m g e n e s by Takeuchi et al. (1958), and the potent curative effect of blasticidin S on rice blast was found by Misato et al. (1959). Thereafter benzylaminobenzene sulfonate of blasticidin S was reported to be least phytotoxic to the host plant without reduction of antifungal activity against Pyricularia oryzae, the pathogen of rice blast (Asakawa et al., 1963), and this salt has been industrially produced for agricultural use. Three other members of the blasticidin family, blasticidins A, B, and C, have been isolated &om the same strain (Fukunaga et al., 1955; Kono et al., 1968), but these were found to be rather ineffective for practical use. 1 . Chemistry

The chemical structure of blasticidin S has been studied extensively by Yonehara and his co-workers, and the final structure assigned blasticidin S is 1-(1’-cytosinyl)-4-[ ~ - 3-amino-5’ ’ -( 1”-N-methylguanidino)valerylamino] 1,2,3,4-tetradeoxy+3-~-erythro-hex-2-eneuronic acid, as shown in Fig. 1 (Otake et al., 1966;Yonehara and Otake, 1966).The structural interpretation of blasticidin S-hydrobromidehas been provided by X-ray diffraction analysis (Onuma et al., 1966). The molecule of blasticidin S comprises a novel nucleoside designated cytosinine and a new p-amino acid named blastidic acid (E-N-methyl+arginine). The Occurrence of 2,Sunsaturated amino sugar in nature prompted new studies in an attempt to synthesize nucleosides with unsaturated carbohydrates, and Goody et al. (1970) reported the total chemical synthesis of the unsaturated amino sugar of blasticidin S, methyl 4-amino-2,3,4trideoxy~-erythro-hex-2-enopyranosiduronic acid.

USE OF ANTIBIOTICS IN AGRICULTURE

63

FIG. 1. Structure of blasticidin S.

Seto et al. (1966, 1968a) studied the biosynthesis of blasticidin S by the producing organism using 14C-labeled suspected precursors. The results showed that the pyrimidine ring of the antibiotic came from cytosine directly and the sugar moiety from glucose; arginine served as the precursor for blastidic acid, and the N-methyl group of blastidic acid came &om methionine. A metabolic intermediate on the biosynthetic pathway of blasticidin S was found to be leucylblasticidin S, and its biosynthetic role was confirmed by the use of washed cells of S. griseochromogenes (Seto et al.,

1968b). 2. Biological Activity Blasticidin S has a wide range of biological activities. Besides its significant inhibitory effects on the growth of P. oryzae, it also exhibits other antimicrobial (Takeuchiet al., 1961)and antiviral (Hirai and Shimomura, 1965), as well as antitumor (Tanaka et al., 1961) activities although medical application is impeded by its toxic properties. The LDS0by intravenous injection to mice is 2.82 mglkg. Fortunately, it can be used in the paddy field since toxicity to fish is rather low.

3. Mode of Action Misato and his co-workers have studied the biochemical properties of blasticidin S on P. oryzae. They found the curative effect of blasticidin S on rice blast to be due to a strong inhibitory action of mycelial growth of the pathogen, and reported that the antibiotic markedly inhibited the incorporation of 14C-labeledamino acid into protein in the cell-free system ofP. oryzae (Huang et al., 1964a), while metabolic pathways-including glycolysis, the succinic dehydrogenase system, the electron transport system, and the oxidative phospholylation system-or incorporation of 32Pinto the nucleic acid were not inhibited by blasticidin S (Misato et al., 1959, 1961a,b). Yamaguchi and Tanaka (1966) reported that the addition of blasticidin S to the cell-free protein-synthesizing system of extracts &om Escherichia coli and Bacillus megaterium remarkably inhibited the incorporation of leucine and phenylalanine into polypeptide, and that increased concentration of tRNA in this system reversed the inhibition. They suggested that blasticidin

64

TOMOMASA MISATO, KEIDO KO, A N D ISAMU YAMAGUCHI

S acts in the step of peptide transfer from peptidyl-tRNA on the donor site with the incoming amino acyl-tRNA on the acceptor site without effect on the preceding steps in protein biosynthesis. On the other hand, Coutsogeorgopoulos (1967) observed that the inhibition of polypeptide synthesis by blasticidin S could not be reversed with increasing concentration of phenylalanyl-tRNA, and blasticidin S did not act as a competitive inhibitor, with respect to phenylalanyl-tRNA, in polypeptide synthesis. Since blasticidin S markedly interfered with the puromycin reaction (Yamaguchi and Tanaka, 1966), it is also possible that the action of blasticidin S involves the formation of peptidyl-blasticidin S on the 50 S ribosome in a manner similar to the peptidyl-puromycin reaction, though, unlike the case of puromycin reaction, peptidyl-blasticidin S may remain bound to the ribosomes instead of being released. The mode of action of this antibiotic on the molecular level is not known in detail with any certainty, but a process related to peptidyltransferase activity is inhibited by blasticidin S (Coutsogeorgopoulos, 1969; Yukioka et al., 1975). In studying the mechanism of the acquisition of resistance against blasticidin S, Huang et al. (1964b) found protein synthesis in the cell-free system from a resistant strain of P. oryzae or from its susceptible parent strain to be equally inhibited by the antibiotic. From the results obtained, they supposed that the acquisition of resistance of P. uryzae against blasticidin S might be due to the reduction of mycelial permeability of the antibiotic into the cell.

4 . Use against Plant Diseases Rice blast, the most harmful disease of the rice plant in Japan, had been mainly controlled since 1953 by organic mercury compounds sprayed on the plants. Since then, various cases of mercury poisoning in the environment have been reported in the world. Although it was shown that these were not due to the agricultural use of organic mercury compounds, the use of these chemicals for agricultural purposes was forbidden in 1968 in the interests of public health. On the other hand, research for new chemicals for rice blast control to replace organic mercury compounds started by the authors in 1953 under the guidance of Dr. Sumiki, Honorary Professor of Tokyo University, resulted in the discovery of a new antibiotic, blasticidin S, in 1959. Blasticidin S was evaluated in field trials in 1959 and 1960, and since 1961 it has been in practical use for rice blast control instead of organic mercury compounds. For spraying in the field to protect rice blast, the effective concentration of blasticidin S is usually 10-20 ppm (1-3 gm of blasticidin S/lOa), but it occasionally causes chemical injury on rice leaves when sprayed at higher concentrations. Such phytotoxic effects of blasticidin S on rice leaves are also

USE OF ANTIBIOTICS IN AGRICULTURE

65

influenced by frequency of application, application time, rice variety grown, atmospheric conditions such as temperature and moisture, soil type, and fertilizer used. Among crops, the tobacco plant is the most susceptible to blasticidin S, followed by eggplant, tomato, and potato; mulberry plants are rather susceptible; grape, pear, and peach plants are resistant, and water melon, cucumber, and rice plants more resistant to chemical injury by the antibiotic. Hashimoto et al. (1963) found that free amino acids, especially tryptophan, and RNA content increased markedly in injured leaves of rice plant. When blasticidin S is added to sweet potato, phenylalanine ammonialyase activity is severely inhibited, and polyphenol synthesis also stopped (Minamikawa and Uritani, 1965).

5 . Hazard to Mammals Application by dusting occasionally causes conjunctivities if the dust accidentally comes in contact with the eyes, although no accident has been reported as a result of spraying wettable powder or solution. Blasticidin S also induces severe inflammation of mucous membrane or of injured skin exposed to the antibiotic (Ohta, 1963), and this toxic effect on mammals is a most unfavorable characteristic of blasticidin S. Many attempts have been made to remove this defect of blasticidin S. One of these involves chemical modification of the antibiotic; Kawana et al. (1972) synthesized aminoacyl derivatives of cytosinine that contain either alanine or phenylalanine in the amino acid portion instead of blastidic acid. Other replacements of the amino acid portion of blasticidin S were tried, but more favorable results as expected have not yet been obtained (Otake et al., unpublished observations). Biological transformation of blasticidin S was studied by Seto et al. (1966), and they reported that a strain of Aspergillus fumigatus transformed the antibiotic into four substances; the main compound was shown to be deaminohydroxyblasticidin S, which is significantly less toxic to mammals though the antimicrobial activity is also fairly reduced. Recently, Yamaguchi et al. (1975) isolated the enzyme catalyzing the deamination of cytosine nucleus of blasticidin S, from Aspergillus terreus. The enzyme was shown to have properties similar to those of cytidine deaminase in some respects, but was well separated from the coexisting cytidine deaminase by electrophoresis. In addition, they found that the enzyme acts on some derivatives of blasticidin S, but not on cytosine, cytidine, and other well known nucleosides. Yonehara et al. (1968)discovered a new group of compounds designated detoxin; these were produced by Streptomyces caespitosus var. detoxicus and by several strains of Streptomyces mobaraensis. It exhibited unique biological activities as a selective antagonist: it negated the inhibitory action

66

TOMOMASA MISATO, KEIDO KO, A N D ISAMU YAMAGUCHI

of blasticidin S selectively against Baci22us cereus, but did not do so against P. oryzae. Moreover, it has been observed that phytotoxicity to the rice plant was depressed markedly without a decrease in curative effect of the antibiotic, and the combined preparation of detoxin complex and blasticidin S produced less eye irritation. Separation of components of the detoxin complex and their characterization were performed by 6take et al. (1973), and the chemical structures of main components (Detoxin D group) were elucidated (Kakinuma et al., 1972; 6take et al., 1974). While troublesome defects of blasticidin S, such as toxic effects on both plants and mammals, were reduced by the addition of detoxins, some dif€iculties have been remained, so that they are not yet in use. On the other hand, Sugimoto (1972) found a simpler method to alleviate eye irritation caused by blasticidin S; the addition of calcium acetate to blasticidin S dust (5%addition) specifically reduced the eye trouble without influencing the antiblast effect, but other mammalian toxicity or phytotoxicity of the antibiotic is also not affected. This improved dust is now in agricultural use.

6. Environmental Metabolism Takagi et al. (1970) reported the residual amount of blasticidin S in the unpolished rice cropped in the field to be estimated as less 0.05 ppm by biological assay. The behavior and fate of blasticidin S in the environment were investigated using radioactive compounds prepared biosynthetically from [14C]cytosine and [14C]-~-methionine (Yamaguchi et al., 1972). The sprayed antibiotic was located on the surface of the rice plant, and little was diffused or transported into the tissue. From the wound or infected part, however, the compound was incorporated, and it was translocated mainly to upper part. The compound located at the plant surface was decomposed by sunlight and gave rise to cytosine as the main degraded product. A considerable quantity of the blasticidin S sprayed fell to the ground and was tightly adsorbed onto the soil surface. Furthermore, significant generation of [14C]-carbondioxide from the ['4C]-blasticidin S-treated soil was observed, and several microbes usually inhabiting the paddy field were found to lower the biological activity of blasticidin S. From the results obtained, Yamaguchi et al. supposed that, after application to the crop at a very low concentration, the antibiotic might be rapidly broken down in the environment, so that there would be no danger of environmental pollution and food contamination. C. KASUGAMYCIN Kasugamycin is a water-soluble and basic antibiotic produced by Streptomyces kasugaensis (Umezawa et al., 1965). Developed after blasticidin S,

USE OF ANTIBIOTICS IN AGRICULTURE

67

kasugamycin has been used as an agricultural antibiotic for rice blast control in Japan since 1965. This antibiotic controls rice blast disease at a concentration as low as about 20 ppm (Ishiyama et al., 1965). It can be safely used on crops without any toxicity and has very low toxicity to mammals. These advantages are the main reason why blasticidin S is losing ground to kasugamycin. However, the virulence of a kasugamycin-resistant strain in the paddy field recently, has raised a serious problem concerning rice blast control by kasugamycin. 1 . Chemistry

The chemical structure of kasugamycin was studied by Suhara et al. (1966, 1968)using chemical methods and by Ikekawa et al. (1966) with X-ray diffraction analysis. As shown in Fig. 2, the molecule of kasugamycin is constituted by three moieties: D-inositol, kasugamine (2,3,4,6-tetradeoxy-2,4diaminohexopyranose), and an iminoacetic acid side chain. In regard to biosynthesis of kasugamycin, the incorporation of several saccharides and amino acids into the kasugamycin fraction were tried by Fukagawa et al. (1968a). They suggested that the incorporation of ["C]glucose (U) and ['4C]-mannose (U) into kasugamycin was 5-fold that of [14C]-maltose. These saccharides were mainly incorporated into the kasugamine moiety. ['4C]-Myoinositol (U), [14C]-glycine(l), and [14C]-glycine(2) were incorporated into kasugamycin mostly in the part other than kasugamine. From the dilution test of D-inositol, D-inOSitOl was proved by Fukagawa et d . (1968b) not to be incorporated into the D-inositol moiety of kasugamycin. Radio-gas chromatography of [''C]-kasugamycin showed the conversion of ['4C]-myoinositol (U) to the ['4C]-D-inositol moiety of kasugamycin. Furthermore, Fukagawa et al. (1968~) suggested that mass spectroscopic analysis

OH

FIG. 2. Structure of kasugamycin.

68

TOMOMASA MISATO, KEIDO KO, A N D ISAMU YAMAGUCHI

showed that the nitrogen atom of glycine is incorporated into the imino nitrogen of the carboxyformidoyl group. Nakajima and his associates studied the synthesis of kasugamycin and succeeded in synthesizing kasuganobiosamine and related compounds (Nakajimaet al., 1968; Kitahara et al., 1969);this achieves the total synthesis of kasugamycin by the introduction of the oxalimidyl group into kasuganobiosamine.

2. Biological Activity Kasugamycin selectively inhibits the growth of P. oryzae and some bacteria, including Pseudomonas species, and shows little or no activity against other fungi tested. At a concentration of 100 ppm it did not inhibit the growth of species of Fusarium, Glomerella, Gloeosporium, Celletotrichum, Helminthospwium, Ophiobolus, Gibberella, Pelliculuria, Penicillium, and Botrytis. Kasugamycin inhibits the growth of P. wyzae in acidic media (pH 5.0), but hardly inhibits it in neutral media (pH 7.0) (Hamada et al., 1965). On the contrary, kasugamycin showed stronger inhibition against Pseudomonas at pH 7.0 than at pH 5.0 or 6.0. The antibiotic did not show acute or chronic toxicity to mice, rats, rabbits, dogs, monkeys, and human beings. The oral LDS0for mice was 2 gmkg. At a concentration of lo00 ppm there was no toxicity to fish. 3. Mode of Action Kasugamycin enters into the plant tissue and shows both protective and curative action. It does not inhibit spore germination even at a concentration of 120 pglml. Its effect against P. uryzae is expressed in the plant and in uitro only at low pH (Ishiyama et al., 1965). Tanaka et al. (1966)reported that kasugamycin inhibited protein synthesis in a cell-bee system of P . oryzae. Kasugamycin inhibits protein synthesis in Escherichia coli by interfering with the binding of aminoacyl-tRNA to the mRNA-30 S ribosomal subunit complex. The compound does not cause miscoding. 4 . Use against Plant Diseases

Kasugamycin is in large-scale use against rice blast. It controls rice blast when sprayed at about 20 ppm in aqueous solution. For practical disease control, kasugamycin is mainly applied as a dust containing 0.3% of active ingredient. No injury was observed to many other plants. After kasugamycin-resistant strains had been detected in the field, the combined formulations of kasugamycin and chemicals with different action mechanisms came into practical use. Coating the seed with 2% wettable powder protects against rice blast in the field beds for a month; therefore the antibiotic may be used also as a seed disinfectant.

USE OF ANTIBIOTICS IN AGRICULTURE

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5 . Development of Resistance Ohmori (1967) obtained kasugamycin-resistant strains of P . wyzae from colonies growing on a culture medium containing 100 ppm of kasugamycin. These resistant strains were not different &om parent susceptible strains in sporulation and hyphal elongation ability, but infectivity of the resistant strains was remarkably reduced. Rice blast caused by the resistant strains was not affected by kasugamycin at a concentration of 30 ppm. Uesugi et al. (1971) reported kasugamycin-resistant strains of P . oryzae obtained by selecting from colonies growing on a culture medium containing 500 ppm of the compound. Rice blast infected by the resistant strains was not affected by the antibiotic even at a concentration of 200 ppm. Huang et al. (1969) studied the mechanism of resistance to kasugamycin using resistant strains of P . wyzae obtained by Uesugi et al. and found that ribosomes from resistant cells were not susceptible to kasugamycin. Resistant strains were not detected in the field for several years after kasugamycin came into practical use in 1965. Following the increase of kasugamycin application in the paddy field, the effectiveness of kasugamycin on rice blast gradually decreased. In 1972, problems arising from virulence of kasugamycin-resistant strains occurred at several places in Japan where kasugamycin was mainly used for rice blast control, However, the population of the kasugamycin-resistant strains has gradually decreased since the successive application of kasugamycin was stopped.

D. POLYOXINS The polyoxins, a new group of peptidylpyrimidine nucleoside antibiotics, are producted by Streptomyces cacaoi var. asoensis (Suzuki et al., 1965; Isono et al., 1965). Polyoxins are composed of thirteen components (A-M) of some closely related “peptidic nucleosides,” so designated by Isono and Suzuki (1968b). They can be safely used with no toxicity to man, livestock, fish, and plants. Such excellent characteristics may be due to the fact that polyoxins selectively inhibit the synthesis of cell-wall chitin of sensitive fungi, as was reported by Misato and his co-workers (Sasaki et a l . , 1968a; Endo and Misato, 1969; Ohta et aZ., 1970; Hori et al., 1974a). Polyoxins have been widely used for protection against some pathogenic fungi, such as Alternaria kikuchiana, Pellicularia sasakii, and Cochliobolus miyabeanus in Japan since 1967. 1 . Chemistry

The isolation and chemical elucidation of polyoxins were mainly attributed to the excellent work by Suzuki and Isono et al. (Isono et al., 1965, 1967, 1968, 1970; for review, see Suhadolnik, 1970).

70

TOMOMASA MISATO, K E I W KO, A N D ISAMU YAMAGUCHI

Structure of all polyoxins were given by Isono et al. (1969) as depicted in Fig. 3. Among polyoxins, the C component is the smallest; though it lacks antifungal activity, it was a key compound in elucidating the structure of polyoxins, since hydrolytic degradation of all the polyoxins afforded polyoxin C or its analogs. Isono and Suzuki (1968a) assigned the structure 1-p(5’-amino-5’deoxy-~-allofuranutonosyl)-5-hydroxymeth to polyoxin C on the basis of chemical and physical techniques, and a single-crystal X-ray diffraction analysis of N-brosylpolyoxin C confirmed the structure (Asahi et al., 1968).The identification of nucleoside with 5-aminohexuronic acid as its sugar group is the first rare example in nature. This prompted the elegant work of total synthesis of polyoxin J by Kuzuhara et al. (1973). Prior to total synthesis of polyoxin J, they prepared deoxypolyoxin C (Ohrui

Polyoxin

R,

R3

CH20H

OH

CH20H HO

OH

COOH

HO

OH

COOH

HO

H

COOH

OH

CH20H HO

I

2:

CH,OCONH, H

CH3

J

CH3

K

H

L

H

HO

OH

M

H

HO

H

OH

OH

OH

R HO I

HO

H

OH

FIG.3. Structure of polyoxins.

72

TOMOMASA MISATO, K E I W KO, A N D ISAMU YAMAGUCHI

iting growth; polyoxins caused a marked abnormal bulbous phenomenon on germ tubes of spore and hyphal tips of the pathogen at low concentration, and this abnormally swelled spore became noninfectious. It was also reported that the incorporation of ['4C]-glucosamine into cell wall chitin of Cochliobolus miyabeanus was markedly inhibited by polyoxin D, without inhibitory effect on respiration and synthesis of macromolecules such as protein or nucleic acids (Sasaki et al., 1968a). Endo and Misato (1969) showed in their kinetic studies of the cell-fiee system of Neurospwa crassa that polyoxin D strongly inhibits the incorporation of N-acetylglucosamine (GlcNAc) into chitin in a competitive manner between UDP-GlcNAc and polyoxin D. Ohta et al. (1970)also observed that UDP-GlcNAc was accumulated in the polyoxin D-treated mycelia of C . miyabeanus. More recently Hori et al. (1974a) reported the relation between polyoxin structure and inhibitory activity on chitin synthetase. According to their kinetic analysis, the carbamoylpolyoxamicacid moiety of polyoxins would help to stabilize the polyoxin-enzyme complex, and the pyrimidine nucleoside moiety of the antibiotics would also fit into the binding site of the protein. Therefore, the excellent characteristics of polyoxins may be due to the fact that the antibiotics inhibit the cell wall synthesis of sensitive fungi but have no influence on other organisms, including mammals, since there exist no cell walls in animal cells. 4 . Use against Plant Diseases In order to control rice sheath blight, many organoarsenic chemicals have been used since 1958. However, the trouble with them was their phytotoxic effect; treatments with organoarsenates after the period of young ear initial resulted in delayed blooming, chlorosis of ear, poor extraction of ear, detriment to ripening of grain, etc. Therefore treatment with organoarsenate was accompanied by strict restrictions during application. Meanwhile in pot tests to prevent infestation of sheath blight on rice, polyoxin complex showed better efficacy than organoarsenate, methanearsonic acid-ferric-ammonium salt complex (Sasaki et al., 1968b). Trials to control the sheath blight in the field confirmed the efficacy of polyoxins and their persistence on rice. Application could be made at any stage of plant growth without producing phytotoxicity. Moreover, it was noted that the more the number of applications or the higher the concentration, the greater the tendency to increased yield. Besides sheath-blight control, polyoxins showed high control efficacy against diseases caused by Alternaria spp. of top fruit orchards, such as black spot of pear and Alternaria leaf spot of apple (Eguchi et al., 1968). Polyoxin complex has been used in practice in duplicate forms: a polyoxin D-rich fraction for the sheath-blight control, and a B-rich fraction for diseases caused by Alternaria spp.

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et al., 1971)and N-hydroxysuccinimide ester of 3,4-di-0-benzyloxycarbonylamino-5-O-carbamoyl-2-deoxy-~-xylonic acid. The addition of the latter to the triethylammonium salt of deoxypolyoxin C gave a mixture of the coupled product and N-hydroxysuccinimide. After the mixture was hydrogenated, it was subjected to chromatography on Avicel to afford crude polyoxin J, which was then treated with carbon. The purified specimen was identical with the authentic one both in chromatographic behavior and biological activity. In the course of the study on chemical modification of polyoxins, Isono et al. (1972)found a new mechanism of 5-carboxyuracilderivatives to react with aqueous sodium bisulfide under mild conditions, resulting in facile decarboxylation to give corresponding 5-decarboxy-5,6-dihydrouracil-6-sulfonate and uracils in good yield. A mechanistic feature of this reaction implied the initial nucleophilic addition of bisuEte across the 5,6-double bond. The biosynthesis of polyoxins would be very attractive since they contain a unique amino acid and 5-substituted pyrimidine ring in their molecules. Isono and Suhadolnik (1973)reported the biosynthesis of the 5-substituted uracil base of the polyoxins from uracil and C-3of serine by a new enzyme system, which differs from thymidylate synthetase. It was also shown that 5-fluorouracil could replace uracil to form the aberrant 5-fluoropolyoxins (Isono et al., 1973). Furthermore, they found a new metabolic role for L-isoleucine as a precursor for 3-ethylidene-~-azetidine-2-carboxylic acid (polyoximic acid). The distribution of 14Cin this unique cyclic amino acid proved that the intact carbon skeleton of L-isoleucine is utilized directly in its biosynthesis (Isono et al., 1975). 2 . Biological Activity Polyoxins inhibit the growth of some fungi but are inactive against bacteria and yeast. All the polyoxins except C and I showed selective antifungal activity against various plant pathogenic fungi (S. Suzuki et al., 1966). Among polyoxins, polyoxin D was most effective for rice sheath-blight pathogen, Pellicularia sasakii, whereas polyoxins B and L were effective for pear black-spot fungus and apple cork-spot fungus at 50-100 ppm. As for its toxicity, oral administration at 15 gmkg and injection at 800 mgkg to mice did not cause any adverse effect, nor was it toxic to fish during 72 hours of exposure at 10 ppm. Moreover, foliar sprays of 200 ppm of polyoxins have produced no phytotoxicity on most crops, and no injury to the rice plant was observed even at 800 ppm application (Isono et al., 1965,1967; Sasaki et al., 1968b). 3. M o d e of Action In studying the mechanism of fungicidal action of polyoxins, Eguchi et al. (1968)observed a specificphysiological action againstAlternuria spp. in inhib-

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5. Development of Resistance Recently, Nishimura et al. (1973) reported the discovery of polyoxinresistant strains of A. kikuchiana in some orchards of Tottori Prefecture, Japan. Hori et al. (197413) suggested that the resistance is caused by a lowered permeability of the antibiotic through the cell membrane into the site of chitin synthesis. Mitani and Inoue (1968) found that inhibition of mycelial growth of P . sasakii by polyoxins was protected by glycyl-L-alanine, glycyl-DL-valine, and DL-danylglycine. Therefore, the peptides may act as antagonists to the incorporation of polyoxins into the cell of the fungus.

E. VALIDAMYCIN Validamycin A (VM-A) is a new antifungal antibiotic recently developed in Japan for the control of rice-sheath blight (Iwasa et al., 1970, 1971a,c). It was isolated from the culture filtrate of Streptomyces hygroscopicus var. limoneus, which also produced five additional components designated validamycins B to F, together with validoxylamine A and B (Iwasa et al., 1971c; Horii et al., 1972). VM-A can be used without injury to plants and with very low toxicity to mammals (Hosokawa et al., 1974). Almost no toxicity was observed for birds, fish, and insects. 1 . Chemistry

The chemical structure of validamycin A was determined by Horii, Kameda, and their co-workers to be N-[(lS)-(1,4,6/5)-3-hydroxymethyl-4,5,6-trihydroxy- 2-cyclohexenyl][0-p-D- glucopyranosy~-( 1+3) -(B)-( 1,2,4/3,5)-2,3,4trihydroxy-5-hydroxymethylcyclohexyl]amine as shown in Fig. 4 (Horiiet al., 1971a,b; Horii and Kameda, 1972; Kamiya et al., 1971). VM-A can be seen to have two kinds of new hydroxymethyl-branched cyclitos in its molecule, Valienamine HOH,C

HO

Validoxylamine

D-Glucose

FIG.4. Structure of validamycins.

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TOMOMASA MISATO, KEIDO KO, A N D ISAMU YAMAGUCHI

and these cyclitol moieties are quite different from known aminocyclitols, such as streptamine, .%deoxystreptamine,actinamine. VM-A is, therefore, a unique aminoglycosideantibiotic and one of the peculiar examples in the field of pseudosugar chemistry. Validamycins A, C, D, E, and F contain validoxylamine A as a common moiety in their molecules (Horii et al., 1971a, 1972), but they differ from one another at least in one of the following characteristics; the configuration of anomeric center of glucoside, the position of glucosidic linkage, and the number of D-glucose molecules. On the other hand, validamycin B contains validoxylamine B in its molecule, which yielded hydroxyvalidamine instead of validamine derived from VM-A, by hydrogenolysis followed by acid hydrolysis (Horii et al., 1971a). Kameda et al. (1975) studied microbial transformation of validamycins, and they observed that a-or p-glucosidic linkage of validamycin was selectively cleaved by microbial hydrolysis; especially the conversion of validamycin C into VM-A by Endomycosis spp. or Candidu spp. has important significance because validamycin C is about lo00 times less active than VM-A against P . sasakii in the “dendroid-test” established by Iwasa et al. (1971b). Semisynthesis of a new vdidamycin, fl-D-gdactosylvdidoxylamine A, using Rhodotmla glutinis was also reported by Kameda et al., though unfortunately it showed less activity than VM-A.

2. Biological Activity Antimicrobial activity of VM-A against about 3000 species of hngi and bacteria was not detected with ordinary methods (Iwasa et al., 1971a,b),nor was disturbance of microflora on rice plant and crop field observed (Matsuura et al., 1971; Wakae et al., 1974). Wakae and Matsuura (1973)found no phytotoxicity for over 150 species of plants sprayed with VM-A even at a concentration of 10oO ppm. Furthermore, acute and subacute toxicities to mammals were markedly low; in oral administration of validamycin A at a dose of 10 gmkg to mice and rats, or in subcutaneous and intravenous administration at the dose of 2 gmkg to mice, all animals examined survived without change for 7 days (Iwasa et al., 1971a). In addition, Iwasa et al. observed no irritating effects on the skin and the cornea in the rabbit, and the LDS0for killifish was found to be greater than 1000 pg/ml. Oral subacute toxicity of validamycin A for 4 months in beagle dogs was investigated by Hosokawa et al. (1974); they observed no significant abnormalities of the morphological and biochemical parameters accompanyingthe daily administration of 200 mgkg of VM-A. 3. Use against Plant Diseases VM-A is a main component of the validamycin complex and is specifically effective against certain plant diseases caused by Rhizoctonia spp., such as

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web blight, bud rot, damping-off, seed decay, root rot, and black scurf of several crops and southern blight of vegetables as well as sheath blight of rice plant (Wakae and Matsuura, 1975). Although the antibiotic showed neither cidal nor static action to Phizoctonia spp., it caused an abnormal branching at the tips of hyphae of the pathogen, followed by cessation of further development (Iwasa et al., 1971a). When it was applied in the early logarithmic phase of lesion expansion on rice plant, sufficient control was achieved by one spraying of 30 ppm of VM-A solution (Wakae and Matsuura, 1973). VM-A has been commercially used upon sheath-blight disease since 1973.

4 . Mode of Action Although validamycin A did not significantly suppress the growth of P . sasaki on a nutritionally rich medium, it caused specifically extensive branching of hyphae and the cessation of colony development on a wateragar, as mentioned above. Therefore, the question arises whether VM-A inhibits the growth of the hngus in quantity or causes morphological changes on the fungus under certain conditions. Nioh and Mizushima (1974) concluded that the antibiotic does not inhibit the fungal growth in mass and no difference in the amount of protein, nucleic acid, and cell wall components occurs, but it alters the morphology of the fungus. Meanwhile, Wakae and Matsuura (1975) observed that VM-A has no need of continual contact with the pathogen in a nutrientless condition for effective control of fungal growth. This point is important in the real state of disease occurrence, since P . sasakii is one of the most typical examples of those that grow rapidly by transporting nutrients from basal part to tip part through long stretches of hyphae; this type of growth provides the tip part rather a condition of poor nutrition. Thus, when VM-A is applied directly to the fungus, there is little concern about residue. As for the mode of action of validamycin A, W a k e and Matsuura (1975) showed that VM-A inhibits the biosynthesis of inositol in P. sasakii, and they supposed that inositol might be indispensable for the normal growth and pathogenic activity of the fungus. Although pathogenicity reduced by VM-A was remarkably recovered by the premixing of inositol in their experiment, further investigation will be required to sort out the specific site and type of action of VM-A.

5. Environmental Metabolism Validamycins have been shown to be susceptible to microbial attack, and their addition to the soil resulted in complete loss of biological activity by soil microbes (Nishi et al., 1973). Their half-life in soil was less than 4 hours. Microbial degradation of VM-A by Pseudomonas denitrijkans gave rise to D-glucose and validoxylamine A, which was further decomposed into valienamine, validamine, and another lower compound (Kameda and Horii,

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1972). The metabolic fate of validamycins in the animal has not yet been reported, but orally administered validamycins are expected to be decomposed by enteric bacteria. According to a personal communication by T. Kanai, VM-A was excreted to feces without being adsorbed into the animal body through the intestinal lining, and in the case of intravenous injection it was rapidly excreted intact in the urine. Furthermore, no cross-resistance with medicinal antibiotics, such as streptomycin and kanamycin, was detected in certain human pathogens. Validamycin A has been used to protect sheath blight of the rice plant in formulations of 3%solution or 0.3% dust. Residues in rice grains and straws were less than each detectable limit by gas chromatography (Kameda and Yamamoto, 1970). Thus, validamycin A is considered to be one of the ideal chemicals with respect to safety and to pollution, though additional toxicological studies, including teratologic, carcinogenic, and chronic toxicities, remain to be done.

F. TETRANACTIN Tetranactin, a new miticidal antibiotic, was isolated as crystalline rhombic prisms from the filter cake of fermented broth of Streptomyces aureus strain S-3466 (Ando et al., 1971a). The antibiotic exerted remarkable pesticidal activity specifically against the adults of carmine mite and showed very weak toxicity to warm-blooded animals. Also it showed no phytotoxicity to apple, mandarin orange, and tea when sprayed at high concentration (Hirano et al., 1973). The 'miticidal property of tetranactin in fields of apple and tea have been evaluated in Japan since 1968, and tetranactin has been used as a miticide for plants since 1974. 1 . Chemistry and Mode of Action

Ando et al. (1971a) isolated the active principle in crystalline form by extracting the mycelial cake of S. aureus with acetone followed by silica-gel column chromatography. They also showed that S. aureus produces, along with tetranactin, two other structurally related macrotetrolide antibiotics, i.e., dinactin and trinactin, in minor production (Ando et al., 1971b). From the studies on the chemical characteristics of tetranactin, it was found that the antibiotic also belongs to the class of macroterolide antibiotics and is a cyclic polyester composed of four units of homononactic acid, as shown in Fig. 5 (Ando et al., 1971b). The stereochemical structure was clarified with the use of X-ray crystallography by Iitaka et al. (1972). As depicted in Fig. 5, the molecular shape of free tetranactin is fairly flat, elongated, and twisted, and the outline of the molecule resembles that of a propeller, whereas the molecular conformation

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FIG.5. Structure and molecular conformations of tetranactin. Middle left figure is the projection of Rb+-tetranactin complex and upper right one is that of uncomplexed tetranactin. Lower figures are the projections of the corresponding32-membered rings showing the outlines of the molecules. (Reproduced with permission from Iitaka et al.. 1972.)

of the rubidium-complexed one is rather globular and like a ball. The surbce of the ball is covered with lipophilic moieties, such as ethyls, methyls, and methylenes. Therefore, the complex readily dissolves in most organic solvents, such as chloroform, dichloromethane, benzene, and ethyl acetate; this is important because, being enveloped with tetranactin, the cation in the living cells may become lipid soluble, as has been reported in other macrotetrolide antibiotics (Pressman, 1970; Morf and Simon, 1971; Duax et d., 1972). In fact, Ando et al. (1975)observed that tetranactin is an uncoupler in cockroach mitochondria and supposed that the antibiotic caused the leakage of alkali cation, such as K+, through the lipid layer of the biomembrane in mitochondria, followed by uncoupling. Meanwhile, based on this complexforming property, Suzuki et aZ. (1971) presented a method for quantitative determination of tetranactin, which determined the antibiotic with accuracy and simplicity in the assay range of 1 4 0 pgIml.

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2. Biological Properties

Specificity in biological activity is a unique property of tetranactin; it exerted potent pesticidal activity against the adults of a carmine spider mite alone, the LDS0for which is 4.8 pg/ml with the spray method (Sagawaet al., 1972). Azukibean weevil and larva of mosquito were moderately sensitive to the antibiotic, whereas other pests, such as the house fly and cockroach, were insensitive. In addition, it was observed that the ovicidal activity of the antibiotic against sensitive mites is not so significant, this appears to be one of the weak points of tetranactin. The miticidal activity, however, was confirmed in field trials. Tetranactin suspensions were sprayed on apple trees on the leaves of which Kanzawa spider and European red mite were naturally parasitic; proliferation of both mites was completely retarded during 32 days of the experiment. The results obtained indicated the superior activity over a positive control agent, Dicofol. When the powder of tetranactin adheres to the mites, they are never killed because penetration of the antibiotic seems to be negligible in this condition. Therefore, the mites were safe on the dry film of tetranactin on the leaves of a host plant. However, when water was sprayed on the mites on leaves covered with the antibiotic thin film, significant miticidal activity was observed (Hirano et al., 1973). It is suggested that the antibiotic exerts its miticidal action only when an aqueous suspension makes contact with the mites directly; that is, water is an essential factor for the generation of miticidal activity of tetranactin. Tetranactin is not phytotoxic to the young leaves of apples, mandarin orange, and tea when sprayed at lo00 ppm, as mentioned earlier. No systemic miticidal activity was observed in the system in which the mites were transferred on the host plants cultivated in an aqueous medium containing tetranactin. Also it is relatively stable to pH, heat, sunlight, and weathering, since no loss of activity was observed at pH 2-13 for 5 hours at room temperature, at 60°C for 15 days, on exposure to sunlight for several days, and on weathering for 10 days (Hirano et al., 1973). Another characteristic of tetranactin is its safety. Ando et al. (1971a) reported that mice tolerated intraperitoneal administration of 300 mgkg and oral administration of 15 gmkg. They also observed that acute toxicity of the antibiotic is very low; the oral LDms are more than 2 gmkg to rats, guinea pigs, quail, and rabbits (Ando et al., 1975). They suggested that the low toxicity is partly attributable to poor absorption by animals. When [“CItetranactin prepared by biosynthesis was administered orally to mice, it was revealed that the antibiotic was little absorbed, so that the distribution in various organs was negligible and almost all radioactivity was recovered in feces 72 hours after administration (Ando et al., 1975).

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Although several miticidal antibiotics have been reported, most of them are highly toxic to warm-blooded animals, so their usefulness is strictly limited. Thus, low toxicity of tetranactin is favorable for agricultural use.

G. CELLOCIDIN Cellocidin is an antibiotic produced by Streptomyces chibaensis (Suzuki et al., 1958; Suzuki and Okuma, 1958). It is an acetylenedicarboxyamide containing only four carbon atoms. As its chemical structure is so simple, it is easy to synthesize chemically. Technical grade cellocidin for commercial formulations is now synthesized from fumaric acid or butynediol. Cellocidin has an excellent preventive effect against rice bacterial leaf blight when sprayed on rice plants at 100-200 ppm (Okimoto and Misato, 1963a). Its toxicity when injected intravenously is high (LD5,, to mice, 11 mgkg), but in oral administration and skin application it is not so highly toxic (LD,, to mice, 89.2-125 mgkg and LD5,, to mice 667 mgkg, respectively). Its toxicity to fish is lower than that of DDT. Cellocidin has been in practical use since 1964. However, its consumption has been remarkably decreased because of its phytotoxicity. The antibacterial action of cellocidin was antagonized by cysteine or glutathione, which indicates interaction with SH-groups. A study of several metabolic systems in Xanthomonas oryzae revealed that cellocidin selectively inhibited NAD-requiring dehydrogenase, especially in the pathway from a-ketoglutaric acid through succinyl-CoA to succinic acid at the minimum growth-inhibitory concentration of 10 ppm (Okimoto and Misato, 1963a,b).

H. EZOMYCIN Ezomycins are antifungal antibiotics produced by a strain of Streptomyces very similar to S . kitazawaensis. Takaoka et al. (1971) isolated a complex of the antibiotics from the culture filtrate of the producing organism and reported that the complex has unique biological activity in suppressing the growth of very limited species of phytopathogenic fungi, such as Sclerotinia and Botrytis spp. Since the complex showed remarkable antimicrobial activity against Sclerotinia scbrotiorum de Bary, which causes stem rot in kidney bean plants (Phaseolus vulgaris L.), isolation and characterization of each component of ezomycins were carried out by Sakata et al. (1974a). According to Sakata et al., ezomycins are new pyrimidine nucleosides, and the presence of L-cystathionine in the ezomycin molecule is responsible for specific antifungal activity. Recently, they elucidated the chemical structure

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yz HOOC-C-CHz-S-CHz-CH2I

H

H I C-COOH

0 -C,

I

,NHz

NB

I

HO

FIG. 6. Structure of ezomycin A,

of all the ezomycins (Sakata et al., 1974b, 1975a,b); Fig. 6 shows the chemical structure of ezomycin A. This antibiotic was registrated in 1970 as an agricultural antibiotic for the control of stem rot of kidney bean, but it has scarcely been on the market since then.

I. OTHER PROMISINGANTIBIOTICS

1 . Milbemycins

The milbemycins, a new group of macrolide antibiotics, are metabolites produced by Streptomyces B-41-146 strain and exhibit insecticidal activity (Mishima et al., 1975). Since milbemycins are at the early stage of product evaluation, their full spectra of pesticidal activity, especially in fields, have not been yet available. Mishima et al., however, observed a remarkable pesticidal activity of the antibiotics against mites, such as the two-spotted spider mite and citrus red mite, and insects, such as a rice leaf beetle and a tent caterpillar, in their laboratory tests, without any phytotoxicity toward many varieties of crops at effective dosage. Milbemycins consist of 13 components ( L Y ~ - ~ , pl-& ,, a3and PI components were converted into the urethan derivatives containing a heavy atom, and each structure was determined by X-ray analysis (Mishima et al., 1975). The structures are in common composed of three basic units: a sixteenmembered lactone, a spiroketal ring system consisting of two six-membered rings, and cyclohexenediol or phenol. On the basis of the established structures of ar3 and p1components, Mishima et al. determined the structure of other constituents by chemical correlation andor comparison of spectral data. 2 . NK-049: An Anisomycin Analog Yamada et al. (1972) found a strain of Streptomyces that produced two plant-regulating substances, which were later identified as anisomycin (Nishimura et al., 1956) and toyokamycin (Sobin and Tanner, 1954). They

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observed that anisomycin exerted strong growth-inhibitory activity on the roots and shoots of all the plants tested (rice, barnyard grass, crabgrass, lucerne, and tomato) at 12.5 and 50 ppm, respectively. These results led to the investigation of effects of compounds having a p-methoxyphenyl group (p-anisolederivatives) on plant growth-regulating activity, and many anisole derivatives were synthesized and their activities were tested (Yamada et al., 1974a,b). This resulted in the finding of interesting plant growth-regulating activities of p-methoxydiphenylmethanes and p-methoxybenzophenones. Especially, remarkable herbicidal activity was confirmed for 3,3’-dimethyl4-methoxybenzophenone (NK-049) in paddy field tests. NK-049 completely induced chlorosis in barnyard grass and provided a satisfactory herbicidal effect at 4 kg/ha application, although weak chlorosis was occasionally observed in rice stem at 6 kg/ha (Ishida et al. , 1975). According to Ishida et al., NK-049 is quite a stable substance, but it is gradually decomposed by sunlight. In paddy fields, it also seems to be susceptible to microbial attack; the concentration of NK-049 in the soil reached a maximum of 2.16 ppm 7 days after application, but decreased to 0.018 ppm after 30 days and to below 0.004 ppm after 60 days. While the metabolic fate of NK-049 in the environment is presently under investigation, thirteen metabolites have so far been identified; the methoxy group was transformed into the hydroxy group and the benzophenone skeleton was decomposed to m-toluic acid and 4-hydroxy-m-toluic acid. In addition, the acute toxicity of NK-049 to mice and rats was found to be more than 4 gmkg independent of routes of administration (Ishida et al., 1975). Therefore, NK-049 is considered to be a promising herbicide with a high level of safety for use in the environment. 3. Antiviral Antibiotics

One of the most serious problems of plant disease control is the virulence of virus diseases. Trials to develop antiviral antibiotics have been enthusiastically conducted by many workers. Consequently, many antibiotics have been revealed to be effective in inhibiting the multiplication of several plant viruses by in uitro test and pot test. They include blasticidin S, laurusin, bihoromycin, miharamycin, citrinin, and aabomycin A. However, there is no antibiotic in practical use for controlling a plant virus disease. a. Blasticidin S . The antiviral activities of blasticidin S on rice stripe virus and tobacco mosaic virus were reported, respectively, by Kitani and Kiso (1963) and Hirai and Shimomura (1965). By Hirai et al. (1968) it was revealed that blasticidin S inhibited TMV-RNA synthesis without affecting host RNA synthesis. However, blasticidin S was phytotoxic to the tobacco plant when a concentration of more than 2 ppm was sprayed on the plant. Afterward, Yonehara et al. (1968)found that a substance, detoxin, which was isolated from culture broth of Streptomyces spp., was able to suppress the

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phytotoxicity with only slight antiviral activity and made the application range wider. b. Laurusin. Laurusin was found to be an antibacterial substance for controlling bacterial leafblight of rice plant. It was isolated from a culture broth of Streptomyces lavendulae, No. 6600 Gc-1, by Aizawa et al. (1965).Huang et al. (1966) showed that laurusin remarkably inhibits TMV multiplication in tobacco tissue and also inhibits the local lesion formation caused by TMV on pinto bean leaves. However, phytotoxicities were occasionally observed on test plants. c. Bihwomycin. Bihoromycin was isolated from a culture broth of Streptomyces filipinensis var. bihwoensis by Misato et ul. (1967). The biological activity of bihoromycin was observed on inhibiting local-lesion formation of pinto bean caused by TMV, and inhibitory activity on TMV multiplication was rather low. This antibiotic was occasionally phytotoxic on tobacco plant. d . Miharamycin A. Miharamycin A was isolated from a culture broth of Streptomyces miharuensis by Noguchi et al. (1968). They claimed that the antibiotic was effective in controllingTMV, CMV, PVX, and RSV. However, there were phytotoxicities observed on some host plants when miharamycin A was sprayed at a concentration of more than 10 ppm. e. Aabomycin A. Aabomycin A was isolated from culture broth of S hygroscopicus var. aabomyceticus by Aizawa et al. (1969). Yamaguchi et al. (1969), with a leaf disc dipping method, showed aabomycin A to have about 80% inhibition on TMV multiplication in tobacco tissues. Aabomycin A is effective not only in inhibiting disease development of TMV, but is effective also in inhibiting that of CMV and AMV, etc., with pot test. f. Citrinin. Citrinin was isolated from a culture broth of Penicillium citro-viride Biourge, the causal fungus of yellows of rice grains, as a toxin. Yasuda et al. (1969) claimed that the local-lesion formation caused by TMV on Nicotiana glutinosu was remarkably inhibited, while the TMV multiplication in tobacco leaf tissues was not affected. Therefore, citrinin may not be put into the category of antiviral substances, V. Future Prospects The future shape of agriculture in the entire world will depend largely on the availability of the proper kinds of pesticides in adequate quantities. The following considerations might be important for the future development of new agricultural antibiotics. 1. Establishment of screening directions. The development of a new antibiotic is a lengthy and expensive process. Some effective antibiotics, however, were discovered shortly after establishing a new screening project. Examples of these are cellocidin against rice bacterial leaf blight and

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polyoxins against rice-sheath blight. It is expected that new antibiotics will be discovered in the search for new applications, for example, as herbicides, insecticides, and antiviral agents. 2. Establishment of novel screening test methods. Introduction of a novel screening test method may carry with it the possibility for development of new effective antibiotics. Kasugamycin was discovered by replacing the assay system using the cup method with direct application of culture broth to young rice plants. 3. The relationship between chemical structure and biological activity. Modifications of existing antibiotics provide new potential chemicals. The best examples of these are synthetic penicillin and modified kanamycin. Structures of more than one thousand antibiotics have been now determined. These compounds should be subjected to more intensive biochemical study as a source of new bioactive compounds, and attention should be focused on finding the relationship between chemical structure and biological activity. 4. Tolerance of pathogens to antibiotics. Kasugamycin, polyoxins, and validamycin show very selective toxicity to pathogenic fungi. They are not toxic to humans, mammals, fish, and plants and are rapidly decomposed by soil microorganisms. In recent years, however, an Occurrence of resistance of plant pathogenic fungi to kasugamycin and polyoxins has been noted in Japan. When pathogenic microorganisms resistant to antibiotics have emerged, the alternate or combined application of chemicals with different action mechanisms should be made. These procedures are effective in controlling the development of resistant pathogents. In this article the present status of agricultural antibiotics has been described. The development of antibiotics in Japan has brought about successful discoveries of blasticidin S, kasugamycin, polyoxins, and validamycin. Recently, studies on agricultural antibiotics have not been limited to controlling plant pathogenic microorganisms, but extended more widely and more actively to various objectives, such as use as antiviral agents, insecticides, herbicides, and plant regulators. It is expected that many potential antibiotics will be developed and applied in agriculture in the near future. REFERENCES

Aizawa, S., Hidaka, T., Otake, N., Yonehara, H . , Isono, K . , Igarashi, N . , and Suzuki, S. (1965). Agric. Biol. Chem. 29, 3 7 5 3 7 6 . Aizawa, S . , Nakamura, Y., Shirato, S . , Taguchi, R . , Yamaguchi, I . , and Misato, T. (1969). J . Antibiot. 22, 457-462. Akai, S., Shishiyama, J . , Watanabe, Y., and Kuwabara, M. (1962a). Ann. Phytopathol. SOC. J p n . 27, 90 (abstr.) (in Japanese).

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Enzymes Involved in P-Lactam Antibiotic Biosynthesis E. J. VANDAMME Laboratory of General and Industrial Microbiology, University of Gent, Gent, Belgium 1. Introduction ............................ 11. Structure of 8-Lactam Antibiotics. . . . . . . . . . 111. Biosynthesis Mechanisms of &Lactam Antibiotics and Their

...................... A. Precursor

89 89 92 92 93

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Cephamycin Biosynthetic Pathways . . . . . . . . . . . . . . . . . . . IV. Terminal-Sta Biosynthes .. A. Involv Biosynthesis ....................................... B. Involvement of Acylases, Esterases, and Transferases in Cephalosporin Biosynthesis . . . . . . . . . . V. Concluding Remarks References ............................................

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97 112 117 119

1. Introduction Compounds known to possess a p-lactam ring are infrequently encountered in nature, although they now appear to be more common than previously supposed. So h r , the following p-lactam ring structures have been found: the alkaloids pachystermine A and B (Kikuchi and Uyeo, 1967), the antimetabolites tabtoxin (wildfire toxin) (Stewart, 1971; Taylor et al., 1972)and (S)-alanyl-3-[cw-(S)-chloro-3-(S)-hydroxy-2-oxo-3-azetidinylmethyl](S)-alanine (Scannell et al., 1975), the antibiotics bleomycin, phleomycin (Takita et al., 1972), nocardicin A and B (Aoki et al., 1975, 1976), and what might now be called the classical P-lactam antibiotics-the penicillins, cephalosporins, and cephamycins (Abraham, 1974a,b; Gauze et al., 1972; Nagarajan et al., 1971; Stapley et al., 1972). This last group of compounds, which will be discussed here, belongs to the peptide antibiotics, the biosynthesis of which is quite different from that of protein synthesis (Abraham, 1974a; Bodanski and Perlman, 1969; Demain, 1974; Katz, 1971; Kurahashi, 1974; Maier and Groger, 1972). II. Structure of P-Lactam Antibiotics The penicillin structure may be conceived as a condensation product of L-cysteine, D-valine, and an acyl side chain. Residues of the two amino acids 89

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are present in a bicyclic dipeptide, 6-aminopenicillanic acid (6-APA), which is the nucleus of all penicillins. From the chemical point of view, this nucleus may be described as a fused lactam and thiazolidine ring system. The structure of the cephalosporins can be considered as an oxidative condensation product of acetate, L-cysteine, D-dine, and D-a-aminoadipic acid. The nucleus, composed of a /3-lactam ring fused with a dihydrothiazine ring, coupled to an acetoxygroup is named 7-aminocephalosporanic acid (7-ACA). The cephamycins are 7-methoxycephalosporin derivatives. /3-Lactam antibiotics are now known to be produced by both eukaryotic and prokaryotic microorganisms, i. e., filamentous hngi and streptomycetes. Fungi, belonging to the genera Penicillium (P. notatum, P . chrysogenum), Aspergillus (A. flavus, A . nidulans, A . ochraceus) and their sexual forms (Elander, 1975; Merrick and Caten, 1975),Trichophyton (T.mentagrophytes, T. gypseum, T . interdigitale), Microsporium, and Epidermophyton ( E . interdigitale, E . floccosum) (Cole, 1966; Elander et al., 1969; Uri et al., 1957), Mulbranchea (Aragozziniet al., 1970), Thermoascus, Gymnoascus, and Polypaecilum (Kitano et al., 1975) produce penicillins. Several hydrophobic penicillins, only differing in their side-chain structure, are produced, together with the hydrophilic isopenicillin N (Cole and Batchelor, 1963; Flynn et al., 1962). The different penicillins, normally encountered in unsupplemented fermentation broths of these fungi, are called the natural penicillins. However, by adding specific side chain precursors to the media, penicillins can be produced in a selective way: they are named the biosynthetic penicillins. In precursor-free media, accumulation of the penicillin nucleus, 6-APA, occurs (see Table I). Fungi, belonging to the genera Cephalosporium (Acremonium), Emericellopsis, Paecilomyces, Diheterospora, Scopuluriopsis, Arachnomyces, Anixiopsis, Spiroidium produce penicillin N, cephalosporin C, deacetyl-, deacetoxy-, N-acetyldeacetoxycephalosporin C, andlor related metabolites (Abrahamet al., 1974a,b; Elander et al., 1961; Fujisawaet al., 1973; Higgens et al., 1974; Kanzaki et al., 1974a; Kitano et al., 1974; Liersch et al., 1974; Newton and Abraham, 1955; Pisano and Vellozzi, 1974; Queener et al., 1974; Traxler et al., 1975). Certain Streptomyces species produce penicillin N (Miller et al., 1962), usually accompanied by a variety of 7-methoxycephalosporin-derived antibiotics (cephamycins) andlor deacetyl-3-0-carbamoylcephalosporin C (Nagarajan et al., 1971; Stapley et al., 1972; Albers-Schonberg et al., 1972), deacetoxycephalosporin C, and deacetylcephalosporin C (Higgens et al., 1974). The only penicillin type produced by these microorganisms is the hydrophilic p e n i d i n N (see Table I).

ENZYMES INVOLVED I N

P-LACTAM ANTIBIOTIC BIOSYNTHESIS

91

TABLE I NATURALAND BIOSYNTHETIC PENICILLINS H

R

Penicillins

O C H , - C O

a. Natural 6-Aminopenicillanic acid (6-APA) Benzyl (G) p-Hydroxybenzyl (X) 2-Pentenyl (F) n-Amy1 (dihydro-F) n-Heptyl (K) L-a-(bAminoadipy1) (iso-N)

HO+CH,--CO

CH3C HZC H 4 H C Hz-CO CH&HZCHzCH&Hz-CO CH3CHzCHzCHzCHzCHzCHz-CO B-HOOCCH(NHz)(CH&-CO L

D-a-(bAminoadipy1) (N)

6-HOOCCH(NHZ)(CHZ)3-C0 D

b. Biosynthetic Phenoxymethyl (V) Butylthiomethyl (BT) Allylthiomethyl (0) 3-Chloro-2-butenylthiomethyl (S)

O C- ,H C-OJ=(

CH3(CHZ),SCH,-CO CHz=CHCHzSCHz-CO CH3-C=CHCHzSCHz---CO c1

In contrast to the wide variety of side-chain structures of the natural penicillins, up to the moment, no natural cephalosporins or cephamycins are found other than those with a D-a-aminoadipyl side chain, except N-acetyldeacetoxycephalosporin C (Traxler et al., 1975). 7-ACA, the cephalosporin nucleus, has not been detected in fermentation broths of these strains. The substituting group at the C-3 position of the cephalosporim can be an acetoxy group (cephalosporin C), a -H-group (deacetoxycephalosporin C), a -OH-group (deacetylcephalosporin C) or a carbamoyl group, depending on the strain involved. The substituting group at the C-3 position of the cephamycins is variable, but seems also to be determined by the Streptomyces species involved (see Table 11).

92

E. J . VANDAMME

TABLE I1 NATURALCEPHUOSPORINS AND CEPHAMYCINS

COOH ~~

~

Cephalosporin or cephamycin'

Producing microorganisms

R3

Rz

Cephalospwium acremonium Cephalospwium, Emen'cellopsis, Paecilomyces, Scopulariopsis, Diheterospora, Spiroidium, Anoriopsis, Arachnomyces Streptomyces lipmanii, S . claouligerus NRRL 3585 Streptomyces claouligerus NRRL 3585

Cephalosporin C Deacetylcephalosporin C, Deacetoxycephalosporin C

-H -H

-0COCHI -OH

-H

-H

Deacetyl-3-0 carbamoylcephalosporin C 7-Methoxycephalosporin C

-H

-0CONHZ

-0CH3

-0COCH.q

S. lipmanii NRRL 3584

-0CH3

-OCO-C=CH

S . gdseus NRRL 3581

Cephamycin A

ACH,

Cephamycin B

-OCH,

-ocO-C=CH I OCH,

Cephamycin C

--0CHS

-0CONHZ

OH

S. griseus NRRL 3581

Streptomyces lactamdurans NRRL 3802,S. chVuZigems NRRL 3585, S. griseus NRRL 3581

"For all compounds, R, = D-~-HOOC-CH(NH~)(CH&-

Ill. Biosynthesis Mechanisms of p-Lactam Antibiotics and Their Enzymes A. PRECURSOR FORMATION

The penicillins, cephalosporins, and cephamycins are now believed to be derived fi-om heterocyclic tripeptides, which are synthesized fiom the build-

ENZYMES INVOLVED IN P-LACTAM ANTIBIOTIC BIOSYNTHESIS

93

ing blocks: L-a-aminoadipic acid, L-cysteine, and L-valine (Abraham, 1974a,b; Arnstein and Morris, 1960a,b; Trown et al., 1963; Whitney et al., 1972). Indeed, the side-chain precursor of (iso)penicillin N, cephalosporin C, and the cephamycins was found to be L-a-aminoadipic acid, and L-cysteine and L-valine are precursor molecules of the nucleus of the antibiotics. Phenylacetylvaline and phenylacetic acid were shown to behave as direct precursors of the phenylacetyl side-chain moiety of penicillin G. The 0-acetyl group of cephalosporin C comes from acetate, and the 7-methoxy group of the cephamycins is derived from methionine (Amstein and Morris, 1960a,b; Abraham, 1974a; Whitney et al., 1972). In eukaryotic /3-lactam producers, a-aminoadipic acid is an intermediate in lysine biosynthesis; in the prokaryotic producers it is derived from lysine (Kirkpatrick et al., 1973). On the basis of structural analogy and labeling experiments, a tripeptide, 6-(a-aminoadipylcysteinylvaline), which has been isolated from p-lactam producing fungi and streptomycetes, has been interpreted as an essential intermediate in the biosynthesis of penicillins, cephalosporins, and cephamycins (Arnstein and Morris, 1960a,b; Fawcett and Abraham, 1975; Loder and Abraham, 1971a,b; Whitney et al., 1972). The tripeptide was shown by Amstein and Morris (1960a,b) to be present in small amounts in the mycelium of Penicillium chrysogenum, and it has been produced by cell-free extracts of Penicillium chrysogenum from its amino acid components in the presence of an energy-generating system (Bauer, 1970). However, these workers did not establish the sequence of incorporation of the amino acids nor their optical configuration. A mixture of related peptides was obtained by Loder and Abraham (1971a,b) from the mycelium of Cephalosporium acremonium C 91. The major component of this mixture was found to be the tripeptide 6(L-cY-aminoadipyl)-L-cysteinyl-D-valine,and two minor components appeared to be tetrapeptides, both containing an additional glycine residue and one containing a residue of P-hydroxyvaline in place of valine. Recently, Fawcett and Abraham (1975) reported that similar peptides are present in the mycelium of a 7-methoxycephalosporin producing Streptomyces cluvuligerus, and Adriaens et al. (1975a,b) found the same LLDconfiguration for the P. chrysogenum tripeptide. B. TRIPEFTIDEFORMATION The enzyme mechanism by which the amino acids are combined and cyclized to form the p-lactam antibiotics is not completely understood. An enzyme, &(a-aminoadipyl)cysteinylvaline synthetase, which catalyses the

94

E. J . VANDAMME

formation of the tripeptide only from 8-(L-a-aminoadipyl)-L-cysteine and L-valine has been isolated from C. acremonium. It is mainly present in the 20,000 g particulate fraction of ultrasonic-treated and homogenized mycelium (Fawcett and Abraham, 1975). Protoplasts and their lysates have been prepared from Cephalosporium acremonium and Penicillium chrysogenum (Abraham, 1974a; Anne et al., 1974; Kohsaka and Demain, 1976; Duncan and Newton, 1970; Fawcett et al., 1973) using combined enzyme systems, such as Cytophaga enzymes or cellulase from Oxoporms plus gut juice from Helix pomutia or enzymes from Streptomyces grarninofaciens ATCC 12705 or Streptomyces venezuehe RA. Aerated protoplast suspensions of Cephalosporium produce penicillin N and cephalosporin C, and Penicillium protoplasts produce penicillin G. Lysed protoplast preparations synthesize labeled tripeptide from 14C-labeled a-aminoadipic acid, L-cysteine, and L-valine in the presence of an energygenerating system (Abraham, 1974a; Fawcett et al., 1973). Huang et al. (1975) obtained evidence for the nonparticipation of a,P-dehydrovaline intermediates in the formation of the tripeptide, although such a tripeptide derivative might be a common intermediate in the biosynthesis of penicillin and cephalosporin antibiotics (Demain, 1966; 1974).

C. RING FORMATION Addition of the labeled isomers of 8-(a-aminoadipyl)cysteinylvaline to Cephalosporium protoplast lysates indicated that only the LLD form is the precursor of penicillin N. In fact, the 50,000 g supernatant fraction of these cell-free extracts contained the responsible enzymes (Abraham, 1974a). These findings are consistent with the hypothesis that the LLD tripeptide is converted to penicillin N with optical inversion of the aminoadipic acid residue, but without the involvement of an intermediate in which the asymmetry of the D-Vdine residue is lost. It also indicates that the D-configuration of the 8-(a-aminoadipyl-)side chain of penicillin N and the cephalosporins must arise at a late stage, probably during ring formation. Abraham (1974a) also obtained further evidence that the tripeptide from Cephalosporium is indeed a precursor of penicillin N and cephalosporin C from the fact that the tripeptide began to be rapidly labeled from [14C]-valine at that moment in the fermentation cycle where rapid antibiotic production started. Using biochemical genetics as a tool toward pathway elucidation, Lemke and Nash (1972)demonstrated that C. acremonium mutants, unable to form cephalosporin C or penicillin N, fell into two classes: one type blocked in the synthesis of the tripeptide and the other blocked in the conversion of the tripeptide into antibiotic. Subsequently, they found that these two classes were complementary; i. e., heterokaryons formed from a peptide-negative

ENZYMES INVOLVED IN

p-LACTAM ANTIBIOTIC BIOSYNTHESIS

95

and a peptide-positive mutant were capable of antibiotic synthesis (Nash et al., 1974). All these data indicate that the tripeptide is indeed an intermediate in cephalosporin C and penicillin biosynthesis. Aspects of the ring-closure mechanism have also been investigated by following the incorporation of labeled cysteine and valine into antibiotics by intact mycelium (Abraham, 1974a; Aberhart and Lin, 1974; Kluender et al., 1973; Neuss et al., 1973). The 13Clabel of 2s (3S)-[4-13C]valinewas incorporated by an aerated mycelial suspension of C. acremonium exclusively into the exocyclic methylene carbon of cephalosporin C and into the a-methyl group of penicillin N (Kluender et al., 1973), and the 13C label of 2RS(3R)[4-13C] valine was found in the C-2 position of cephalosporin C and in the P-methyl group of penicillin V (Neuss et al., 1973). By the use of deuterated valines, it was established that the methyl groups remain intact during formation of the ring system. Incorporation of the carbon atoms of the valine isopropyl group into penicillin and cephalosporin C is stereospecific, and incorporation into the penicillin ring system occurs with retention of configuration at C-3.

D. DIVERGENCE OF PENICILLIN FROM THE CEPHALOSPORIN AND CEPHAMYCIN BIOSYNTHETICPATHWAYS Incorporation of chiral [4-I3C]valinein both cephalosporin C and penicillin

N supports the hypothesis, that a common dehydrovaline derivative of the tripeptide is the precursor of penicillin N and cephalosporin C and that oxidation of the methyl group (that becomes the &methyl of the penicillins) is followed by the formation of the cephalosporin C ring system. In fact, a dehydrovaline intermediate, at a pivotal position, could yield cephalosporins or could cyclize spontaneously to give penicillin N, as compounds of this type have been reported to form penicillin nonenzymically (Wolfe et al., 1969). It remains an open question whether the immediate precursor of the penicillin ring system contains a D-P-hydroxyvalineresidue, and whether the first cyclic product is a derivative of “cyclic cysteinylvaline” rather than one containing the p-lactam ring (Sjoberget al., 1965).As a result of doublelabeled [3H]-~-cysteine and [3H]-DL-valineincorporation experiments with P. chrysogenum Wis. 49-2105, Adriaens et al. (1975a,b) recently found that P-lactam antibiotic formation occurs by ring closure between carbon 3 of cysteine and the nitrogen of valine, and that the tripeptide can be transformed into isopenicillin N. According to them, an a,p-didehydrovaline intermediate formation remains possible, but seems unnecessary. As the same tripeptide acts as an essential intermediate in the biosynthesis of the structurally related penicillins and cephalosporins, and many cephalosporin-producingmicroorganisms can produce penicillin N, it is con-

96

E . J . VANDAMME

ceivable that some stage in the biosynthesis of the two ring systems might be reached by a common pathway and a necessary step then toward cephalosporin biosynthesis should be ring expansion. This idea is consistent with the fact that mutants of Cephalosporium acremonium unable to synthesize cephalosporin C, still can produce penicillin N; they are presumably deficient in the enzyme system involved in the ring expansion step (Lemke and Nash, 1972). However, Dennen et al. (1971) described an arylamidase in C. acremonium, which has specifically P-lactamase activity toward cephalosporin C, while penicillin N was not affected. This is an unlikely but possible reason for naturally occurring strains that produce only penicillin N. On the other hand, it is still possible that branching may occur before formation of the S-containing ring. However, Kohsaka and Demain (1976) demonstrated that cephalosporin production by protoplast lysates of Cephalosporium acremonium was stimulated by penicillin N. They also obtained indications that an oxygenase is involved in the conversion of penicillin to cephalosporin. These findings suggest a real intermediate role for penicillin N in cephalosporin biogenesis. Queener et al. (1974) isolated Cephalosporium acremonium mutants blocked in cephalosporin C synthesis and investigated these strains for accumulation of other p-lactam compounds. One of the blocked mutants (MH63) accumulated mainly deacetoxycephalosporin C, deacetylcephalosporin C, and penicillin N, while the parent strain (M8650-4)produced high levels of cephalosporin C, deacetylcephalosporin C, and penicillin N , but only traces of deacetoxycephalosporin C. These accumulated patterns led to the interpretation that none of the mutations in the isolates appeared to simply affect a single enzyme in the proposed pathway, involving sequentially: penicillin N, deacetoxycephalosporin C, deacetylcephalosporinC, and cephalosporin C. These pleiotropic effects might indicate strong genetic and/or biochemical interactions such as polar mutations in an operon, or defective protein subunits in a large enzyme complex, but can also be explained by a mere block in the last-step enzyme. The co-occurrence of cephamycin C and deacetylcarbamoylcephalosporin C in Streptomyces clavuligerus broths suggests that the methoxy group at C-7 is introduced at a late stage in the biogenesis of the cephamycin antibiotics (Albers-Schonberg et al., 1972). Demain (1966, 1974) proposed a biosynthesis pathway, suggesting that Amstein’s tripeptide would yield isopenicillin N, which is present in Penicillium chrysogenum broth and mycelium. Isopenicillin N would then be converted by an acyltransferase in the presence of precursors to penicillins, or to 6-APA by the action of a penicillin acylase if no precursors are available. He also considered the possibility that the two activities are functions of the same protein molecule. Cephalospwium acremonium would then possess a

ENZYMES INVOLVED IN

P-LACTAM ANTIBIOTIC BIOSYNTHESIS

97

racemase that converts the isopenicillin N to penicillin N, which might be the precursor for cephalosporin (and cephamycin) production (KohsAka and Demain, 1976). Recently Abraham and Loder (1972), Abraham (1974a), and Fawcett et al. (1975)found that both 6-APA and isopenicillin N can be converted to penicillin by Penicillium extracts. These results are consistent with, and a confirmation of, the earlier proposed suggestions of Demain (1966). The proposed pathways involved in penicillin, cephalosporin and cephamycin biosynthesis are represented in Fig. 1. At this moment, the isolation of the LLD-tripeptide molecule from Penicillium, Cephalosporium, and Streptomyces mycelia and its synthesis by their cell-free extracts, have been achieved, and its intermediate role is now well established. How it is further transformed into the corresponding p-lactam antibiotics remains to be elucidated. Enzymes potentially involved in terminal-stage reactions of p-lactam biosynthesis have been studied rather extensively during the last few years. They are discussed in the following sections.

IV. Terminal-Stage Enzyme Reactions in P-Lactam Antibiotic Biosynthesis A.

ACYLASESAND TRANSFERASES PENICILLIN BIOSYNTHESIS

INVOLVEMENT OF

IN

1 . Acylase

Fungal and microbial enzymes that hydrolyze penicillin into 6-aminopenicillanic acid (6-APA)have been described. This type of enzyme activity was originally reported by Sakaguchi and Murao (1950) in mycelial extracts of Penicillium chrysogenum. Subsequently, penicillin acylase was found to be widely distributed among microorganisms and is used on an industrial scale to catalyze removal of the side chain from biosynthetic penicillin to yield 6-APA. This product is the essential intermediate for the industrial synthesis of the semisynthetic penicillins. This enzymic coupling can be effected by the synthetic activity of penicillin acylases (Carrington, 1971; Chain, 1971; Vandamme and Voets, 1974b). The enzyme has been given different names: penicillin amidase, penicillin acylase, penicillin amidohydrolase, penicillin splitting and synthesizing enzyme, penicillin deacylase and penamidase-but it is here referred to as penicillin acylase, (EC 3.5.1.11) (Hamilton-Miller, 1966; Vandamme and Voets, 1974a,b). Three types of penicillin acylase can be recognized according to the penicillin they preferentially hydrolyze: penicillin G acylase, penicillin V

98

E. J . VANDAMME

a

9

c

co

ENZYMES INVOLVED IN

P-LACTAM ANTIBIOTIC BIOSYNTHESIS

99

acylase, and ampicillin acylase, described by Nara et al. (1972) and Okachi and Nara (1973) in Pseudomonas melanogenum and Pseudomonas ovalis. The hydrolytic activity of penicillin acylase is optimal at alkaline pH values (7.5-9.0), while synthetic activity is displayed at an acid pH optimum (5.06.0), although a few exceptions to this general rule have been reported (Holt and Stewart, 1964; Nara et al., 1971a,b; Vandamme et al., 1971a,b). The cellular location of the penicillin acylases is quite diverse, depending on the microorganism concerned. Bacillus megaterium ATCC 14945 and Streptomycetes lavendulae BRL 198 produce extracellular penicillin acylases, but in general bacterial strains produce the enzyme intracellularly. Whether the intracellular enzyme is cell-wall bound, located in the cytoplasmic membrane, or in the cytoplasm, has not yet been elucidated (Cole, 1969a,b; Kaufmann, 1964; Pruess and Johnson, 1965; Vandamme and Voets, 1974a,b,). Most of the penicillin acylases produced by fungi are intracellular (Baumannet al., 1971; Vanderhaegheet ul., 1968),although some extracellular fungal acylases have been described (Uri et al., 1963, 1964). Penicillin acylases in general have a low affinitytoward their substrates, as is indicated by their relatively high K , values (Table 111). Comparison of the substrate specificity of purified penicillin G acylases from Escherichia coli ATCC 9637 (Bondareva et al., 1969a,b; Self et al., 1969),E . coli NCIB 8743A (Vojtisek and Slezak, 1975; Cole, 1964, 1969a,b), E . coli ATCC 11105 (Kutzbach and Rauenbusch, 1974),Kluyvera citrophila KY 3641 (Shimuzi et al., 1975), Bacillus megaterium ATCC 14945 (Chiang and Bennett, 1967), and Neurosporu crassa (Rossi et al., 1973)revealed that the enzymes are relatively nonspecific, hydrolyzing aliphatic and aromatic amides and removing phenylacetyl, or other nonpolar acyl groups, not only from the penicillins, but also from the N-acyl derivatives of a variety of amino acids and of 7-ACA (Bauer et al., 1960; Cole, 1964, 1969a,b,; Huang et al., 1960, 1963; Kaufmann and Bauer, 1960, 1964; Rossi et al., 1973). On the contrary, purified penicillin V acylases from Erwiniu aroideae, Fusarium, or Penicillium all display a high specificity toward the penicillin molecule only (Baumann et al., 1971; Spencer and Maung, 1970; Thadhani et al., 1972; Vandamme and Voets, 197413, 1975; Vanderhaeghe, 1975). Baumann et al. (1971) studied the substrate specificity of the penicillin V acylase of Fusarium semitectum BC 805 and found that the intact mycelium was able to hydrolyze not only penicillins, but also phenoxyacetyl derivatives of several amino acids. However, they clearly demonstrated that two distinct enzymes were involved: a penicillin acylase, specifically acting on penicillins and an amidase, hydrolyzing N-acyl amino acid derivatives. It has been reported that several phenoxyacetylamino acids are hydrolyzed by Penicillium chrysogenum mycelium (Brunner et al., 1966a,b; Brunner and Rohr, 1965), but this enzymic-activityis also different from that responsible for the deacylation of penicillin.

TABLE 111 K, VALUES OF PURIFIEDAND CRUDEPENICILLINACYLASES

K, value Enzyme source Penicillin G acylase Kluyoera citrophila KY 3641 Bacillus megaterium ATCC 14945 (enzyme") Eschmkhia coli E . coli E . coli NCIB 8743A E . coli ATCC 9637 (immobilized enzyme) E . coli ATCC 11105 (enzyme) Penicillin V acylase Eminia aroideue (enzyme) Streptomyces lavendulae BRL 198 Fusarium semitectum F . semitectum (enzyme)

MW

(mM)

63,000

1.4

120,000 -

-

4.5 1.35-1.59 4.00 30

70,000

7.7 0.02

62,000 -

67,000

F . moniliforme AYF 255 Penicillium chrysogeum (enzyme) ~~

'Enzyme: indicates purification to homogeneity.

35 10.3 2.5-2.75 4.75 5.75 16.7

References

Shimuzi et al. (1975) Acevedo and Cooney (1973) Brandl (1965, 1972) Holt and Stewart (1964) Cole (1961a,b), Warburton et al. (1973) Bondareva et al. (1969a,b), Self et al. (1969) Kutzbach and Rauenbusch (1974) Vandamme and Voets (1974b, 1975) Batchelor et al. (1961b) Brandl (1965, 1972) Baumann et ul. (1971), WaldschmidtLeitz and Bretzel (1964) Vandamme et al. (1971a) Spencer and Maung (1970)

ENZYMES INVOLVED IN P-LACTAM ANTIBIOTIC BIOSYNTHESIS

101

The activity of penicillin acylase is extremely poor on penicilloic or penilloic acids, and substitution of the 6-APA moiety by 7-aminocephalosporanic acid (7-ACA) in most cases reduces enzyme activity. The physiological role of penicillin acylases, in the case of organisms that do not produce p-lactam antibiotics is still largely a matter of speculation. This subject has been reviewed recently by Vandamme and Voets (1974a,b). Acylases from P-lactam-producing microorganisms were first described by Rolinson et al. (1960). Cole and Rolinson (1961) demonstrated penicillin acylase activity in the mycelium of Cephalosporium sp. and Emricellopsis minima, after they found 6-APA in the fermentation broth of these molds during penicillin N production. Acylases from Penicillium chrysogenum A 9342 and Cephalsporium CMI 49137 were found to preferentially hydrolyze penicillin V (Claridge et al., 1963). Omission of side-chain precursors from fermentation media of Penicillium chrysogenum W-5120 (Erickson and Dean, 1966) and of Emericellopsis minima I M I 69015, Cephalosporium amemonium and Cephalosporium salmosynnemutum, results in the formation of 6-APA (Cole and Rolinson, 1961). Uri et al. (1963) and Cole (1966) demonstrated the presence of 6-APA in precursor-free growth media of penicillin-producing dermatophytes (Trichophyton and Epidemphyton) and also observed penicillin acylase activity. Erickson and Bennett (1965) demonstrated the presence of acylase activity in Penicillium chrysogenum W-49-408, a mutant strain unable to synthesize either penicillin or 6-APA, and in a high-producing strain P-5009. Both acylases were found to be very similar, thus indicating that penicillin acylase production is not always accompanied by penicillin formation, although all penicillin-producing fungi do display acylase activity (Cole, 1966, 1967; Kitano et al., 1975). The acylase of the nonproducing W 49-408 strain was studied in detail by Vanderhaeghe, et al. (1968). Aliphatic penicillins were the best substrates, and this acylase was not able to hydrolyze either penicillin N or isopenicillin N (Vanderhaeghe, 1975). Also, the penicillin acylase preparation, purified from Penicillium chrysogenum 51-20F3, displayed a high specificity toward the penicillin molecule (Spencer and Maung, 1970). In fact, acylase hydrolysis of penicillin N and isopenicillin N has never been observed (Vanderhaeghe, 1975).Table IV gives a survey of P-lactam antibiotic producing microorganisms and the occurrence of penicillin acylases. The substrate spectra of these acylases are represented in Table V. As only traces of 6-APA and no 7-ACA at all are found in Cephalosporium-type fermentations, the very low (if any) corresponding acylase activity might contribute to the hilure of these microorganisms to produce cephalosporins or penicillins other than those with a D-a-aminoadipic side chain. Nevertheless, Cole and Rolinson (1961) and

TABLE IV #%LACTAM ANTIBIOTIC-PRODUCING MICROORGANISMS AND THE

OCCURRENCE OF PENICILLIN ACYLASES~

~~

~

~~~

Antibiotics Microorganisms Penicillium chrysogenum Q 176, W 48701, W 39133 W 501247, W 5120 Penicillium chrysogenum sp. Penicillium chrysogenum A-9342 Trichophyton mentagrophytes Epid-phyton interdigitale Trichophyton gypseum Trichophyton interdigitale Asperigillusflavus Malbranchea pulchella Penicillium chrysogenum W-49-408 Penicillium chrysogenum P-5009 Penicillium chrysogenum SC-3576 Trichophyton mentagrophytes BRL 569, BRL 579 Epidewphytonfloccosum BRL 623, BRL 722 Aspergillus ochraceus BRL 731 Penicillium chrysogenum BRL 781, BRL 803 Penicillium sp. BRL 735, BRL 736, BRL 737 Penicillium sp. BRL 733 Penicillium BRL 807

Cephalosporins

Penicillins

6-APA

Acylase

+

+

+

iso-N

+ + + + + + + + + + + + + + +

?

+ + + + + ?

+ + ? + -

+ + +

?

+ + + + + + + + + + + + + + + + ?

References Batchelor et al. (1959, 1961a,b) Cole and Batchelor(l963), Flynnet al. (1962) Claridge et al. (1963) Uri et d.(1963, 1964)

Shimi and Imam (1966) Rode et al. (1947, Kitano et al. (1975) Erickson and Dean (1966), Erickson and Bennett (1965), Vanderhaeghe et al. (1968)

Cole (1966)

Thermoascus, Gymnoascus, Polypaecilum Penicillium chrysogenum 51-20F3, 50935, 1951, 47-638, 48-701, 49-2166 Penicillium chrysogenum D6/347/13 Penicillium chrysogenum ATCC 12690 Penicillium chrysogenum SC-6041 Cephalosporium sp. CMI 49137 Emericellopsis minima (Stolk) I M I 69015 Cephalosporium salmosynnematum MDH 3590A (Emericellopsis salmosynnemata) Emmicellopsis taricola var. glabra Paecilomyces persicinus Cephalosporium acremonium M8650-1-3, ATCC 11550 (Acremoniumchrysogenum) Streptomyces sp. Actinomyces cinereorectus

-

-

I

+ ?

+ +

? ?

? ?

f

+ + + -

+ cc

Deacetylcephalosporin C

Cephalosporium 52-54 Cephalosporium, Emericellopsis, Paecilomyces carneus, Dihetorospora, Scopulariopsb, Arachnomyces, Anixiovsis. Sviroidium ,

+ +

Desacetoxycephalosporin C

+ + + +

Kitano et al. (1975) Gatenbeck and Brunsberg (1968),Pruess and Johnson (1967), Spencer (1968) Brunner et al. (1968) Bauer (1970) Fawcett et al. (1975) Cole and Rolinson (1961)

? ?

? ? ?

Pruess and Johnson (1967) Pisano and Vellozzi (1974) Dennenet al. (1971),Lemke and Nash (1972)

N N ?

-

-

? ? ?

Miller et al. (1962) Chugasova et al. (1974) Fujisawa et al. (1973)

N

-

?

Higgens et 01. (1974), Kitano et al. (1974), Queener et al. (1974)

N N N

+ -

.

(Continued)

TABLE IV (Continued) Antibiotics Microorganisms Streptomyces claouligerus NRRL 3585

Streptomyces lactamdurans NRRL 3802, Streptomyces chouligerus NRRL 3585 Streptomyces lipmanii NRRL 3584

Cephalosporins

Penicillins

6-APA

Acylase

References

Deacetyl-3-0carbamoylcephalosporin C, deacetoxycephalosporin C Cephamycin C

N

-

?

Higgensetal. (1974),Nagarajanetal. (1971), Stapley et al. (1972)

?

?

Stapley et al. (1972)

?

Gauze et al. (1972)

7-Methoxycephalosporin C, deacetoxycephalosporin C Cephamycin A, cephamycin B

Streptomyces griseus NRRL 3851, S . chartreusis, S . cinnamonensis, S. fimbriatus, S . halstedii, S. rochei, S. oiridochromogenes Streptmnyces rubiginosoheloolus, S . laoendulae, S . aureofaciens, S . griseus

N N

?

Unidentified p-lactam antibiotics ~~

“Symbols and abbreviations: -, no synthesis,

~

~~~~~

+, synthesis, ?,

unknown, Cc, cephalosporin C, N, penicillin N, iso-N, isopenicillin N

ENZYMES INVOLVED IN

P-LACTAM ANTIBIOTIC BIOSYNTHESIS

105

Cole (1966)demonstrated that Cephalosporium-type acylases hydrolyze different biosynthetic penicillins, but not penicillin N. The synthetic tripeptide, L-a-aminoadipyl-L-cysteinyl-L-valine was also not hydrolyzed by a Cephalosporium preparation (Loder et al., 1969), although Cephalosporium enzymes were able to hydrolyze the dipeptides L- and Da-aminoadipylcysteine (Loder and Abraham, 1971a,b). It is' not known whether Penicillium acylase is able to effect this hydrolysis. In view of the recent finding that Streptomyces species are able to produce p-lactam antibiotics (see Tables I1 and IV), these particular strains deserve further attention with respect to their acylase activity. Many actinomycetes display acylase activity (Batchelor et al., 1961b; Dennen et al., 1971; Haupt and Thrum, 1967; Nara et al., 1971b), but have not been checked for p-lactam antibiotic production (see Table VI). The extracellular penicillin acylase of Streptomyces lauendulae BRL 198 hydrolyzed heptyl, 2-pentenyl, and phenoxymethylpenicillin readily at pH 9.0 (Batchelor et al., 196lb). Streptomyces erythreus and S. netropsis displayed penicillin V acylase at pH 7.5 at 28°C; these strains possessed an aspecific amide synthetase activity (Haupt and Thrum, 1967). Also Nara et al. (1971a,b) found penicillin acylase activity in Streptomyces ambofaciens SPSL-15 and in Nocardia globerula ICY 3901, a genus already described in 1960 by Huanget al. (1960) to be able to produce penicillin acylase. It is not known whether these acylases can hydrolyze penicillin N, cephalosporin, or cephamycin structures or acylate their nucleus; so far these strains have not been reported to produce p-lactam antibiotics. To what extent the penicillin acylases, synthesized by p-lactam producers, are involved in 6-APA or antibiotic biosynthesis is not exactly known. According to Erickson and Bennett (1965) and Vanderhaeghe et al. (1968), a possible origin of 6-APA in fermentation media is the deacylation of the natural aliphatic penicillins such as F, dihydro-F, and K by the action of penicillin acylases. More specifically, the detection of penicillin acylases able to synthesize penicillins &om 6-APA and an appropriate side-chain structure among p-lactam antibiotic-producing fungi has led to the assumption that these enzymes might be involved in the biosynthesis process of the antibiotics (Cole, 1966; Erickson and Dean, 1966; Spencer and Maung, 1970). Indeed, Erickson and Dean (1966) demonstrated that acylase preparations of Penicillium chrysogenum W-49408 and SC-3576 were able to synthesize at pH 6.8 and at 25°C penicillin V or G, starting from 6-APA and phenoxyacetic acid or phenylacetic acid, respectively. Cole (1966) demonstrated this reverse reaction also with a Penicillium chrysogenum BRL 807 strain. These penicillin acylases were endowed, soon after their discovery, with a potential biosynthetic role, this belief arising from the isolation of 6-APA

TABLE V PENICILLINS HYDROLYZED INTO 6-AMINOPENICILLANIC ACID (6-APA) BY ACYLASESFROM Microorganism P-Lactam antibiotic producers

i

Emericellopsis m i n i m

(Stolk) IMI 69015 Penicillium chrysogenum A-9342

ANTIBIOTIC-PRODUCING MOLDS

Penicillin

Hydrolysis"

Penicillin V Penicillin G Penicillin G Penicillin V 3-Carboxy-3-methylbutylpenicillin l-Amino-3-methylbutylpenici~h

+++

3-Carboxypropy lpenicillin Ampicillin

2-Carboxy-3-decen ylpenicillin 2-Carboxy-1-indolyleth ylpenicillin

2-Carbox yprop ylpenicillin Cephalospm'um acremonium CMI 49137 Penicillium chrysogenum P-5009

P-hCTAM

Phenethicillin Penicillin V Penicillin V Penicillin K Penicillin F Penicillin G

+ + +++ +++ +++ ++ ++ + + + +

++ +++ ++ ++ +

References Cole and Rolinson (1961) Claridge et al. (1963)

Claridge et al. (1963) Erickson and Bennett (1965)

Aspergillus flavus Dermatophytes Mutant, no antibiotic production

Penicillium chysogenum W49-408

E

-l

"+++, ++, +: extent of hydrolysis.

Penicillin G Penicillin V Penicillin G n-Undecylpenicillin n-Nonylpenicillin n-Heptylpenicillin (K) Penicillin V n-Pentylpenicillin Penicillin G n-Propylpenicillin CBH&HZOOC-(CHZ)4-CO-APA C,HSCHZOOC-(CHZ)3-CO-APA CzH5OOC-(CHz),-CO-APA L-C,H,CH,OOC-$H-(CH,),-CO-APA NHCOOCH,C,H, L-C,H~CH,OOC-$H-(CH~),-CO-APA N,

++ +++ + +++ +++ +++ +++ ++ + + + + + +

4

Shimi and Iman (1966) Cole (1966),Uriet al. (1963) Vanderhaeghe et al. (1968)

108

E. 1. VANDAMME

TABLE VI ACTINOMYCETES DISPLAYING PENICILLIN ACYLASEACTIVITY Microorganisms

References

Penicillin G acylase activity Mycobacterium phlei Nocardia F D 46973, ATCC 13655 Streptomyces ambofaciens SPSL-15

Claridge et al. (1960) Huang et al. (1960) Nara et al. (1971b)

Penicillin V acylase activity Nocmdia globerula KY 3901 Streptomyces lawendulae BRL 198 S. netropsis 2814, S. erythreus JA 4143 S . ambofaciens SPSL-15 Acthoplanes utahensis Unidentified actinomycetes

Nara et 01. (1971b) Batchelor et al. (196lb) HauptandThrum(1967) Nara et al. (1971b) Dennen et al. (1971) Nara et al. (1971b)

from fermentation broths and by its ability to couple side-chain groups to 6-APA. Although some evidence has suggested that 6-APA and, connected with it, the acylase enzyme might have a role in the biosynthetic process of penicillins, their significance in the scheme has not yet been established, especially since acylases do not hydrolyze isopenicillin N (Abraham, 1974a; Demain, 1974; W O Eand Arnstein, 1960). The possibility exists that free 6-APA is only a shunt product of the overall biosynthetic process, being formed by the hydrolytic action of acylse on some preformed natural penicillins. The role of 6-APA as an initial precursor and its subsequent acylation into penicillins is now in disagreement. However, 6-APA might be a cryptic intermediate; it can be produced as a transitory stage during hydrolysis and reacylation of isopenicillin N, as is suggested by recent experiments with P . chrysogenum extracts (Abraham, 1974a; Fawcett et al., 1975). 2. Transfwases

The transacylases or penicillin acyltransferases are enzymes (usually from penicillin-producing molds) that are capable of transferring an activated side-chain acid, or the side chain from a penicillin, to 6-APA. In this last case, the penicillin can be considered as an activated form of a side-chain molecule (Abraham, 1974a; Brunner et al., 1968; Gatenbeck and Brunsberg, 1968; Spencer and Maung, 1970). In 1967, Pruess and Johnson described a penicillin acyltransferase in extracts of Penicillium chrysogenum. This soluble enzyme catalyzed at pH 8.0 the exchange of acyl groups from penicillin V, G, K, X, and dihydro-F with

ENZYMES INVOLVED IN

P-LACTAM ANTIBIOTIC BIOSYNTHESIS

109

6-APA in the presence of sulfhydryl compounds, However, the acyl group of penicillin N was not exchanged with 6-APA. The enzyme did not appear to act as a penicillin acylase, and the course of acyltransferase production followed that of penicillin synthesis. They also confirmed the findings of Peterson and Wideburg (1960)that this strain catalyzes isotopic exchange of 35S between penicillin G and penicillin V. The penicillin N-producing Emericellopsis terricola var. glabra displayed no acyltransferase activity, but it was present in the nonpenicillin-producing Penicillium chrysogenum W-49-408 strain. The increased level of activity in high penicillin-yielding mutants suggested that the enzyme is involved in penicillin biosynthesis. Spencer (1968)prepared an extract from Penicillium chrysogenum 51-20F3 and demonstrated in a 60-fold purified preparation two activities: one exchanging the acyl group of penicillin G with 6-APA, and the other producing penicillin G starting from 6-APA and phenylacetylcoenzyme A. Brunner et al. (1968) demonstrated in Penicillium chrysogenum D6/347/ 13a side-chaincoenzyme A-(CoA)ligase,activating side-chain groups, and also a direct acylation of 6-APA with CoA derivatives of phenyl- and phenoxyacetic acids (transacylase). The first enzyme catalyzed the synthesis of phenylacetyl-CoA and phenoxyacetyl-CoA according to the reaction BCOOH

+ CoA + ATP

CoA-SCOR

+ AMP + PPi

where R is the phenylacetyl or phenoxyacetyl radical. It was found in Penicillium chrysogenum just prior to and during the stage of rapid penicillin production (Brunner et al., 1968). Phenylacetyl-CoA ligase was obtained as a crude extract from freeze-dried mycelium, after disintegration with glass beads. The resullting supernatants were treated with DEAE-cellulose, and the filtrate was fractionated with (NH4),S04. The enzyme preparation is maximally active in a pH range of 6.0 to 7.0, and it catalyzes the activation of phenylacetic, phenoxyacetic, and acetic acids with similar degrees of activity (Brunner and Rohr, 1975). The “reverse” enzyme, hydrolyzing activated side chains into an acetic acid radical and CoA, according to the reaction Phenylacetyl-CoA + H 2 0 + phenylacetic acid

+ CoA

has been recognized in extracts of a member (W-51-20F3)of the Wisconsin family of high-yielding mutants of Penicillium chrysogenum (Spencer and Maung, 1970). This phenylacetyl-CoA hydrolase was purified 130-fold and was active in the presence of EDTA and glutathione. The enzyme is 5 times as active against phenoxyacetyl-CoA as compared to phenylacetyl-Cod, and this compares to a similar ratio between the acylase activity of this preparation in hydrolyzing penicillins V and G. The purified preparation also hydrolyzes n-nitrophenyl acetate and phenoxyacetylglycine at about the same

110

E . J . VANDAMME

rate as that of phenylacetyl-CoA. The hydrolase activity shows a pH optimum of 7.6-7.8 and a K , of 3.95 mM. The molecular weight by Sephadex gel filtration is 25,000. The enzyme is sensitive to sulfhydryl inhibition and to oxidation (Spencer, 1975). These findings indicate that this ligase is involved in the formation of natural and biosynthetic penicillins. The above-mentioned findings of Brunner et al. (1968) that mycelial extracts of P . chrysogenum contain acyl-CoA:6-APA acyltransferase (transacylase) catalyzing the following reaction Acyl-CoA

+ 6-APA + penicillin + CoA,

led Brunner et al. (1968) to the conclusion that the final step in penicillin biosynthesis might involve the direct N-acylation of 6-APA by the CoAactivated side-chain precursors, formed from acetyl radicals and CoA by the phenylacety1:CoA ligase. They succeeded in this way in synthesizingpenicillins G and V, using 6-APA and the appropriate side-chain groups in the presence of ATP and CoA at an optimal pH of 7. They proposed a scheme for penicillin biosynthesis showing the acylation of 6-APA with the CoA derivative of a side-chain acid. The origin of 6-APA was not discussed, however. Gatenbeck and Brunsberg (1968) also isolated and purified 150-fold an acyl-CoA:6-APAacyltransferase from Penicillium chrysogenum ATCC 12687. Cells were ground with sand; the resulting extract was treated with ammonium sulfite, passed through a Sephadex G-25 column, eluted from a hydroxyapatite gel and a Sephadex G-200 column with phosphate buffer at pH 7.8. The purified enzyme was incubated at 30°C in the presence of 6-APA, phenylacetyl-[1-14C]-CoA,dithiothreitol and a buffer solution, pH 8.4, containing 0.2 M Tris, 50 mM phosphate, 0.2 M NaC1, 1 mM EDTA), and radioactive benzylpenicillin was produced. This enzyme preparation can utilize a number of acyl-CoA derivatives, although at different rates, e.g., phenoxyacetyl-CoA, p-methoxyphenylacetyl-CoA, octanoyl-CoA. The enzyme displayed no penicillin acylase activity; its optimum pH was approximately pH 8.5, and its activity was stimulated by the presence of thiol compounds (Gatenbeck, 1975). 3. Discussion

Whether penicillin acylase is also involved in one of the above enzymic reactions to supply 6-APA or is identical with one of the mentioned enzymic activities, is not entirely clear and deserves further investigation. In particular exchange reactions with and hydrolysis of penicillin N and isopenicillin N by different purified acylases, transferases, and acyltransferases should be studied further. Abraham and Loder (1972) and Fawcett et al. (1975) demonstrated that both isopenicillin N and 6-APA stimulate penicillin G biosynthesis, whereas penicillin N and isocephalosporin C were without effect. They found that a

ENZYMES INVOLVED I N

8-LACTAM ANTIBIOTICBIOSYNTHESIS

111

soluble protein fraction from Penicillium chrysogenum converted isopenicillin N into penicillin only in the presence of phenylacetyl-CoA. This is consistent with a real involvement of ligase and transacylase activity in penicillin biosynthesis (Fawcett et al., 1975). However, the use of a crude extract eliminated the need for phenylacetyl-CoA as a substrate for the conversion of isopenicillin N into penicillins, suggesting the action of a penicillin acyltransferase (which might be particle or membrane bound). They concluded that isopenicillin N is a substrate for an acyltransferase in Penicillium chrysogenum. Whether there is a transient formation of free 6-APA during acyltransferase action, or whether 6-APA is formed that remains coupled to an enzyme complex, is not known. In this context, the conversion of this 6-APA into penicillins by crude Penicillium extracts would be consistent with a concerted action of acylase, transacylase, and acyltransferase in penicillin biosynthesis. In this respect, the short communication by Spencer and Maung (1970) is of interest. They observed four enzymic activities in Penicillium chrysogenum 51-20F3: penicillin acyltransferase, which catalyzes exchange of side-chains from penicillins to 6-APA; 6-APA transacylase, which synthesizes penicillin from acyl-CoA and 6-APA; penicillin acylase, which hydrolyzes penicillin into 6-APA and the side-chain acid; and phenylacetyl-CoA hydrolase. Disruption of the mycelium, precipitation of the enzyme with (NH4).$04, and fractionation on Sephadex G-100 and DEAE-cellulose yield a 130-fold purification of each of the activities. The ratio of the activities was constant throughout purification and was detected in the same fraction. On polyacrylamide gel electrophoresis, all four activities were found in the same band. The influence of temperature and inhibitors on each enzymic activity was identical. Spencer and Maung (1970) claimed that the four activities are one and the same thiol-dependent enzyme. This suggests that ligase, acylase, transacylase, and acyltransferase form an enzyme complex, with multiple activities in penicillin biosynthesis. These suggestions find support in the recent experiments of Fawcett et al. (1975). Indeed, it was previously demonstrated that the synthetic capacity of penicillin acylases was enhanced by using energy-rich side-chain precursors, such as phenylacetylglycine and analogs (Bauer et al., 1960; Cole, 1964, 1969b), and for this reason the acylase could be considered to act as an acyltransferase or transacylase. It was suggested by Lemke and Brannon (1972) that the difference between penicillin acylase and acyltransferase is that the latter is an intracellular or particle bound form of the former. Up to the moment, no acyltransferase activity has been described among the Cephalosporium types; this is consistent with the fact that only a-aminoadipic acid structures have been found as side chains of their antibiotics. Isopenicillin N, but not penicillin N, is a substrate for Penicillium

112

E. J . VANDAMME

acyltransferase (Fawcett et al., 1975), although this compound does not act as substrate for penicillin acylases. However, the i c t that a crude penicillin acylase preparation from Penicillium chrysogenum, was able to produce some 6-APA when the carboxyl groups of the side chains of penicillin N and isopenicillin N were esterified, might suggest that these or related analogs of these penicillins are also substrates for acyltransferase activity (Vanderhaeghe et al., 1968). It seems that a lack of purified acyltransferases, transferases, and acylases from P-lactam producers and their poor characterizationstand in the way of a straightforward conclusion about their particular or concerted role in p-lactam biosynthesis. The biosynthetic activity of the acylases, transacylases, and acyltransferases is represented in Table VII. Quite another approach to the elucidation of the possible involvement of penicillin acylases or transferases in penicillin biosynthesis was advanced by Cooper (1972). Cooper (1972) prepared chemically a derivative of L-cysteinyl-D-dehydrovaline (thiazoline-azetidinone) from penicillin and suggested that this product is a model of a possible intermediate in the biosynthesis of penicillin and cephalosporin antibiotics. In this hypothetical pathway, the cysteine moiety would be derivatized with a-aminoadipic acid or attached to a protein surface. The proposed course of events would be amidation with L-valine, followed by p-lactam ring closure (Fig. 2). The resulting thiazoline-azetidinonemight be converted by acylase, transacylase, or acyltransferase action of Cephalosporium or Penicillium into cephalosporins and/or penicillins.

B. INVOLVEMENT OF ACYLASES,ESTERASES, AND TRANSFERASES IN CEPHALOSPORIN BIOSYNTHESIS 1 . Acylases

Microbial acylases capable of hydrolyzing cephalosporin C specifically into 7-ACA and D-a-aminoadipic acid have not been detected so far. Although cephalosporin C acylases, splitting off the a-aminoadipic acid, have been found in species of Brevibactaium, Achromobacter, and Flavobacterium, during their hydrolytic action the acetyl group of cephalosporin is also lost, so that deacetyl-7-ACA is produced rather than 7-ACA (Walton, 1964). The a-aminoadipic acid moiety of the cephalosporins can also undergo oxidative deamination by the action of pig kidney D-amino acid oxidases (Mazzeo and Romeo, 1972), but so far no microbial enzymes have been reported to carry out this reaction. Some semisynthetic cephalosporins can be split into their side-chain acid and 7-ACA by a few bacterial penicillin G acylases, but generally the rate of hydrolysis is lower than that of the corresponding penicillins. This is shown

TABLE VII PENICILLIN SYNTHESIS BY ACYLASES, TRANSACYLASES, OR ACYLTRANSFERASES FROM fl-hCTAM-PRODUCINGMICROORGANISMS

Microorganisms Penicillium chysogenum W49-408, SC-3576 Penicillium BRL 807 Penicillium chysogenum w49-408 Penicillium chrysogenum 51-20F3 Penicillium chysogenum D61347113 Penicillium chrysogenum ATCC 12687 Penicillium chysogenum

Penicillium chysogenum sc-6041

Enzyme preparation

Phenoxyacetic acid or phenylacetic acid Phenylacetic acid Intact cells Penicillin V,G,K,X, Soluble extract dihydro-F Purified enzyme Penicillin G Purified enzyme Phenylacetyl CoA Penicillin

Intact cells

Crude extract Purified enzyme Crude extract

Phcnylacetyl CoA or phenoxyacetyl CoA Phenylacetyl CoA Phenylacetic acid or phenylacetyl CoA

Crude extract

Crude extract Soluble extract

Penicillins produced

Substrates

Phenylacetyl CoA penicillin G Phenylacetyl CoA

+

Enzyme($ involved

References

+ 6-APA

Penicillin V or G

Acylase

Erickson and Dean (1966)

+ 6-APA

Penicillin G Penicillin V, G, K,X, dihydro-F Penicillin G Penicillin G Penicillins

Acylase Acyltransferase

Cole (1966) Pruess and Johnson (1967)

Acyltransferase Transacylase Acylase, transacylase acyltransferase Transacylase

Spencer (1968) Spencer (1968) Spencer and Maung (1970)

Transacylase

Gatenheck and Brunsherg (1968)

Acylase (?), transacylase (?j, acyltransferase Acylase (?), transacylase (?), acyltransferase Acylase (?), acyltransferase Transacylase, acyltransferase

Abraham (1974a,b), Loder (1972)

+ 6-APA + 6-APA + 6-APA + 6-APA + 6-APA + 6-APA

Penicillin G Penicillin V Penicillin G

+ 6-APA or

Penicillin G

isopenicillin N 6-APA or isopenicillin N

Solvent soluble penicillins

+ Isopenicillin N + Isopenicillin N

Penicillin G Solvent soluble penicillins

Brunner et al. (1968)

Abraham (1974a,h), Fawcett et al. (1975)

114

E. 1 . VANDAMME

R

B

B

j,

\

I

COOH Thiazolineazetidinone

COOH Cysteinyl-valine derivative

Cysteine derivative

R

"AXs

Cephalospovium acremaium and/or Penlcillium chrysogenum enzymes

R'CONH

1

--qk 0

o + ~ c H 2 0 c o c H 3 COOH Cephalosporins

/,

COOH R'CONH

+

0 5=CWH N

Penicillins

FIG.2. Biosynthetic pathway of penicillins and cephalosporins according to Cooper (1972). Reprinted with permission from the]ournal of the American Chemical Society 94,1018. Copyright by the American Chemical Society.

in Table VIII. Cephalosporin acylases from Bacillus megaterium and Erwinia aroideae were found to hydrolyze, respectively, the phenylacetyl and phenoxyacetyl derivatives of 7-aminodeacetoxycephalosporanic acid (7ADCA) into the side-chain and 7-ADCA (B. J. Abbott, personal communication). These two strains have previously been described for their penicillin G and penicillin V acylase activity, respectively (Vandammeand Voets, 197413). It is not known whether the penicillin acylases of P-lactam producers also display this activity. In contrast to this lack of hydrolytic cephalosporin C acylases, Japanese workers have detected bacterial enzymes able to synthesize cephalosporins, starting from 7-ACA or 7-ADCA and an appropriate side-chain ester (Takahashiet al., 1972). As side-chain structures, a-D-phenylglycine methyl ester and analogs (glycine ethyl ester, D-danine ethyl ester, D-leucine ethyl ester, and the methyl esters of D-a-cyclohexenylglycine, D-CX-P-hydroxyphenylglycine, and D-a-cyclohexylglycine) were coupled to 7-ACA or 7ADCA at pH 6 by enzymic action in microorganisms, includingXanthomonas oryzae IF03995, Acetobacter pasteurianus ATCC6033, Acetobacter turbidam ATCC9325, Gluconobacter suboxydans ATCC621, Pseudomonas naelanogenum IF012020, Mycopha dimorpha IF013212, and Protaminoaluoflauus IF013221 (Takahashi et al., 1974). This type of enzymic activity was subsequently found among a wide range of bacteria: Aeromonas, Escherichia, Staphylococcus,Arthrobacter, Proteus,

115

ENZYMES INVOLVED IN P-LACTAM ANTIBIOTIC BIOSYNTHESIS

TABLE VIII ENZYMIC PRODUCTION OF 7-ACA FROM SEMISYNTHETIC

CEPHALOSPORINS~ ~~

Penicillin acylase-producing microorganism Escherichia coli BRL 351 Nocardia FD 4697, Proteus rettgeri FD 13424 Escherichia coli NCIB 8734A, E . coli BRL 351 Kluyoera citrophila KY 3641

Substrate Benzylcephalosporin N-Phenoxyacetyl-7-ACA, N -Phenylmercapto-7-ACA Cephalothin, cephaloridine Cephalothin

References Sjoberg et al. (1967) Huang et al. (1963) Sjoberg et al. (1967); Cole (1969a) Nara et al. (1971b); Shimuzi et al. (1975)

aFrom Vandamme and Voets (1974b).

Coynebacterium, Achromobacter, Flavobacterium, Clostridium, Spirillum, and Bacillus, but has not yet been detected in cephalosporin producers. That enzymic activity was also clearly different from that of penicillin acylase, although some penicillin acylases are able to acylate 7-ACA or 7-ADCA (Marconi et al., 1975). However, the enzymic synthesis of cephalosporin C starting from 7-ACA and a-aminoadipic acid or its derivatives was not reported. As neither cephalosporin acylases nor 7-ACA have ever been found in cephalosporin-producing microorganisms, these enzymes and 7-ACA seem to have no role in the biosynthetic process of the cephalosporins. If, in the future, analogous enzymes are detected in cephalosporin or cephamycinproducing microorganisms, it is hard to believe that they could open additional perspectives toward p-lactam antibiotic biosynthesis, as it will be difficult to interpret the findings because of the still obscure role of the well-known penicillin acylases in penicillin biosynthesis. 2 . Esterases Enzymes that deacetylate cephalosporins are widespread in nature. Jeffery et al. (1961) isolated from orange peel a cephalosporin acetylesterase. This enzyme hydrolyzes cephalosporin C to deacetylcephalosporin C, other cephalosporins containing an 0-acetyl group to the corresponding deacetylcephalosporins, and 7-ACA to 7-aminodeacetylcephalosporanic acid. The enzyme is an esterase that shows highest activity with esters of acetic acid as substrates with a pH optimum between 6.0 and 6.5. A survey of microbial sources revealed that many bacteria, actinomycetes, and yeasts are capable of deacetylating cephalosporins (Demain et al., 1963; Nuesch et al., 1967). The enzyme was also found in liver and kidney tissue (O’Callaghan and Muggleton, 1963). Abbott and Fukuda (1975) reported on the purification and immobilization of a Bacillus subtilis NRRL-B-558 cephalosporin esterase. A molecular weight of about 19O,OOO, a narrow pH

116

E. J . VANDAMME

optimum at pH 7.0, and an optimum temperature between 40" and 50°C were its characteristics. In addition to cephalosporins and 7-ACA, the enzyme can hydrolyze mono- and triacetin, a-naphthyl acetate and glucose pentaacetate. The occurrence of deacetylcephalosporin C in Cephalosporium acremnium fermentations was first demonstrated by Jeffery et al. (1961). Huber and co-workers (1968) and Brannon et al. (1972) concluded that deacetylcephalosporin C found in cephalosporin C fermentations is merely a nonenzymic degradation product of cephalosporin C. However, the isolation of the carbamoyl p-lactam antibiotics from Streptomyces fermentations raised the question of the origin of the novel carbamate moiety (Nagarajanet al., 1971). Occurrence of enzymic 0-carbamoylation had already been demonstrated in Streptomyces polychromogenes (Tanaka and Sashikata, 1963). Brannon et al. (1972) found a cephalosporin C acetyl esterase in the culture broth of the carbamoyl-cephalosporin producing Streptomyces clavuligerus NRRL 3583, able to deacylate cephalosporin. The enzyme is inhibited by diisopropyl fluorophosphate, known to be an effective inhibitor of acetyl esterases (Jansen and Balls, 1952). The enzyme was only detected extracellularly. No evidence was obtained for a possible biosynthetic pathway in Streptomyces clavuligerms for cephalosporin C through deacetylcephalosporinC to the carbamoylcephalosporin. Also, it is not known which enzymes are involved in the terminal stages of cephamycin biosynthesis. Fujisawa et al. (1973) isolated mutants of Cephalosporium sp. M8650 and 52-54 strains, some of which produced only deacetylcephalosporin C (mutants 20, 29, 36, 40), while another mutant (81) initially produced cephalosporin C, which was subsequently converted into deacetylcephalosporin C. They found that strain 81, but not the other mutants nor the wild type, produced a cephalosporin C esterase, with optimum pH at 8.0, and concluded that, in the mutants 20, 29, 36, and 40, deacetylcephalosporin C is produced de novo and in strain 81 by enzymic decomposition of cephalosporin C. If so, cephalosporin C should be synthesized by enzymic acetylation of deacetylcephalosporin C. 3. Transferases Indeed, by incubating cell-free extracts from the wild type or mutant 81 in the presence of acetyl-CoA, deacetylcephalosporin C, and Mg2+ at pH 7.0, cephalosporin C was produced (Fujisawa et al., 1973, 1975). The responsible enzyme was named acetyl-CoA:deacetylcephalosporinC acetyltransferase. This transferase activity was not present in cell-free extracts of strains 20,29, 36, and 40 or in the cephalosporin C esterase fraction from strain 81. Accordingly, deacetylcephalosporin C accumulation in those mutants was due to the lack of acetyltransferase activity. Kanzaki et al. (1974b)and Fujisawa and Kanzaki (197513) concluded that the terminal biosynthetic step proceeds

ENZYMES INVOLVED IN P-LACTAM ANTIBIOTIC BIOSYNTHESIS

117

through a step catalyzed by the acetyltransferase, that deacetylcephalosporin C is an intermediate in cephalosporin C biosynthesis, and that racemization of the L-a-aminoadipyl moiety takes place before deacetylcephalosporin synthesis. These researchers also discovered some novel Cephalosporium metabolites with a potential role as intermediates in cephalosporin C biosynthesis (Fujisawa and Kanzaki, 1975a; Kanzaki et al., 1974a). Cephalosporin Cnegative mutants, obtained from Cephalosporium acremonium ATCC 14553 by N'-methyl-N '-nitro-N-nitrosoguanidine treatment, were shown to accumulate deacetylcephalosporin C accompanied by a cephalosporoate. This latter compound accumulated only in culture broths of mutants defective in acetyltransferase, and it is probably a degradation product of deacetylcephalosporin C. They proposed a biosynthetic scheme for cephalosporin C as follows: Tripeptide + deacetoxycephalosporin C

C + cephalosporin C

4 deacetylcephalosporin

Queener and Capone (1974) and Liersch et al. (1974) came to the same conclusion and reported on an enzyme catalyzing the hydroxylation of deacetoxycephalosporin C to deacetylcephalosporin C. Hence, Cephalosporium or Streptomyces strains, blocked in their hydroxylating (and acetylating) enzymes, produce deacetoxycephalosporins. Deacetoxycephalosporin C may be used in the industrial preparation of new cephalosporin antibiotics. Chemical deacylation removes the aminoadipyl side chain to yield 7-aminodeacetoxycephalosporanicacid (7-ADCA). This compound can be reacylated chemically or enzymically with cephalosporin acylases to produce new antibiotics (Takahashi et al., 1972). In strains lacking acetyltransferase activity, deacetylcephalosporin C can be considered as an end product of the p-lactam antibiotic biosynthetic pathways. However, deacetylcephalosporin C found together with cephalosporin C in many fermentations is most likely a degradation product of preformed cephalosporin C. Although, the acyl-CoA acetyltransferase has a definite role as the terminal-stage enzyme in cephalosporin C biosynthesis, whether a similar enzymic activity is involved in cephamycin biosynthesis is still an open question. V. Concluding Remarks Experiments with broken-cell systems, especially with protoplasts and their lysates (Abraham, 1974a; Kohsaka and Demain, 1976), have been encouraging with respect to the elucidation of p-lactam antibiotic biosynthesis. Such systems eliminate permeability barriers, but still give rise, as do all experiments with cell extracts, to modified enzyme activities caused by loss of membrane supports or conformational changes. The availability of cell lysates derived from high-producing strains of Penicillium, Cephalosporium,

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and Streptomyces used in the fermentation industries would certainly help throw more intense light on this complex problem. However, the question is still there whether the biosynthesis of p-lactam antibiotics can be achieved by soluble cytoplasmic enzymes or whether it requires, at least in some steps, the integrity of membrane- or particle-bound enzymes or enzyme complexes. On the other hand, biosynthetic mechanisms can be studied in intact cells using the techniques of biochemical genetics (Nash et al., 1974). Specific mutations that influence the availability of the building blocks influence p-lactam biosynthesis and can resolve the relationships between primary metabolism and antibiotic synthesis. Nonantibiotic-producing mutants of Penicillium, Cephalosporium, and Streptomyces mutants are also gaining importance as potential tools for the elucidation of intermediates and enzymes involved in p-lactam antibiotic synthesis. It should be noted that Penicillium chrysogenum and Cephalosporium acremonium species, from which all modern industrial strains are derived, contain viral particles that could influence the complex biosynthetic processes in the cell (Banks et al., 1969; Day and Ellis, 1971). However, there seems to be no relationship between antibiotic biosynthesis and the virus titer of the strain (Lemke and Nash, 1974). Viruses for the p-lactamproducing streptomycetes have not yet been reported. There are also indications that plasmid genes are involved in the production of certain antibiotics by streptomycetes (Akagawa et al., 1975; Kirby et al., 1975). However, this phenomenon has not yet been studied in P-lactam-producing streptomycetes. Hyphal differentation of Cephalosporium into arthrospores coincides with active antibiotic formation, indicating an interrelation between p-lactam antibiotic synthesis and stages of cell differentation (Nash and Huber, 1971; Queener and Ellis, 1975). Gordee and Day (1972) found that addition of exogenous penicillin V, G, or 6-APA during penicillin fermentation inhibited further accumulation of the antibiotic, suggesting that penicillin is a negative regulator of its own biosynthesis. All the above potential implications stress the overall complexity of p-lactam antibiotic biogenesis. Nevertheless, in recent years much progress has been made toward a fundamental understanding of these biosynthetic processes (Abraham, 1974a; Demain, 1974; Fawcett et al., 1975). ACKNOWLEDGMENT This review was born during a postdoctoral stay at the Sir William Dunn School of Pathology, University of Oxford, England. The author wishes to thank Professor E. P. Abraham, University of Oxford, and Professor A. L. Dernain, Massachusetts Institute of Technology, Cambridge, Massachusetts, for their helpful discussions, criticism, and stimulating advice during the preparation of the manuscript. The author is indebted to Professor J. P. Voets, University of Gent, for encouragement and a leave of absence.

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Information Control in Fermentation Development D. J. D. HOCKENHULL Glaro Laboratories Ltd., Ulverston, Cumbria, England I. Introduction . . . . . , . . ....................... A. Information for Process Documentation . . . . . . . . . . . . . . . . B. Information for Technical Management . . . . . . . . . . . . . . . . 11. Research and Develop 111. Project Initiation Reques Program) . . . . . . . . . . . . . ...................... IV. Formal Program . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Periodical Reports.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Weekly Report.. . .............. B. Monthly Report . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , C. Quarterly and Half-Yearly Reports . . . . . D. Half-Yearly Project Re ........................ E. Annual Report . . ....................... VI. Laboratory and Plant Protocols . . . . A. General Management Material B. Equipment Operating Procedur ............... VII. The Standard Operating Procedure . . . . . . . . . . . . , . . . . . . . . . . VIII. Direct Experimental Records . . . . . . . . . . . . . A. Experimental Instructions . . . B. Laboratory Notebooks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Program Job Packs.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , D. Pilot Plant Records ....................... E. Analytical Records . . . . . . . . . . . . . . . . . . . . . . . . . . . F. Computer Storage . ....... G. Plant Graphs.. , . . . .............. IX. Miscellaneous Informatio .................

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XII. Making the Best Use of Data . . . ............ References . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. Introduction New products, and increasingly efficient means of producing them, are what industry asks of research and development (R&D). The know-how involved has to be communicated to the manufacturing end of the business; indeed, the main output from R & D is technical information. The form and content of this should be satisfactory to, and usable by, whichever sector of the Company needs it (and is perhaps paying for its time). The first stage in the process of informing is the generation of data. These can be defined, for the purpose of this review, as being raw experimental results, such as instrumental readings, analytical figures, and other firsthand observations. The second stage in the process is the converting of these into 125

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patterns that have useful meaning. The data are systematized, compared, collated, and otherwise worked upon to gain maximum interest summarized in the smallest compass. Thus is achieved the clearest possible view of those experimental relationships with which the informant should be most concerned. Following from this emerges the third stage-producing instructions for carrying out the most profitable methods that have been developed. In this study I shall discuss the various ways of handling information. I shall consider the various vehicles at our disposal for storing and publishing information. I shall look critically at these in the light of the particular jobs they are supposed to do. I shall also give some indication of how such information vehicles fit in with the major needs of industry. Are they as useful as they might be in supporting the internal organizational needs of the laboratory and pilot plant? And do they contribute to rapid economic exploitation of new discoveries on a manufacturing scale? Many people are involved in R & D activities. Investigations consume many man-hours and generate astronomical amounts of data. There is too much to deal with sensibly without reducing it all to a pattern simple enough for one brain to think about. In Ashby’s terminology (1961), there is too much variety. Edwards and Lees (1973)put it thus: “Unduly high informational load is an external agent of stress.” Miller (1960),as quoted by them, has listed the mechanisms that are used by man to reduce such stress: 1. Omission-temporary nonprocessing of material 2. Error-processing incorrect information, which may enable the system to return to normal processing afterwards 3. Queuing-delaying the response during a period of high overlap of input information in the expectation that it may be possible to catch up in a lull 4. Filtering-neglecting to process certain categories of information while processing others 5. Cutting categories of discrimination-responding in a general way to the input but with less precision than would be done at lower rates 6. Escaping from the task-making no decision, guessing, or tossing a coin Inevitably, most of these result in performance deterioration. The reduction of data to manageable proportions is carried out by summarizing, selection, and categorization. We exclude only what is unnecessary to our current purpose in order to build the data into a new informational structure. If we do this effectively, the value of the reduced d a t a - o r information, as we may now call it-should be much greater. We must be careful, however, not to throw out potentially useful facts. The specific aims of our current experimentation may well be superseded as a result of other ways of looking at the data. Some time in the future we may

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want to look at aspects we had not hitherto considered important. So we need to store the data in a retrievable form. This may be done either as primary unprocessed results or after systematic data reduction. Deciding how much of each kind we should store depends on the cost of the original data, the cost of storage space, and the cost of reduction. These are the main criteria that should decide our day-to-day policy. Another factor is the fluidity of the field-where development is rapid, information may become obsolete in five years or even in one year; e.g., when surface culture of mold fungi was superseded by deep culture, only the most general of conclusions were relevant to the new order. However, my main preoccupation here will be to list and describe the collection and reporting of results. I shall describe the various vehicles for doing this and discuss their several advantagesfor the different tasks involved in the preparation of information for the direction of technical development. Before continuing with the study, let us look at an example. Figure 1 describes a typical experimental procedure taken from the field of systems engineering (MacFarlane, 1964). The example given is a very simple one. It entails subjecting a real combination of things-physical system-to a series of arbitrary known changes, observing how the instrumental readings vary in response (measuring system); setting out some sort of descriptive hypothesis (model system); and further regularizing this by describing the behavior of the system in mathematical terms (mathematical system). The description is tested against further experimental variations of the physical system and, as a result, the model and mathematical systems are further modified to give a truer approximation to reality. Ideally, the measuring system should mirror the behavior of the physical system exactly. In real life, however, especially in biological work, it does so only imperfectly; either our methods are clumsy or we cannot make measurements without interfering with the experimental conditions by doing so. The model system is basically a description of what is happening in abstract terms; i.e., it is a collection of abstract objects derived from the consideration of the relationships “between measurements performed on the physical system in terms of state, work, power energy, signal and information.” Finally, the mathematical system is a set of abstract mathematical objects together with defined sets of relationships between them. It is used together with the model system to predict the behavior of the physical system. The example just described may concern very simple investigations as well as more complex ones. Typically, the task could be (a) to find the relationship of load and extension in a stretched spring; (b) to plot the pH characteristics of the titration of acetic acid with caustic soda; or (c) to find the relationship between the growth rate and temperature of a bacterium. The most basic type of experiment is subject to drastic reduction of data and

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physical system

physical system

and predicted system behavior

t Adjustment of measuring, model, and mathematical systems to give an accurate comparison between observed and predicted system behavior

FIG. 1. Experimentation charted as a series of system interrelationships.The arrows direct the sequence in which the various systems are developed and modified. From MacFarlane (1964). Reprinted with permission from “Engineering Systems Analysis,” p. 1. G. Harrap, London.

summarization. In biological work, this is more diEcult. The complexity of the experiments is high, and the number of covariants very great; the number of different physical systems we can choose from any set of experiments is high also. Many experiments may be required to establish a single set of relationships. Or, taking different experimental series together might reveal other relationships and answer other questions. Or, perhaps late in an investigation we may realize that certain relationships could be significant. This boils down to the need, on the one hand, to have procedures that reduce the data we have to manageable proportions and, on the other, to store a great deal of the original material to keep our options open and, in case it comes in useful, to back up further systematization. For instance, we may have to use the results for comparing the costs of different processes, perhaps years after the original work was done. We may need the results as older data for comparison with new data, or for testing out experimental models that did not exist when the original results were acquired. These possibilities all emphasize the need to store data adequately and to have simple means for their retrieval. At the same time, data reduction must be effective enough for us to manage the ongoing activities without being snowed under with detail-for example, to answer questions and to communicate necessary innovations to the production area. Different kinds of research and development units require different kinds of informational structures. The information generated may be specific to the

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activities and investigations. Equally, different information will be needed for their control. Briefly, the data must be treated in a manner appropriate to the way in which they will be used and to whom they may be useful. This is not to be thought of as a permanent structuring, even in one area of activity. As a field develops, new needs grow because technologies advance and establishments grow in size and diversity. I shall, however, draw from my experience in the pharmaceutical industry. I shall describe various informational and communicational vehicles that have been used over a period. I shall point out their purposes and their effectiveness in serving these ends. Similar problems have been discussed elsewhere, especially in the chemical industry. My study, however, will deal with the special problems of the biological industries. It may also throw light from a different direction on common problems. My hope is that this study will provide the basis for a flexible approach to information handling, so that the efficiency of a process can be altered and improved in response to altering needs. The conclusions developed should be relevant whatever the style of management or whatever the nature of the scientific and technical activities considered. Technical information is used in two principal ways. First, for the definition of improved methods of production and, second, for the better management of R & D. We may define the objectives more specifically by listing a number of functions subsumed under these two headings. A. INFORMATIONFOR PROCESSDOCUMENTATION

1. If processes are not adequately described, they will not be repeatable or translatable to another scale (particularly, the production scale), nor will they provide a basis for future work. 2. The Company’s interests have to be protected against its competitors. Reliably documented information has to be furnished for obtaining patent protection. Evidence of prior use has to be established against the corresponding patent application of business rivals. 3. Technical information has to be communicated to other working groups. The program of work may have relevance to what other individuals are doing. The methods used may be useful to others. Findings on one organism may illuminate the behavior of another. Such information can minimize duplication of effort. It may explain phenomena encountered in other areas. It may put into appropriate words some thoughts that until then were vague or ambiguous. Such information may often be qualitative or descriptive as well as mathematical and concrete. It may suggest new approaches in theory or technology. Finally, such information will reveal the existence of knowledgeable individuals with whom common problems may

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be discussed. In return, other groups may help with the solution of the problems brought to their attention. 4. One must provide a memory store for results and for the reasoning and phenomena leading up to them. It is especially important to identify good ideas, in spite of their having come to nothing in the past. The current technology may have been too weak to support them. After the lapse of a few years, new techniques and the ability to look at the problem from a different angle have often been proved to have revived such ideas and brought them to successful fruition.

B.

INFORMATION FOR

TECHNICAL MANAGEMENT

1. No department can run at maximum efficiency if the decisions as to long-term and short-term aims are based on unsound information. The amount of effort-in men, equipment, and cost-must be adjusted to give the greatest return in actual or potential process development and in the creation of new entrepreneurial opportunities. The accurate assessment of progress in each area is then essential to check the results against the targets previously set. As a result of such comparisons, new targets may be set, new projects started, unpromising lines curtailed, and resources redeployed. 2. The right kind of informational system is necessary for adequate regulation of responsibility. Regular information in an impersonal form from his submanagers tells a boss what is going on at regular intervals and warns him when his visits should be “social” or when he should intervene in the work. The discipline of producing regular written information improves the under-managers’ powers of self-criticism. The objective viewpoint thus taken strengthens greatly their ability to do their jobs. Written reports make it easy to ask for help or guidance in future schemes. They are also aidesmemoire to help the boss work out which major problems, technical or managerial, he should discuss and with whom he should discuss them. Gellerman (1973) sums this up as follows: “Nothing, literally nothing, is more valuable than reliable information. To know what is really happening, what is really possible and what other people believe about both is fir more important than having money, factories or armies of workers. To know what is happening enables a manager to put even limited assets to decisive use: not to know may cripple even the mightiest Company.” Thus there are many reasons for creating an informational system in R & D. Its completeness and efficacy will depend on the needs of the situation and on the motivation of the human elements composing it. By and large, both the strength and the weakness of an efficient information system is that it lays the protagonists open to criticism. This human problem can best be

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dealt with by fair and enlightened management. Technical successes also make life easier by engendering trust. It is not a good sign when written reports are neglected or when their place is taken by verbal ones. This is especially true of the more bequent ones, such as monthly or fortnightly section reports. The absence of downward communication can have a similarly bad effect on morale. Gellerman (1973) also says, “Higher levels keep information from the lower, partly to limit their exposure to criticism and partly on the basis of a wholly inappropriate military model of communication (where, for the sake of secrecy, information is given only to those “with a need to know”-which is typically interpreted as meaning hardly anyone). But lower levels also keep information from the higher, partly in the adolescent belief that what they do not know will not hurt them and partly to protect a sort of territorial right to information that gives them prestige and power (or the illusion of both).” In short, the value of information must be recognized at the top, and the promulgation of adequate systems must be passed on down. There must be a means €or those at the top to gain access to every part of the organization should necessity dictate, but it should not be so difficult that it interferes with their main preoccupations-which are strategic. A manager should not have to waste his subordinates’ time to find out what they are doing-that information should come to him at regular intervals. He should be lefl free to discuss new possibilities, their impact on present conditions, possible expansion of resources, intensification of activity, and so on. 3. A third point is that writing is a good discipline. Describing what one has done and fitting it into one’s frame of reference is useful. Good also is writing it up to fit into contexts in which different use is made of the data. There is good also in examining the pros and cons of the various ways of looking at the data. Writing, like hanging, is a wonderful clearer of the mind. Provided that the writer is free to indulge in a reasonable amount of speculation, the act of committing his observations to paper can often display novelty where it exists. Thus the projection of future work can be made much more certain. The discipline of regularly writing-up work can make the task of data reduction much easier. Use breeds facility, and continuous application spreads the labor thin. The ongoing fitting of every experiment into the historical scene as one goes along helps to keep the detail down to manageable levels. In this way, the problems are kept free from clutter so that the major issues can be decided first and the less important details made to follow from the master plan. However, we must be careful not to be dominated by that to which we have become accustomed. We must not be dis-

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couraged from enterprising and original experiments because they are not in the traditional canon or because our plans are made with a too rigid and logical basis. Let us not forget, however, that most of the results are pragmatic. We often know that the phenomenon follows from the conditions imposed on it, but we cannot give a rational explanation. Often, we can only describe the conditions in qualitative terms. This does not matter so long as we have enough information to repeat the experiment as often as we please. Our aim is to fit the results of our work into the technology, not necessarily into the basic science-to help control the universe, not explain it. Accordingly, the underlying theory is not very important to us post &to. In the longer term, however, we do not neglect the basics. Those experimental results we have must be interpreted in the light of theory to give understandable patterns and to systematize our findings. With the help of a good fundamental knowledge of the sciences we use, we can look forward to the discoveries of tomorrow. Furthermore, unless experimentation is based on both rational and creative speculation, an investigation drops to the level of “bung another newt in the cauldron, dearie.” However, we should not forget that the end of experimentation is to be able to carry out newer and better processes that will be judged in financial terms. A danger in any R & D organization-consequent to some extent upon the basic university training of its members-is to be concerned with proving a particular point regardless. That is, trying to show oneself, as an expert, to be backing the right theory so hard that it blinds one to obvious opportunities, such as introducing arbitrary changes. Such blindness leads to doing experiments in the head rather than on the plant. And, finally, it leaves no room for serendipity. So, having expanded upon the kinds of use we may make of processed information, let us proceed to a study of the various ways of storing and promulgating information. The examples will be drawn as usual from the field of research and development in the pharmaceutical fermentation industry. The types of work involved are experimental process development, strain improvement (applied genetics), analytical methodology, and backup work of an explorative nature (biochemistry, microbiology, enzymology, molecular biology, etc.). As indicated above, I shall describe the various forms of storing and processing information. These descriptions will give a concrete basis for discussion of the jobs they are intended to do and their effectiveness in doing them. I shall not be able to dot every i and cross each t, because each R & D organization is different from every other. Differences of topic, managerial style, the areas of search, the state of particular projects and their previous

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histories, the kind of staff and their collective expertise, the Company mores, all introduce their quota of variety into the decision-makingbetween alternative informational forms. I do, however, hope to give a picture of what is available. Especially I want to bring out the necessary differencm in form and content of each device and its appropriateness in satisfying particular needs.

II. Research and Development Budget This document may be produced by the appropriate Manager of Hesearch or of Development or both. It is a statement of what products are in need of development, how much improvement can reasonably be expected, and how much return on expenditure this is likely to yield. The computations are based on statements from the Production Management and the likely outcome of work in progress. The principal factor is, however, the existing commitments of the plant. The more cash flow there is from a product, the more development may have to be done on it. This viewpoint is modified with new and experimental products or when marketing information clearly indicates that a lowering of price can affect the market situation to an exceptional degree. The R & D plans for the year are finally decided upon by the Development and the Production Directors together. By and large, the budget concerns itself only with the broadest elements of control. The ways in which the money available to each product is spent are generally left to the discretion of the Development Manager, who is, in the short term, limited by his resources and staf€. The budget is completed by a construction and maintenance statementthis, according to custom, in my Company is a part of the factory annual budget. The statement explains, however, what money is available for new equipment and how this will be spent. Ill. Project Initiation Request (Research or Development Program)

This brings us down to practical work-to the particular deployment of effort as opposed to the general long-term strategy of the R & D Budget. The request document is produced before work may begin in a particular area. It sets out the aims of the investigation in a concrete way. These aims should not be diffuse but, rather, have a well defined and limited prior objective. The objective should be reflected in the title, which must not be too catholic. The document then states what is to be done, what knowledge and theory exist currently, the benefits likely to accrue from the work, criteria of pro-

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gress, the expected duration of the investigation, and the cost in terms of time, manpower, and equipment. It sets dates for review and for interim reports of the project. As an example, we may take such a title as “Investigation of the possibility of applying continuous carbohydrate feeding to the production of penicillin.. . .” The benefits likely to accrue follow fiom the possibility of using cheaper carbohydrates, like sucrose and glucose, instead of the expensive lactose, which was in use at the time that project was initiated. Further benefits which might also accrue include the possibility of carrying on extended fermentations (hitherto limited by the top level of lactose that could be included before inoculation) and by the use of less pure, cheaper sugar sources, such as crude starch hydrolyzate. Special equipment might be needed for sterile additions of nutrient, and the designing of this might have to be included in the project. Some idea might be obtained as to the financial return on some of the process changes possible, and that, in turn, would affect the size of the project. On this particular project a good deal of background knowledge would, in fact, already exist. Hypotheses would have been made and discussed in the literature as to how lactose was assimilated and used. There would aIso be some knowledge of continuous fermentation techniques. Theoretical concepts of the relationship between growth and product formation between primary and secondary metabolism would also be useful if they existed at the time. (Of course, the example is “made up.” The history of this project is that it actually started in a University laboratory in Wisconsin, and its development took place piecemeal through the industry over a number of years. But the example should nevertheless have utility.) Such a document, if well drafted, is of great value. It is particularly so to its originator. It keeps him to the point. He does not dissipate his energies by too frequent a change of line. It gives him a yardstick by which to measure his progress, and it gives him objectives to meet. It is also useful to the R & D manager (and, to a great extent, to the director above him). It enables them to manage the work. In particular, the manager has a useful means of assessing what is to be expected from his team and whether he has available the right kind of skills and equipment. Frequent informal review is ensured and attention is drawn to current difficulties. Thus the manager is put in a better position to allocate his resources to the various projects he has running. Such formal programming is useful in relating each branch of activity to the general departmental budget. It is also useful in establishing a common understanding with the production units so that they are aware of what kind of developments to expect in the future and are advised of how “their” money is being spent.

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All formalizing procedures have dangers. Experimental plans may be followed too inflexibly. This can lead to overperseverance with unsuccessful techniques. It may cause the suppression of a promising “lead on grounds of irrelevance (although it could equally result in setting up that “lead with a project of its own). On the other hand, a definite plan of campaign enables one to stand off and look at it from time to time with a cold, critical eye. The circulation of such a document is important. On the one hand, it should be distributed widely enough to bring in the maximum of technical support. On the other hand, the list should be selective enough to omit carping and negative busybodies. Programs designed to help with particular plant problems, as, for example, the feasibility of using a cheaper grade of ingredient, the development of more uniformly behaving inocula, the testing of improved strains of microorganisms, or the development of prototype equipment, all gain by discussion with the plant managers. Such talks will be more valuable at the drafting stage and, even more so, later when financial and technical help has to be asked for. On the other hand, the selfsame individuals might be obstructive to a project if they thought that it was too theoretical and “way out. ” The project initiation request might then be the wrong way to sell them on the line of work chosen. In sum, the initiation procedure is useful in setting aims and methods. It must be used with a light hand, or it will restrict the emergence of novelty. It ensures the regular review of subprojects. It should abort frustrations and unprofitable areas of endeavor. It enables work to be coordinated inside the R & D Department and in contiguous areas such as the Company’s “Research Institute” or the production units. Finally, the Project Initiation, or whatever it is decided to call it, needs to be a formal document with a standard format. In fact, some companies using this technique provide specially printed manuscript paper to help maintain uniformity. The most important point is that the case shall be well presented. The statements made must therefore be clear and unambiguous. However, when the production of such documents is general, the writers soon get to know what is expected of them.

IV. Formal Program This description is used rather than “project report” to avoid confusion occasioned by the organization of some R & D departments on a project basis. Under those circumstances, the activities on a particular product are united under a single head, who may therefore be responsible for a number of programs of research side by side. These can be of similar types or require very different facilities. The usual way to report such activities would be in

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the monthly report, but they may also be the subject of project six-monthly reports and will be discussed later under that heading). When discussing the initiation of a program I mentioned that dates should be set for the interim and final reports of the work. These should take the form of Project Reports. Similar reports should also be issued when some aspect of the work has been brought to a stage at which it makes a good story and has generated firm conclusions. The Project Report is a formal document consisting of Summary, Introduction, Experimental, Discussion, Bibliography, and Circulation list. There should again be a standard of quality as regards style and content. The objects are (1)to state progress; (2) to ensure ready retrieval of experimental information. One copy is always deposited in the Library or other suitable center; (3)to publish techniques and results to other experimental groups; (4) to assist in technical direction of the work; (5) to establish continuity in spite of any staff changes. Project reports should be available to all parties-with due regard, of course, for security. Summaries should therefore be widely circulated while complete reports would naturally have a restricted circulation. Reports should go to the Production Department where the work has likely application. In that direction, the observations made in the report should be related to their possible utility in the production process, and by special memoranda attention should be drawn to the applied aspects of the work. The reports should also be circulated to other research and development units that might be affected by the conclusions, might be interested in the methods used, and might contribute ideas and further development in the same area. Certainly, if the work is an extension of that carried on by another group, the fact should be acknowledged and the information duly communicated back. Again, if new speculations arise from the discussion section, these should be communicated in case they might be useful as a basis for future work. We have mentioned standards of production: there are also standards of content. The write-up should be adequate-with due recourse to other reported material, e.g., standard operating procedures and previous project reports-to enable the work to be repeated with similar results. That is to say, the experimental section should be not only adequate, but accurate. The most important part of a report is its summary. This, as noted, can have a much wider circulation than the complete report. Generally, the summary is all that the most senior officials of the company, directors anyway, get to read. The summary should therefore give some idea of the way the work affects the current commercial process. That is not always easy to do. It may not even be possible with certain kinds of laboratory work, especially that concerned with getting background information or of a very

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pioneer or speculative nature. It is, however, essential with experimental fermentations or in reporting on the performance of new mutant strains. The latter, especially, would inevitably affect the commercial situation. The general exposure of financial aspects may, however, be inadvisable for security reasons. Even so, if the cost calculations have to be presented “under separate cover” the summary should draw attention when a breakthrough has been made. The possible emergence of a more efficient process is what we all want to know about and this aspect should be discussed. The report should be critical. Here, of course, one is in some difficulty. The writers of project reports wish to present themselves in a good light. They will therefore tend to be optimistic in the interpretation of results. Retaining objectivity calls for a high level of personal integrity on the authors’ part. Human nature being what it is, integrity is not universal. Thus the manager has a weighty responsibility not to accept too cavalier a treatment of data. A subtler perversion of facts is the choice of an inappropriate way of assessing performance. To give a trivial example, the statement that method A gives 20% more product than the control may conceal the fact that in the particular series of experiments the control was 30% lower than the standard plant performance. Quoting the concentration of an antibiotic in the harvested culture may seem to show impressive improvements whereas the total yield per unit of ingredient cost, the output per fermenter or the cheapest process per unit of total expenses are more realistic measures of commercial utility. These criteria of performance are useful in their appropriate contexts. Their current usefulness depends on the state of the order book, the availability of plant, and the commitment to different final products made from the same harvest broth. A good summary should contain some recommendations. If there is an immediate application to the production process, it should be spelled out. The way in which the work should be continued, and where and by whom, should also be indicated. Possible new lines arising from the work should also be noted, and suggestions made for their exploitation. I have already mentioned a great deal of what should go on in the discussion of the experimental results. This discussion is most important. It should be critical. The strength of the evidence for each conclusion should be listed, and use made of statistics where possible. The ideas behind the work should be reevaluated in the light of their practical outcome, and their ratification or rebuttal made the basis of suitable restatements about the nature of the systems involved. The experimental techniques should also be reexamined, and this appraisal be made the opportunity for forging better investigating methods. Suggestions should also be offered, if the methods apply, for carrying on work in related areas, though they should be accompanied by a critical

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analysis of their possible usefulness. Suggestions may also be included for the backup work by other units.

V. Periodical Reports Special reports may be called for from different groups on a regular time schedule-annually, six-monthly, three-monthly, monthly, or weekly. Generally speaking, the functions of these differ a great deal from each other and are aimed at different targets. This will become clear as I discuss each individually.

A. WEEKLYREPORT The weekly report is an informal memorandum. It is generally collected from the leader of each group and collated by the R & D manager. Its circulation is limited. It is aimed at the factory manager in particular, with copies to his deputy, to the production manager, and to the chief engineer. The weekly report is a technical newsletter. It is a summary of current interests in the area of R & D. Each unit contributes a very short statement drawing attention to points of recent interest. The report also includes general points of management-s@ deficiencies, unsatisfactory levels of services, difficulties successfully coped with, and so on. Although a good monthly reporting system renders the technical side of the weekly report redundant, the latter’s brevity and journalistic style tends to make it a better reminder of what is going on. Indeed, because of the informality, it can be less reserved in statement, which may make it more easily used by recipients when they exercise their prerogative of enquiring further into the programs. The weekly report has, however, one property that must not be forgotten. The original information going toward the weekly report has passed through at least two levels of command. The opinions expressed are not therefore those of the original experimenters but are slanted, filtered, or otherwise transmogrified to put the situation from other points of view. Remember, therefore, that the coding and the destruction or reprocessing of information can give a misleading idea of what is actually going on.

B. MONTHLY REPORT The monthly report consists of abridged accounts of all experiments carried out. It is generally sectionalized product by product. The summary draws attention to the experimental advances and the success or failure of particular trials. It also contains references to reports and standard operating procedures (SOP) issued in the month. The body of the report refers by

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number to each experiment thus aiding the retrieval of further data. It also contains critical discussion of each experiment or set of experiments. What are the functions of the monthly report? 1. It shows the current state of progress on each product. This stands out especially when compared with previous monthly reports. 2. It informs the higher command about progress toward the targets set for each product (mainly through the summary). This helps to modify the programs, if necessary, to meet the changing circumstances arising during the course of the year. Hence one can more easily meet altering needs or take advantage of new resources. 3. It enables the management to assess the strengths and weaknesses of the current approaches. 4. It informs other units in R & D of the progress made on other projects and gives them necessary background for their own work. Thus the fate of improved strains in the experimental fermentation unit, suggestions about medium from the laboratory, and similar pieces of information are exchanged. In addition, the document may be circulated to other parts of the organization, such as, say, the Research Institute, where it will be of use. 5. It informs the Production Department of current developments that may be almost ready for plant trial. Thus it enables them to forecast their own needs in terms of equipment and manpower. In addition, because much R & D work is carried out at the direct request of the Production Department, it informs the latter that its needs are being satisfactorily met. 6. The monthly report is the major reference for locating work information in the archives. It is a ready means of retrieval of detailed information because it should report all results, positive or negative, good or bad, well or badly investigated. So one can readily find the series numbers of the experiments and trace them back to the departmental records, notebooks, or whatever. Thus, the monthly report is a check on wild statements as to what happened in the past. It may also lead to more effective experimentation by helping to avoid repeating the mistakes of long ago. It can give the information to answer such questions as: What was the performance of the penicillin process in November 1965? How much of the present improvement over that of 10 years ago is due to improved strains and how much to improved bioengineering? Generally, the monthly report is written in parts by the experimenters themselves and put together by their departmental head. The experimental results are the most important part, their interpretation secondary to this. Nevertheless, discussion and speculation should be included because the report should give guidance on the experimenters’ intentions for both present and future. There is, however, a greater need for objectivity and for the opportunity to show it than in other kinds of report, where the evidence may

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be organized to support one hypothesis or another. This is not, of course, meant to imply that the facts are distorted elsewhere, but that a decision is often asked in such a way that only limited sets of experiments are relevant. The monthly report is, however, a report of all work done: highly relevant to its efficient direction is that the aims are under continual scrutiny. Of course, as in any other report, portions may be “bulled-up” and every jewel burnished, however small. These are things that must be controlled by the director in charge of Development. If the right judgments are to be made on the basis of the monthly reports, the work must be presented objectively. The duty of the writer is, however, to indicate what options he would recommend even though he cannot be absolutely certain about them. The great value of the monthly report is its immediacy. It reports on work in progress. Feedback from its publication is obtained while there is still time to modify the program. (This is an important distinction from sixmonthly or project reports, for those often appear only when the project has closed down or when the avoidable mistakes have been made.) The monthly report includes all the work in progress, and thus it is especially suitable for pilot plant work in which a large number of units are subject to regular scheduling. The monthly report gives an indication of the use made of resources. While there is an area of discretion around how much one should report in the way of operational (e.g., mechanical) failures, their reporting ensures that the standard is kept up. Thus the monthly report is the main information channel for tactical decision, while other vehicles, such as quarterly reports and project reports, are used to define the long-term or strategic aims. The latter reports, however, owe much to the monthly one, for it is the basis of the quarterly report to the Managing Director and for the Annual Report which is used to formulate next year’s policy and t a back up the coming R & D budget. Such an instrument should be dignified with a format suitable to its importance. Usually, a suitable confidential cover is provided. The resultant document is filed as a monthly installment of an ongoing series. Its filing should be under proper security control. As far as is consistent with this it should, however, be widely accessible for anyone to consult. This is the dilemma of industrial security. What is useful to ourselves is useful to our competitors, and vice versa.

C . QUARTERLY AND HALF-YEARLY REPORTS These are reports that are called up for special purposes. In particular, they tell the directors what is going on in a general sense. They are usually, though not always, derived from the monthly reports together with a few explanatory notes with regard to the age and progress of the particular projects. In general, they are highly selective in that they are in effect a

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managerial statement: one man’s view (albeit the most important man) of his departmental activities.

D. HALF-YEARLY PROJECTREPORT When the whole range of activities concerned with a single product is organized together, i.e., when the department is split according to projects rather than specialization, the work may also be reported in that way. The monthly report may, in most instances, be entirely adequate and often is the best vehicle for this. Nonetheless, some kinds of work may be so long-term that reporting by the month gives too fragmentary an idea of what is going on. Such work is best dealt with by reporting the programs as they mature, and in the monthly reports simply indicating the stage of completeness of the work. (This gives a good retrieval potential.) Another way is to report everything on a six-monthly basis, the vast volume of information being then, as we have said, broken down projectwise. Some of the work, particularly studies in biochemistry or genetics, is well suited to this method. It can be rounded off, and the results of a complete series of experiments summarized in a more understandable and compact form. Also one discussion section serves for a good deal of work. Partially completed work and defunct lines of investigation present problems, however, These can, if reported in full, distort the narrative flow of the report. If not reported, they may never be recorded or come to the notice of other workers, contemporary or future, who might profit from them. There is a tendency, because six months work is a lot to write up at a single time, to cut out “irrelevant” material in order to lighten the workload. Besides the omission of work, there is often a degree of oversummarization. Even considering these things, the report is never short. It is a large physical labor to produce it at all. It takes a long time. This is especially true because it is a rather formal document, involving many people and attempting to draw rather firm conclusions (unlike the monthly report, which gives experimental facts and discussion but does not claim the last word on anything). This adds to the publication delay. The oversummarization also suppresses ideas and notions and makes the data appear stale. The delay in appearance after the last work reported, and the lack of “feel” in the data quoted, make the six-monthly report useless for tactical purposes and reduce its use in longer-term planning. Receiving a report on work carried out between January and July 1975 in March 1976 is, to say the least, irritating. Especially so when one spots a neglected opportunity or a bossed shot far, far too late to do anything about it. In those circumstances, criticism becomes carping, since it is too late to make constructive suggestions. All these points should be considered in deciding the frequency of project reports or whether we should use them at all.

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E. ANNUALREPORT The annual report is usually prepared in time for the construction of next year’s budget. Most of its facts come from the monthly reports. However, an equal emphasis is placed on the situation in the production units and the relevance of the R & D effort to this. How many improvements have been adopted in the manufacturing process? How many have been indicated by the development department and are waiting for translation? In what areas has no progress been made? Is this through lack of effort or equipment? Or is it because the aims are not practical? A further aspect that has to be covered in the annual report is the state of the facilities. Are there enough staf€? Or too many? Is the st& of the right kind? Is it correctly distributed and organized? Is there sufficient equipment of the right kind? Is its maintenance efficient? Will any have to be replaced? Are any large capital items of plant needed in the forthcoming year? Further, what outside assistance will be needed? What will be expected of the service departments? And what special work for other parts of the organization will be called for? Finally, a program of the next year’s R & D activities must be suggested to provide agenda for R & D budget meetings and a blueprint for future activities, from which the budget can be drawn up.

VI. Laboratory and Plant Protocols A. GENERAL MANAGEMENTMATERIAL Most companies have a general administration handbook that gives guidance in respect to hours of work, compensation for overtime, traveling expenses, disciplinary matters, and hiring and firing. Similarly, most departments run more or less formalized training schemes for new incumbents. In addition, there are work safety procedures, dangerous chemicals regulations, emergency procedures, and fire precautions. All these are codified and set out in appropriate instructions which should lay down actions, authority, and responsibility.

B. EQUIPMENT OPERATING PROCEDURE (EOP) In areas where large and complicated plant is used regularly, standard procedural handbooks or brochures are produced which, as completely as possible, define the recommended procedures. These are available to the people operating such equipment and to those supervising such staff.

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The exact content differs, of course, with the kind of plant and the kind of laboratory. A typical semitechnical scale unit would contain sketches of, say, the fermenter and its ancillary equipment. It would give specifications of such items as motor HP, the type of impellers, etc. It would contain instructions as to how to carry out each operation. For example, how to check the mechanical condition of the fermenter, how to see that the instruments and other equipment were in good order, and how to make it safe for engineers to work on it. Instructions should be supplied on the procedure for batching and filling up a fermenter (but not the details of medium composition), how to sterilize the vessel, cool it, and inoculate it, how to run the fermentation, what to record, how to operate automatic nutrient supply to the culture, how to sample, sterilize, and use antifoam pots, how to terminate an experiment either by harvesting or by sterilizing and putting a batch to drain. Finally, how to work and clean the apparatus ready for the next run. These instructions should also draw attention to safety hazards and clearly set out what to do in case of emergencies. Generally speaking, this is the invariable part of any establishment, and the instructions are not often referred to in detail. However, they are most valuable in checking for unorthodox and corner-cutting activities, especially by the “old hands.” For instance, mysterious epidemics of contaminated fermentations have been cleared up by carefully going back to the standard procedures. However, although such information is not often carried up the line, it is most valuable when one is comparingprocesses fi-om different establishments, in particular those that one has licensed from outside companies. As this type of document is often not seen outside the section using it, some pressure is often necessary to get it written up adequately and competently. The absence of such documents, moreover, reflects adversely on the managerial staff.

VII. The Standard Operating Procedure This vehicle is most important in research into process development, although it has also a considerable value in any activity that is based on a standard set of procedures (for instance, the isolation and comparison of mutant strains). What are the functions of standard operating procedures (SOPS)?They are as follows: (1)to establish a baseline process or set of methods against which progress can be measured; (2) to establish comparability between different scales of experimental equipment (whether scaled down or scaled up); (3) to record more advanced, more flexible or merely different processes in a regularized standard way so as to permit of repetition by others; (4) to crystallize new standard processes; (5) to provide the hllest data for translation

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onto the plane (6) to provide exact process information for other investigatory groups. Such a document should answer all reasonable procedural questions. The minimum should be left to chance or to the judgment and memory of individuals. One “skilled in the art” should, when armed with an SOP, the necessary organisms, equipment, and staff, be able to reproduce the whole system and obtain the expected results, yields, and behavior. At this point it will be as well to bring the argument down from the realms of abstraction by giving an example. Suppose we are concerned with improving J fermentation process not yet carried out in our own area. The data will be supplied from another group inside or outside the Company. The first thing to be done is to write a draft SOP. This is the first approximation to a process. The details at our disposal are made the basis for scaling up or down onto the corresponding pieces of plant at our disposal. Any gaps in the information are filled arbitrarily or on the basis of experience with other processes. A check list is a good idea at this stage, especially when processes are being licensed from outside companies who have arranged only a limited number of technical visits. The following groups of information are needed: 1. The physical equipment for each of the process stages from the preparation and storage of the master cultures to the final antibiotic production stage. This will include the type of ampoule for lyophilized seed or the soil flask required; the slant tubes or medical flat bottles for growth of the solid media stage; the types of flask and shaker used for submerged spore or vegetative inoculum preparation; the stirred vessels used for the plant or pilot plant stages of inoculum preparation and final production. Vessels should be specified in their entirety-dimensions, baffles, agitators, cooling systems, motors, instruments, air filters, materials of construction, samplers, addition kits, etc. 2. The media employed in each stage will be defined. Exact recipes for their preparation will be given. Generally, composition will be given by percentage together with the actual weights of each material used. This redundancy is deliberate and gives a useful check. In particular, it prevents scaling errors that are easy to make when several sizes of vessels are on the go at the same time. The specifications of all materials should be recordedsource, purity, physical state, etc. Notes are made on the volume changes occurring in the medium preparation, in particular, the amounts of condensate to be allowed for. In large-scale processes most media are described in terms of composition before adding the inoculum. The composition and preparation of nutrient feed are also noted. 3. The ancillary apparatus, feed pumps, shot counters, thermometers, pressure gauges, etc., are specified until this information has become a standard part of the EOP.

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4. Control criteria are laid down. The temperature, pressure, aidlow, agitation rate, power consumption, and rates of nutrient addition are specified for the appropriate periods. Bulk additions of nutrients are scheduled. The sampling routines are established. 5. Standards of acceptability are established for inocula, especially in respect to freedom from contamination. Control routines, such as the addition of sugar at varying rates according to the pH value of the culture, are clearly set out. Any further control observations and instructions are also stated when possible with appropriate explanation. Complete of such a list can define a process with considerable accuracy. Even so, there will be gaps. Furthermore, skill in the art leads us to make use of such a check list with a sense of proportion, balance, or aesthetics. With experience, one comes to recognize what are the variables most likely to have profound effects and to which is the process relatively insensitive or tolerant of variation. However, one should not try to cut corners. When the draft SOP has been followed experimentally and adjusted to give a fermentation that is an adequate base for further development, the process should be written up as in a standard operating procedure (SOP). This should incorporate information from the actual experiments. It should include a graph of the fermentation, filled out as amply as possible. That is, one should indicate changes in the levels of important broth constituents, especially the desired product, the rates of nutrient addition, and such values as pH. The standard operating procedure should be rewritten (a) when the changes made in a process, and their effects on the control instructions, are so many that brief specification becomes impossible; (b) when the performance of the modified process is significantly greater than the original; and (c) when an entirely different kind or pattern of fermentation (e.g., chemically defined medium as opposed to complex) has been evolved, whatever the yield level. The SOP should be written for all scales, including the production units, thus giving bases for introducing modifications into the plant or for testing possible solutions to plant problems in the laboratory. SOPS are especially useful, if adequately maintained over the years, for effecting rapid scale-up to the main production unit and scale-down from it for the purpose of trying radical innovations or testing materials. This is a rather lengthy example, dealing with a particular aspect of fermentation research, albeit an important one. However, similar situations exist outside the semitechnical area. In the laboratories, for example, standardized procedures are highly desirable for testing newly isolated organisms from mutation programs. In fact, the programs themselves may need formal recording. Indeed, when complicated routines are necessary, standard operating

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procedures are essential: first of all, in seeing that the baseline performance is maintained and that we are still using the same baseline as a standard of comparison; second, as a means of achieving standard performance after interruption in program, migration of staff, etc.; third, in providing a line of retreat when a particular set of investigations has come to a dead end. Fourth, in supplying a standard procedure against which true novelty can be recognized. (As an extension of the last thought, the existence of well written processes enables us to judge when an idea has novelty and when it is simply circular verbiage.) Fifth, the SOPs are milestones showing progress or lack of it. (As such they may not be wholly popular with development staff, for the nature of things dictates that, in general, novelty and revolutionary improvements occur less fiequently as products become older and older. Diminishing returns are very much a fact of life.) Sixth and finally, the existence of a series of fully documented descriptions of processes provides a skeleton scheme that can be used for teaching the background and history of the processes in current use. This is especially useful in the instruction of newcomers to the field. In addition, some of the older processes may be used as starting points for different lines of development as they may be more suitable for the introduction of new techniques thaR later and more elaborate methods. Together with the collected series of monthly reports and the special project reports, the SOPs thus constitute an archive of the collective enterprise of the R & D Department. Before leaving this topic, one should stress the value of a formal plan and uniform presentation for SOPs. This not only Ezcilitates storage, retrieval, and use, but also helps to ensure that essential information is not omitted because it is taken for granted by the authors. A formal plan that fits a number of processes also saves time in the collection of facts and the writing of the procedures. Last, as the information is so complete and compactly presented, security measures must be adequate to prevent it falling into the wrong hands. Each copy should be accounted for and, when not actually in use, it should be kept under lock and key.

VIII. Direct Experimental Records The aim of the systems described below is to record all experimental information as it arises. For experimental fermentation the following series is usual: (1)experimental instructions; (2) laboratory notebooks; (3)project job packs; (4) pilot plant experimental records; (5) analytical and control laboratory records; (6) computer-stored data; (7) plant graphs. However, different types of investigation may require some modification of this list.

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A. EXPERIMENTAL INSTRUCTIONS These are particularly relevant to experimental fermentation work for which crews of operators need standard instructions on how to carry out each run. They comprise the batching sheet, which says how the experimental media shall be formulated and prepared, and the experiment report, which fills in the rest of the necessary details. Both documents are set out on standard forms especially designed for the purpose. While the experiment is in progress, further and supplementary messages are communicated by way of the shift logbook which also records any remarkable incident that occurs during each shift.

B. LABORATORY NOTEBOOKS All experimenters keep personal records of a kind. The majority of laboratories try to ensure that such records are complete and accurate, and that the compilation of such records is adequate. To this end, such records are more or less regularly inspected and expected to meet a set of common standards. Often, the notebooks form part of a numbered series, the issue of which is recorded centrally. Their surrender on the departure of their owner from the company should be mandatory. Ideally, each experiment should be written up as a unit. The notebook should set out the experimental aims and the results insofar as they meet these aims. Appropriate comparisons with other work should be drawn. All experimental details should be recorded. For instance, individual weighings should be written into the notebook rather than listing them on scraps of paper, filter papers, or cigarette packets, and then recording only averages. The notes should include calculations, comment, and any observations apparently unrelated to the main theme. The latter may turn out later to be pertinent when the background knowledge has increased sufficiently. The work should be cross-referenced with other projects, with the analytical records, and, if pertinent, with the pilot plant record files. It is important that the work be dated. (This applies also to every piece of paper in the pilot plant and analytical records.) As the notebook is the most crucial document source when making application for patents, the importance of dating it cannot be overrated. Y e t constant vigilance is necessary to ensure that workers do in fact date their work. In this context, should a notebook be passed on to another experimenter or group, the work notes should also be signed on transfer. At this point, perhaps, “ownership” may be discussed. As mentioned, most industrial concerns issue notebooks centrally and consider them to be the property of the Company. They are intended to be returned to a central

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depot, say, the library, when the “owner” moves to another activity within the organization or migrates. However, most people regard their notebooks as personal property and are loath to do this. In fact, considerable pressures often have to be exerted to get people to relinquish them. In some cases, a dual system is practised: “one for blow and one for show.” This can be tolerated to a degree so long as the data are available, adequate for repetition of the work, and valid as a supporting document for patent purposes. Many of the laboratory notebook‘s functions are covered by the analytical and other records, the SOPS, and the Progress Reports. Certainly the information there is often more usable. Most notebook descriptions, without the prop of the originator’s memory, are most unsatisfactory recipes for experimentation. Again, because the notebook is so personal to its “owner,” it is not likely to be overintelligible to another reader. The communication may be obscure and abbreviated, and omit much of the technology that the originator takes for granted. For these reasons, it is especially important to insist upon a high standard. New processes should be written up in detail, regardless of apparent redundancy, so that other people can follow them. To some extent the SOP method is a substitute but, as we have implied elsewhere, it is suitable only for process descriptions. Much laboratory work cannot be written up in SOP format. Summarizing, the improvement of standards of notebook keeping may be directed toward two goals. First, the records should be as complete and accurate as possible. Second, their degree of communication should be adequate. The first goal can be effected reasonably well when the worker is comparatively junior and if the current standards of his contemporaries are high. With more senior staff, dignity can be wounded and resentments arise if completion or accuracy is called into question. The second goal is more difficult. Human motivation is generally at odds with the idea of giving someone else’s career a leg-up at the possible expense of one’s own. This is a problem of general management and depends on adequate recognition of worth and the development of a good team spirit.

C. PROGRAMJOB PACKS An alternative to individual notebooks is the Progress Job Pack. Here a file, or communal notebook, is started for each project accepted by the manager. All documents, results, and notes pertaining to this are collected centrally and the whole pack is centralized with the Project Leader. There would be a reference number for each project or series of experiments. Each document or write-up would be accompanied by the product

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code, the experimental name, the date and the experiment number, and would provide adequate cross-referencing for patent purposes. There are difficulties. The collection of heteromorphous, loose-leaf material can lead to confusion, or loss of some documents. It might also lead to a weaker position in regard to patent procedure, especially in terms of dating for the serial writing-up of one experiment after another, in a conventional notebook, adds confirmation that the work is recorded properly in this respect. For this reason alone, a notebook for the project would be essential. But that in turn would lead to complications if many people were recording data in different individual series of experiments at the same time. The job-pack system, however, has something in common with the compilation of pilot plant records. It has advantages in that the information is all in one place. The necessary sorting out also makes for completeness and ease of retrieval.

D. PILOTPLANTRECORDS This method of generating and storing information may well not be of universal application. As work on the semitechnical scale is, however, a large contributor to biological research and because lessons may be learned from how it is run, I shall treat it rather extensively. My example is the record-keeping procedures of a moderate-sized pilot plant with large and small vessels. The particulars of each fermentation carried out in the larger fermenters (say 500-liter) and of each group of 3, 6, or 12 fermentations in the small units (5- or 50-liter) are collected together in a cardboard folder. The standard documents are (1)the project request form, (2) the batching sheet, (3)the inoculum preparation sheet, (4) and (5) the running-log sheets for inoculum and for the final stage, (6) and (7) the graphs for inoculum and final stages. The layout of these standard documents is most important. Our current form is the result of evolutionary modification over years of practice. Thus the very topology of these documents gives a general idea of how the design and scope of the processes specified therein have changed over the ages. The pro formas, then, are so designed that the practical details can be written in clearly and unambiguously without unsuspected omission. Starting from the basis of an existing SOP, the details of experimental variations are decided upon. Once clear on this, the project request form may be filled out. This refers back to the SOP and indicates the purpose of the experiment and the detailed experimental operating instructions to be substituted while retaining the SOP instructions in the control. The date is

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specified also for the beginning of the experiment so that it may be fitted into a schedule. These instructions are then used by the foreman or technician in making out detailed batching sheets in which the weights of materials to be taken, the medium-making and the sterilization procedures are specified in detail for the process workers who will carry out the operations. So, similarly, are designated the samples to be taken and their destination. The inoculation details are also filled in as they become available. The running sheets are issued together with instructions as to how to control the fermentation and what supplementation is necessary. The instructions are written in a general way on the batching sheet and are particularized by explicatory and amplifying notes in the shift log. (The shift log is the process operator’s communal log book: in it are recorded nonroutine occurrences, special instructions from the foremen and messages from one shift team to those following it.) When the fermentation is under way, certain observations, such as temperature, pressure, airflow, and current consumption, are recorded on a running log. Others of a less routine nature, but more important because they represent the substance of the experiment, are plotted on a graph specially marked out for the purpose with check marks for additions and target lines for the control of such variables as the pH value. The various documents thus described have been designed over the years to act, as far as possible, in the way of check lists. Ingredients have to be ticked off as weighed and as batched: the operations of sterilization have to be carried out exactly and in the right order. The particulars and history of inocula must be recorded. The operations governing the actual fermentation stages are subject to similar checking. Most of the information on the summary sheets tells us that the physical conditions and mechanical state of the equipment are in order-for example, that the temperature controls are working. Most of all, however, it ensures that the operators visit the particular fermentation units as often and for as long as they should and that they have checked that all is running as it ought. Even with automatic recorders the practice ofpilot plant recording is worth keeping up for these reasons. The data-unless the plant is misbehaving-are mostly trivial, however, and may be discarded at the termination of a run, together with the paper charts from the temperature and airflow recorders. On the other hand, analytical data such as pH, sugar level, ammonium concentration, antibiotic titers, are what we want to examine in detail. They are recorded on the graphs, or copied into the experimenter’s notebook. Most of the analytical results are also stored in the analytical departments that issued them. Some figures that are a check on normality are not transferred; if subsequently needed, they can be retrieved.

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E. ANALYTICALRECORDS When analyses are carried out by service departments, the results are entered into records, primarily for the purpose of any subsequent checking of consistency, standard error, unexpected results, and miscalculations. Often, the figures are centralized in books wherefrom the figures can be collected by the “clients.” Many of the results are regarded as indication of normalcy, smoothness of control, experimental competence, etc., and, provided they meet certain criteria, are not carried forward with the rest of the information. The analytical records thus provide the store for this kind of information and permit its retrieval when needed. One can see that considerable doubt might arise as to whether all such analyses should be made, for they all cost money. Certainly, routine plant fermentations, say, require less “redundant” analyses than do experimental batches. I have so far dealt with situations in which the information is produced in long sequences of the same routine type. By and large, the doing of “one-off’ analyses is rather different. Here the retrieval is much more difficult; it cannot be identified by batch number, date, and product, as can fermentations, because it is too individual. Rapid transfer of the data to the experimenter for immediate recording in his notebook or elsewhere is therefore mandatory for the hture recovery of “one-off’ data. An independent record should be kept of such special commissions.

F. COMPUTER STORAGE The digital computer is a grand thing for storing data. Its capital cost is, however, very great. Apart from high capacity and near-immediate recall, it offers very little advantage over human recording. In fact, it may tempt us to store too many raw data rather than process them into manageable size and shape. The computer has considerable advantages when the data are collected on line. Where direct instrumental coupling is not possible, and the reading and copying of data is a necessary preliminary to feeding them into the computer, the proposition is not so attractive. (In the normal way, a considerable amount of data reduction is effected simultaneously with these operations.) Automatic data acquisition on line has been described by George (1973) (see Fig. 2). The system is very good for a highly stereotyped and long series of investigations. Chemical processing might be very well suited by such an approach. When the science is less reductionist, and more concerned with whole organisms or complex systems, the computer method is more difficult of application. Biological work varies not only in form, but in content, and a

152 Signal-Acquisition

D. J. D. HOCKENHULL Information storage -and r e t r i e v a l s y s t e m ~ A ” a l y s i s

-Processing

2. Desk calculator

1. Remote terminal

1. Card punch

3. Magnetic tape 4. Magnetic disk

1. Digital

1. Card reader

1. Tables

3. Magnetic tape reader 4. Magnetic disk

1. Filing

cabinet 2. Computer memory 3. hoseleaf notehok

1. Manual

Statistical 2. Computer

statistical 3. lnhlitive

FIG. 2. Six basic steps used by data systems, automated or manual:(1) signal generation, (2) acquisition, (3) processing, (4) storage and retrieval, (5) analysis, (6) report. From George (1973). Reprinted with permission from Chemical Engineering, December 10, 1973, p. 123.

series of experiments are likely to change in general pattern and setup through the program. Additionally, experiments with living material give rise to nonquantitative observations. Many physiological and topological features have to be noted. True, some of these could be coded and systematized in a numerical system, but great expense would be involved in working out how to do this. For some purposes, then, descriptive techniques have it over the computer. Until such time as programs can be written to deal with such inexact data, we shall have to carry on in the old way. However, computers have so much room for storage of data that they are uniquely fitted to deal with projects that generate large numbers of instrumental readings. George’s article says all that need be said on this topic as far as the current thesis is concerned. G. PLANTGRAPHS Most experimental fermentations are recorded graphically while in progress. The main function of these graphs is to provide visual opportunity for ongoing metabolic control of the processes. They also provide eidetic records of the process. The graphical representation enables one to see the finished fermentation as a gestalt, and gives yet another set of criteria for comparison. The inclusion of a graph (sometimes idealized or smoothed) into SOPS is usual.

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IX. Miscellaneous Information by Memoranda A good deal of information is transmitted in the form of memoranda. These may be directed either at individuals or at committees. They are properly used for the following functions: (1)drawing attention to the existence of information; (2) answering specific technical questions; (3) presenting data for specific purposes (e.g., working out the cost of a new process); (4) giving opinions. In general, memoranda should never be allowed to replace the proper production of formal reports, but they may summarize facts therein and, if necessary, give opinions and judgments. Generally, it may be said that memoranda are useful but unreliable instruments. This is because the standards applied to issuing memoranda are largely at the discretion of the individual producing them. The data are condensed, and this practice often conceals statistical shortcomings. Generalizations may be made on inadequate grounds, and personal opinions and prejudices may color the conclusions. However, with all these disadvantages, memoranda are necessary for adequate communication-and certainly they are more effective than the telephone. Their usefulness depends on the standard of professional integrity demanded. Swift retribution should descend on the perpetrator of misleading or unsound information. X. Minutes In most R & D situations, regular meetings are held, usually every month. These meetings are generally of two kinds. The one category deals with the organization and execution of the work of the department and is aimed internally. The other involves the “customer” departments and is aimed at reporting progress, adjusting the effort to the needs of the plant, and setting forth proposals for future work and for the transfer of pilot results to plant scale. In addition, decisions are sought, delays explained, and misunderstandings ironed out. Both categories of meeting have a great deal in common. Their main advantages over written communication are confrontation, dialogue, and multiple participation. Information can be transmitted rapidly with instant feedback, not only up and down the line but across it, that is, between departments or units. New ideas may be introduced earlier than a formal communication would permit, so that the benefit of a wide collective experience can be brought to bear on them. This at times might constitute a “think tank’ for the development of new ideas. Certainly, specific actions and joint programs of work may be agreed to fulfill the needs of the customer department.

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It is important, therefore, that technical meetings should be adequately and accurately minuted. Not only do the minutes act as a present aid to management; if properly filed they constitute an important historical archive and help in the retrieval of important information. This is especially true because they will, if adequate and accurate, record not only experimental facts but the ideas current at the time, which may not be reported otherwise.

XI. Collection and Flow of Information There are no hard and fast rules about collecting and projecting information. The informational network is the nervous system of the organization. Therefore, the processing and transfer of information is measured by the criteria of how effectively and how quickly it can reach the place where it is needed. We sort data into what is worth keeping and what not. The information arising from this process is either stored or used immediately. Of that used immediately, some is also stored. The new data are compared with information already in store, and the resultant interactions generate new relationships of a more refined type. We have listed the various devices for storage and communication already. Figure 3 gives an idea of how such information stores are related, and how information from a single series of activities may be stored in them. Note that some redundancy is always present: some data may be stored in more than one place. Such redundancy is necessarily present because one

P i l o t P l a n t Records 1 . Request f o r m

1. Equipment SOP

2 . Batching sheet

2. P r o c e s s SOP

3. Running log

3. Weekly r e p o r t s

4. Fermentation g r a p h s

4. Monthly r e p o r t s

5. P r o j e c t r e p o r t s

Experimenter

1

Control Laboratory

1. Laboratory notebook

1. Analytical r e s u l t s

2. P r o j e c t book

2. Sterility data

LJ FIG.3. Filling and deposition of information.

I

INFORMATION CONTROL IN FERMENTATION DEVELOPMENT

155

may need summarized and condensed information, on the one hand, but also, on the other, additional related details for quite different purposes. The

same information may also be expressed in differing ways in the different reports. Figure 4 illustrates the flow pattern from an experimental series. It shows in a very simplified outline what happens to data. It does not, however, show everything that happens to them or every purpose for which they are used. Nor does it trace the transfer of data in detail. Figure 5 amplifies this picture by introducing information from other experiments. In a simple way, it traces information from the experimental interface to the financial one. Synthesis of a new process is very much like an inversion of Fig. 4. Information is brought together &om the following information stores: (1)the equipment operating procedure (Plant Manual), (2) the standard operating procedure, (3)the experiment (project) request sheet, (4) the batching sheet, (5) the collected analytical results (experimental notebook, analytical records), (6) the plant graph; (7) miscellaneous information (vessel observation, shift log, etc.). Although category 7 consists mainly of subjective and descriptive matter, it throws light on the interpretation of the “harder” information. It also

P r o c e s s operatives

Control laboratory

Experimenter

luct

Svecial obsei rations

constituents

Power Running log -

- - - - - - -- ---- -- - - --

I

: Total range of

Sugar, etc.

i information f

P r o c e s s graph

a f t e r com-

I

Product

1 I

I parison

---

Experimental notebook

t -

Total range of information a f t e r comparison of experimental units

New SOPS

FIG. 4. Generation and transfer of information from unit experiments. Continuous lines denote complete transfer of information; discontinuous lines denote selectedabstracted information.

156

D. J. D. HOCKENHULL

Instrument

Analytical data f r o m periodic sampling

I

t

Analytical r e c o r d s

-

1

Statement of intent Experimental findings Supporting evidence

Experimental s u m m a r y in monthly r e p o r t p r o j e c t r e p o r t s in monthly report

Comparison and collation with other experiments

New SOP/or project report

provides an additional set of criteria to ensure that the system behaves as intended. Further, it contributes to what we call process sense or knowing the “feel” of the system. Figure 6 gives a rough idea of the way in which this information is processed in order to attain a new standard process. These exercises depend on the proper functioning of the reporting processing, and storage vehicles available. While the needs of the whole organization, as discussed earlier, influence the total apparatus, the uses in this context greatly influence its structure. This is especially so in the way in which the requirements for differently processed information adapt the physical constraints of the system components. Thus the channel size is modified to deal with the signal frequency. And what is desirable is adjusted to conform with what is possible.

XII. Making the Best Use of Data Data cost money, but only when processed into information do they become useful. Effective processing and storage help us to get the most out of

INFORMATION CONTROL IN FERMENTATION DEVELOPMENT Imposed variables

Experimental data

Reduction (filtering)

157

Standard conditions

Equipment operating procedure Standard operating procedure

Novelty

Reduction (filtering)

Systematization into c u r r e n t technology

Organization Summarization Elimination of experimental error

-

Proposal for new process

-

Comparison with controls

Augmentation

Results of other experiments. p r o g r a m s Additional calculations Cost information

Synthesis (augmentation

C r i t e r i a f o r new p r o c e s s

Augmentation (synthesis)

Information r e t r i e v a l

New p r o c e s s

FIG. 6. Informational aspects of developing a new process.

the data in hand. In addition, however, if the vehicles I have mentioned are used in the right way, they can discipline our thoughts and activities so that we can cut down our generation of new data and substantially reduce the costs of each investigation. One man’s information is another man’s noise. For example, many of the readings one makes during an experiment serve only to indicate that the standard control conditions are being met. Such information may be left at low level, i.e., on the charts of the recording instruments, or its generation

158

D. J. D. HOCKENHULL

may be avoided by the possession of automatic control instruments of absolute reliability. Ashby (1961) has said that one requires to generate variety in order to destroy variety (cf. Beer (1966). That costs money: we must see that it is well spent. The information must be worth processing; i.e., the experiments must be carried out competently. Equally, it must be properly organized and designed to illustrate definite points. It is both difficult and expensive to collect useful information on one topic from an experiment designed to generate information on another. In particular, the analysis of variance carried out on data generated by random variation in incompletely controlled systems is a particularly expensive way of progressing. An adequate information system will avoid such excesses and, ultimately, lead to economies in planning and execution. Furthermore, the grouping, processing, and recording of data in an orderly way means that the Organization as a whole knows where to find them. This may be unflattering to many egos, because achievements or their absence are then often painfully clear. But when error is admitted, redemption has begun. For the Development Director or Manager to know at once that a line is not going well is more important than to let awareness creep upon him. It is not always good policy to kill the bearers of ill tidings. Success speaks for itself; rapid feedback of a failure may ensure that the boss can change from an unprofitable venture before he has become irretrievably committed. Also, there are many situations in which the acquisition of further information for the purpose of making a decision becomes too expensive in time and cost. Good reporting enables management to realize readily when the point is reached for a decision to be made on the available facts. As we have said before, data cost money. If we can process data into useful information, we may get some of the money back. Hopefully, we may even show a profit. But some may say that organizing a written information service wastes paper and the time spent on filling it. This opinion has more support than is generally realized because by producing the right documentation the individual most often does more for other people than he does for himself. Only collectively is the benefit achieved. However, it is perhaps true to say that if one does not waste time on paper work one has twice the time to waste on doing experiments that one should not have done anyway. This is a man-management problem. A further point is that the informational structure must be appropriate to the needs of the activities carried on. The aims of the organization are always changing. The nature of the investigations changes. New working groups evolve; old ones decay. The needs of the different departments also change and, with this change, so do their informational needs. Managers must be prepared to change their ideas, and to change them back if they do not work-a much harder thing to do.

INFORMATION CONTROL IN FERMENTATION DEVELOPMENT

159

This goes especially for the information setup. The decisions as to what form it should take should evolve &om the needs of the group. What is most practical? What can we manage to run efficiently? How usehl will it be? What will it cost? Choosing an attractive means-say a computerized memory bank (wherefore art thou, Romeo?), without due reference to the ends it has to serve is only too easy. One has to judge a system not on what it will do, but on how well it satisfies one’s needs. If one wishes to store and retrieve ten readings per hour it may well be quite irrelevant and extravagant to use a system that will handle ten thousand. In short, the user should produce his own specifications if he wants the right job to be done. However, the manager must manage flexibly. H e must be prepared to see different problems solved in different ways in different work groups. There must be sufficient regional autonomy to deal with the kind of work done and the kind of people doing it. One cannot fit every activity on to a common procrustean information system. The manager must keep his sources of information under constant review. His decisions can be no better than the information reaching him. H e must know how far he can rely on it. He must watch out for distortion resulting either from the constraints of the system (e.g., oversimplification) or from personal bias. H e must maintain as much redundancy as will enable him to check the system’s reliability and decrease the error component to a safe level. A little time spent on the mechanics of information flow will save a good deal of time otherwise occupied in putting right the actions taken on bad grounds. Finally, informational systems are only as good as the men who operate them. The motivation and management of the individuals in each area who hopefully participate therein are, in the last analysis, the most important features of an R & D manager’s life. REFERENCES Ashhy, W. R. (1961). “An Introduction to Cybernetics,” Chapters 7 and 11. Chapman & Hall, London. Beer, S. (1966).“Decision and Control,” p. 277 seq. Wiley, New York. Edwards, E . , and Lees, F. P. (1973). “Man and Computer in Process Control,” p. 20. Inst. Chern. Eng., London. Gellerman, S. (1973). Management Today Casebook 2, p. 97, 98. George, T. J. (1973). Chem. Eng. ( N . Y . ) 80, 123. MacFarlane, A. G . J. (1964). “Engineering Systems Analysis,” p. 1. 6. Harrap, London. Miller, J. G. (1960). Am. J. Psychiatry 116, 695.

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Single-Cell Protein Production by Photosynthetic Bacteria R. H. SHIPMAN,L. T. FAN,AND I. C . KAol Department of Chemical Engineering, Kansas State University, Manhattan, Kansas I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Process Considerations ....... 111. Conceptual Design ..................................... IV. Economic Analysis ........................... A. Capital Investm ........................... B. Annual Operating Costs ............................. C. Total Production Cost and Profitability . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

161 166 172 176 176 178 179 181

I. Introduction Photosynthesis is one of the basic biochemical processes, in which plants, algae, and specialized populations of bacteria convert the energy from sunlight or solar energy into chemical energy for cellular biosynthesis. Man has used this natural process of harnessing solar energy in the development of algal cultivation systems for secondary waste treatment and for the production of human foods, livestock feeds, and fertilizers. Although previous investigators (Hirayama, 1968; Wai, 1971, 1972; Thanii and Simard, 1973; Shipman, 1974a; Shipman et al., 1975) have suggested that photosynthetic bacteria may be a potential feed source for aquaculture systems or livestock, processes for mass-culturing photosynthetic bacteria as a source of human food are undeveloped at this time. Photosynthesis, as it occurs in plants and algae, differs from that of photosynthetic bacteria in several respects as diagrammed in Fig. 1. Plant photosynthesis is basically an aerobic process in which CO, is used as the sole carbon source for cellular biosynthesis. Plants and algae contain different photosynthetic pigments, or chlorophylls, and are capable of hydrolyzing water to derive reducing power (H+)for biosynthesis and molecular oxygen. Bacterial photosyntheses are anaerobic processes in which molecular hydrogen, reduced sulfur compounds, and organic compounds are used as exogenous electron donors. Bacteriochlorophylls, the photosynthetic pigments of photosynthetic bacteria, play a role similar to that of algal chlorophylls. Some photosynthetic bacteria, like plants, may use COz as the sole carbon source for cellular synthesis; however, the overall process requires an inorganic chemical reductant, such as H, or H,S. Other photosynthetic bacteria use light energy for the conversion of organic compounds to cell material. Water is never used as the ultimate electron donor; this 'Biochemical Development Division, Eli Lilly and Company, Indianapolis, Indiana.

161

162

R. H. SHIPMAN, L. T. FAN, AND I. C.

KAO

Green plants and algae

Photosynthetic bacteria

Light

Light

Chlorophyll

0 2

organic compounds NADPH

+ FIG. 1. Comparison of plant and bacterial photosynthesis. Adapted from Pelzar and Reid (1965).

explains why molecular oxygen is never produced as an end product of bacterial photosynthesis (Rose, 1968).The basic photosynthetic reactions of algae and bacteria are depicted in Table I. The pigment system of photosynthetic bacteria plays an important lightgathering function in the assimilation of light energy. This pigment system consists of various bacteriochlorophylls, which differ in absorption spectra, chemicaI composition, and structure, and a number of identical or closely related carotenoid pigments. Bacteriochlorophyll excitation occurs when a bacteriochlorophyll molecule absorbs a quantum of light resulting in the loss of an electron, i.e., BChl -L@L BChl+

+ e-

The released electron then migrates through the photosynthetic unit to transfer its energy to a special reaction-center chlorophyll. In photosynthetic bacteria, electrons flow through a series of intermediates in a specialized transport system, depicted in Fig. 2, with electrons migrating through an unknown intermediate, to ferredoxin, ubiquinone, cytochrome b, and cytochromef terminating with the return of BChl+ to a “ground state.” In the intermediate step between cytochromes b andf, synthesis of adenosine triphosphate (ATP) occurs. This cyclic process, by which solar energy is biotransformed into cellular chemical energy, is designated “cyclic photophosphorylation.” Carotenoid pigments are important participants in the photosynthetic process, where they function primarily as light-harvesting pigments responsible for transmitting absorbed light energy to bacteriochlorophyll. The ef-

163

SINGLE-CELL PROTEIN PRODUCTION BY PHOTOSYNTHETIC BACTERIA

TABLE I BASICPHOTOSYNTHETIC REACTIONSOF PLANTSAND MICROORGANISMS~

I.

Higher plants and unicellular algae solar energy

COZ

+ H20

(CHZO) +

____)

0 2

chlorophyU

biomass

11. Certain algae and bacteria

solar a e r g y

CO,

+ 2H,

(CHZO)

+ HZO

(CH,O)

+ S + H,O

chlorophyll or

bacteriochlomphyll

111.

Photosynthetic sulfur bacteria solar energy

CO,

+ H,S

____2)

bacteriachlorophyll

IV. Purple photosynthetic bacteria solar energy

CO,

+ CHO + HZO orgaluc

(CHZO) + Hz

-w

+ HzO

bacteriachlorophyll

material ~~

~

~

-~

=Adapted from Stanier et ul. (1970).

fectivenessof energy transfer from the carotenoids to the bacteriochlorophyll has been estimated from 3&50% up to 90% in certain species of Rhodopseudomows (Sybesma, 1970). Carotenoid pigments also have an important function in phototaxis by purple photosynthetic bacteria (Clayton, 1953). Finally, a more fundamental function of carotenoid pigments is the protection of light-sensitive bacteriochlorophylls from the harmful photooxidizing effects of bright light. Photosynthetic bacteria are widespread in nature and have been encountered in almost every body of water and in the soil. They are especially abundant in stagnant waters containing decaying organic matter or other organic substances. In lakes and ponds, masses of photosynthetic bacteria frequently develop as stratified layers between water layers containing oxygen and those layers containing H2S. The development of photosynthetic

164

R. H. SHIPMAN, L. T. FAN, AN D I . C. KAO

*_--

__---

,/-

,'

,' Unknown ' Intermediate I I

/

: H h e d o x in I

fI I

I

Ubiquinone I

1

Cy!ochrome b

'. 0-

t

,

I

\\

Cyifchrome f

\

Light at 880 nrn (far red)

'.

FIG. 2. Cyclic photophosphorylation in photosynthetic bacteria. Adapted from Brock (1970).

bacteria depends, as a rule, on the presence of light, the concentration of H2S, and adequate anaerobic conditions (Kondrat'eva, 1965). Photosynthetic bacteria are represented by a rather large number of species differing in morphology, pigmentation, physiological, and biochemical properties. Some of the important physiological, biochemical, and morphological properties of the three major groups of photosynthetic bacteria are shown in Table 11. The purple sulfur bacteria, Thiorhodaceae, are strict anaerobes, photoautotrophic, and are able to oxidize H2S and other organic compounds. Purple sulhr bacteria accumulate droplets of elemental sulfur within their cytoplasm during the initial step of H2S oxidation. The intracytoplasmic sulfur deposits disappear in the second step with the oxidation of sulfur to sulfates and the disappearance of H2S from the cellular environment. The green sulfur bacteria, Chlorobacteriaceae, are easily distinguished fiom the

TABLE I1 MORPHOLOGICAL, PHYSIOLOGICAL, AND BIOCHEMICAL PROPERTIES OF OF PHOTOSYNTHETIC BACTERIA'

THE

THREE MAJORFAMILIES

Green sulfur bacteria (Chlorobacteriaceae)

Purple sulfur bacteria (Thiorhodaceae)

Nonsulfur purple bacteria (Athiorhodaceae)

Major pigment system

BChl c, abs. max. 747 nm BChl d, abs. max. 725 nm

BChl a, abs. max. 820 nm BChl b, abs. max. 1025 nm

BChl a, abs. max. 820 nm BChl b, abs. m a . 1025 nm

Cell morphology

Motile or nonmotile rods, cocci, pleomorphic; some with gas vacuoles

Motile or nonmotile rods, cocci, pleomorphic; some with gas vacuoles

Motile rods or spirals; some multiply by budding

Photosynthetic electron donors

H,S, thiosulfate, H, (organic compounds by some strains)

HzS, thiosulfate, H, (organic compounds by some strains)

HZrorganic compounds (HzS usually toxic)

Sulfur deposition

Always outside the cell

Usually inside the cell, except Ectothiorhodaceae

None

Aerobiosis

Obligate anaerobes

Obligate anaerobes

Facultative; grow in the dark aerobically, with photosynthetic growth anaerobically

Growth factor requirements

B12or none

Blz or none

Usually complex

DNA base composition (I G = C)

48-58

46-67

61-73

Property

~

"Adapted from Pfenning (1967).

A r

g C

3 6

m .c

I66

R. H. SHIPMAN, L. T. FAN, AND I. C. KAO

purple photosynthetic bacteria by their pigmentation. They are photoautotrophic, strictly anaerobic, and also capable of oxidizing H,S and other sulfur compounds. The purple nonsulfur bacteria, Athiorhodaceae, are photoheterotrophic bacteria which use organic compounds as electron donors and as carbon sources; they may also require specific factors for growth. Although these organisms can reduce CO,, they derive most of their cellular material from organic nutrients. A common organic substrate for photosynthesis by nonsulfur purple bacteria is acetic acid. Under anaerobic conditions in the light, almost 90% of the assimilated carbon in the organic substrate is converted into the intracellular reserve material, poly-P-hydroxybutyric acid while only some 10% is oxidized to CO, (Stanier, 1961). Certain species of the nonsulfur bacteria are facultatively aerobic and are capable of developing nonphotosynthetically in the absence of light (Van Niel, 1944). This work is concerned with examination of the technical and economic feasibility of the bacterial photosynthetic process for producing single-cell protein (SCP) from agricultural by-products and wastes.

It. Process Considetations The basic design parameters have been extrapolated from experimental data generated by the present authors (Shipman, 1974a; Shipman et al., 1975) and by Kobayashi et al. (1970). Additional parameters that must be considered in the photosynthetic design are: (1)nutrient source, (2) generation or doubling time, (3)effects of oxygen on pigment synthesis and photosynthetic growth, (4) pH, and (5) optimum temperature. Photosynthetic bacteria may be grown on media of known and simple composition. A number of experimental culture media have been described for the cultivation of photosynthetic bacteria (Hutner, 1950; Sistrom, 1960; Ormerod et al., 1961; Bose, 1963);however, various agricultural by-products may be substituted as an economical source of nutrients. Purple photosynthetic bacteria are frequently among predominating populations of microorganisms in ponds, ditches, and other water sources polluted by sewage and other types of organic matter. Thus cultivating photosynthetic bacteria on a mass-scale in food processing wastes or other agricultural by-products also appears to be a reasonable alternative. Previous studies (Wai, 1971, 1972)have indicated that photosynthetic bacteria may be profusely cultured in extracts of crude carbohydrates prepared from bananas, potato starch wastes, wheat bran, and rice bran. In this and other related works, the use of an infusion prepared from the acid-hydrolysis of wheat bran, containing primarily hydrolyzed wheat starch and proteins, is considered as the nutrient source for photosynthetic cultivation (Shipman, 1974a; Shipman et al., 1975).

SINGLE-CELL PROTEIN PRODUCTION BY PHOTOSYNTHETIC BACTERIA

167

The growth of purple photosynthetic bacteria has been reported on media containing ammonium salts, nitrates, urea, various amino acids, peptones, or yeast autolyzates as nitrogen and carbon sources (Van Niel, 1944; Gest and Kamen, 1949; Cohen-Bazire et al., 1957). In the presence of light and under absolute anaerobic conditions, purple photosynthetic bacteria are able to fix atmospheric nitrogen (Kamen and Gest, 1949). In an atmosphere exceeding 4% oxygen, nitrogen fixation may be completely suppressed (Pratt and Frenkel, 1959). Nitrogen fixation is inhibited by the presence of ammonium salts in the medium and is reversibly inhibited by molecular hydrogen in concentrations exceeding 60% (Gest and Kamen, 1949). Purple bacteria can fix molecular N2 both in organic and mineral media. Investigationsby Gest et al. (1950), have shown that N+S stimulates nitrogen fixation by Rhodospirillum rubrum grown on media containing organic compounds. Studies of nitrogen fixation in an atmosphere enriched with 15N have revealed that the distribution of this isotope in photosynthetic bacterial cells is similar to the distribution of I5N in the cells of Azotobacter. The largest amounts of intracellular I5N have been detected in the glutamic acid and the ammonium fractions (Wagenknecht and Burris, 1950). The vitamin requirements of photosynthetic bacteria may vary considerably. Except for vitamin BIZ, most purple sulfur bacteria do not require vitamins; thus, the supplementation of the culture medium with various vitamins does not result in stimulation of growth (Kondrat’eva, 1965). The addition of some B vitamins is required for the growth of typical representatives of nonsulfur purple bacteria (Hutner, 1944, 1950), and the specific requirement for a particular vitamin may be a distinguishing characteristic for certain bacterial strains, as shown in Table 111. Bacterial photosynthesis is basically an anaerobic process in which the presence of sunlight is an indispensable condition; however, under certain environmental or culture conditions, several species of photosynthetic bacteria may be grown aerobically. Several species of Rhodopseudomonas and Rhodospirillum are facultative aerobes and can grow in the presence of oxygen (Van Niel, 1944; Gest, 1951). All other representatives of the purple photosynthetic bacteria, including the nonsulfur purple bacteria, are obligate anaerobes. Purple nonsulfur bacteria that grow with organic substrates in the dark utilize a purely respiratory metabolism, using the Krebs cycle as a pathway of terminal substrate oxidation, while under anaerobic conditions in the light, an anaerobic light-dependent Krebs cycle is functional (Eisenberg, 1953; Kondrat’eva, 1965). Cultures of photosynthetic bacteria grow rapidly when they are illuminated under anaerobic conditions. In a study examining the effects of 0, on pigment synthesis (Lascelles and Wertlieb, 1971), the generation time of photosynthetic bacteria incubated anaerobically in the light has been found to be ofthe order of 2.5-3 hours, whereas generation times of mutant strains

168

R. H. SHIPMAN, L. T. FAN, AND I. C. KAO

TABLE I11 NONSULFURPURPLE BACTERIA"

VITAMIN REQUIREMENTS OF

Thiamine

Thiamine

+ +

Thiamine

biotin

nicotinic acid

-

-

-

-

-

+b

-

-

-

-

-

+

-

-

biotin

Biotin

p-Aminobenzoic acid

Rhodospirillum rubrum

+

Rhodopseudomonas palustris Rhodopseudomonas capsulatus

Microorganism

+

Rhodopseudomonas gelatinosa

-

-

-

+

-

Rhodopseudomonas spheroides

-

-

-

-

+

'Adapted from Hutner (1946). bFolic acid may be substituted for p-aminobenzoic acid (Hutner and Scher, 1961).

incubated aerobically in the dark varied from 6 to 10 hours. The atmospheric oxygen content is also an important factor in the synthesis and cellular content of the bacteriochlorophylls and carotenoid pigments. The bacteriochlorophyll concentration in purple photosynthetic bacteria normally varies from traces to 25% of the cellular dry weight (Lascelles, 1963). In addition to oxygen concentration, such parameters as light intensity and the relative age of the culture may influence the bacteriochlorophyll content. The greatest concentration of intracellular bacteriochlorophyll may be found during the exponential growth phase. An elevated oxygen tension exerts a unique control over pigment synthesis, as demonstrated by the inhibition of bacteriochlorophyll and carotenoid synthesis in cultures grown aerobically in the dark (Cohen-Bazire, 1963). Introduction of 0, into growing, illuminated cultures will also rapidly inhibit further pigment biosynthesis; however, when illuminated cultures are returned to anaerobic conditions, bacteriochlorophyll synthesis is restored. The repression of pigment synthesis by 4 rather than by an obligatory requirement for light has been suggested as a possible mechanism for the absence of bacteriochlorophyll in nonilluminated, aerobically grown cultures of photosynthetic bacteria (Lascelles, 1960). This theory is supported by the fict that if the atmospheric oxygen content is lowered to 1-6%, photosynthetic bacteria can synthesize bacteriochlorophyll and carotenoids in the dark. Oxygen markedly influences carotenoid pigment synthesis by photosynthetic bacterial cells. Carotenoid synthesis by photosynthetic cells culti-

SINGLE-CELL PROTEIN PRODUCTION BY PHOTOSYNTHETIC BACTERIA

169

vated in light and darkness is almost totally suppressed by vigorous aeration (Goodwin, 1955). Exposure to light also leads to quantitative changes in the cellular carotenoid content; as light intensity increases, carotenoid synthesis decreases. The exact mechanism by which photosynthetic bacteria control pigment biosynthesis is unknown. It may seem paradoxical that photosynthetic pigments, which are formed in the light, are found in greater concentration in bacterial cells grown in dim light than in cells grown in bright light. The ecological explanation for this phenomenon is that in dim light it is of advantage to the cell to have a large amount of light-gathering pigment in order that more of the light reaching the cell can be captured and transformed into chemical energy. When growing aerobically in the dark on organic compounds, pigment synthesis is repressed so that energy made available by oxidative reactions is not diverted into unnecessary materials. Photosynthetic bacteria frequently occur in natural habitats that are reached only by a small percentage of the solar radiation. Although the presence of light is an indispensable condition for photosynthetic growth, illumination is not optional for the bacteria in their natural habitats insofar as their light requirements are concerned. It is not that photosynthetic bacteria prefer environments of low light intensity, but rather that they have adapted to such environments. In nature, purple photosynthetic bacteria can develop under a layer of algae because of the differences in their absorption spectra. While photosynthesis in green plants and algae utilizes light waves mainly in the range of 700 nm, purple bacteria perform photosynthesis by absorbing light waves in the range of 800-900 nm (Kondrat’eva, 1965). Purple photosynthetic bacteria absorb light primarily in the infrared zone of the spectrum, and in some instances light filters (for wavelengths greater than 800 nm) may be used for selectively cultivating photosynthetic bacteria (Van Niel, 1944). Whenever possible, purple photosynthetic bacteria attempt to grow close to the surfice where they are often found at a depth of 20-50 cm (Kondrat’eva, 1965). Several investigators have examined the effects of light intensity on growth rate, cell production, pigment biosynthesis, and C 0 2 assimilation. In studies by Sistrom (1962), the effects of light intensity on growth rate and pigment synthesis have been examined (Fig. 3). The studies indicate that a decrease in light intensity not only results in an increase in pigment content, but also leads to a decrease in growth rate. The maximum specific growth rate has been reported to be approximately 0.75 doubling per 100 minutes, corresponding to a generation time of slightly greater than 2 hours. Other studies have revealed that light intensities below 7 x 103 erg.cm2/ sec are strongly inhibitory to photosynthetic bacterial growth in mineral media (Kondrat’eva, 1965), and that an increase in the light intensity from 7

170

R. H. SHIPMAN, L. T. FAN, AND I. C. KAO

OL

o,:

1000

2000

3000

4000

1 5000

Light Intensity (foot candles)

FIG. 3. The specific growth rate constant and the specific bacteriochlorophyll content of Rhodopseudomonas sphermdes at various light intensities. Adapted from Sistrom (1962). 0-- -0,Growth rate;-., chlorophyll protein.

X 103 to 12 to 15 x 103 erg.cm2/sec markedly stimulates cell growth; however, light saturation ensues at light intensities exceeding 15 x 103 erg * cm2/sec. Photosynthetic growth is affected differently in media containing organic compounds, such as acetic acid, propionic acid, or glucose. In such media, rapid bacterial growth proceeds at light intensities of 2 to 3 x 103 erg cm2/sec. The cell multiplication rate increases with light intensity to an approximate value of 7 x 103 erg.cm2/sec; at light intensities exceeding 12 x 103 erg -cm2/sec, photosynthetic bacterial growth is inhibited (Kondrat'eva, 1965). This phenomenon may be explained by the fact that at low light intensities photosynthetic bacteria behave photoheterotrophically by metabolizing organic compounds. At a higher light intensity, they grow photoautotrophically, biosynthesizing their cellular compounds from CO,. Studies by Hirayama (1968)have shown that illumination of 7ooO-10,oOO lux (650-930 lumens/ft2)is sufficient for normal lipid and pigment synthesis by photosynthetic bacteria. Heden and Levin (1959)have grown photosynthetic bacteria within closed stainless steel fermenters by means of internal illumination. A light intensity of 100 lumensfliter in preliminary glass-flask cultivations has been found to be optimal, higher light intensities actually depressing growth. It has also been found that illumination intensities of 120 lumediter in 1000-liter

SINGLE-CELL PROTEIN PRODUCTION BY PHOTOSYNTHETIC BACTERIA

171

fermenters and 30.0 lumen/liter in 3000-liter fermenters, deviate only slightly from the optimum obtained in glass-flask cultivations. Photosynthetic bacteria have been reported to grow within a wide range of temperatures. Maximum growth and COz reduction occur within a range of 30"40"C (Katz et al., 1942). Van Niel(l944) has indicated that the optimum temperature for most photosynthetic bacterial growth is approximately 37°C. The present authors (Shipman, 1974a; Shipman et al., 1975)have obtained a generation time of 4 hours for photosynthetic growth of Rhodopseudomonas gelatinosa at 35°C. Low temperatures may have deleterious effects on photosynthetic growth. In a study by Dworkin (1959), low-temperature (1°C) cultivation of photosynthetic bacteria led to the destruction of bacteriochlorophyll and subsequent death of bacterial cells. Dworkin concluded that carotenoids lose their protective function at low temperatures as a result of the inactivation of the enzyme system participating in their reduction. The pH and H2S concentration are very important for the predominant development for many forms of purple photosynthetic bacteria. The pH range for the growth of photosynthetic bacteria and the optimal pH for various species of bacteria depend not only on the HzS concentration, which should not exceed 150-200 mg/liter, but also on the presence of other organic and inorganic compounds in the medium and their respective concentrations (Van Hiel, 1931). The range of the optimal pH for various strains and species of photosynthetic bacteria is very wide. The optimum medium pH for most of them range between 7.0 and 8.5; only for a limited number of photosynthetic bacteria does the optimum fall between 6.5 and 6.8 (Pfenning, 1967). Depending on the composition, the culture medium may become more acidic or more alkaline or the pH may remain unchanged during the photosynthetic bacterial growth. Changes in the pH of various media during photosynthetic growth are normally associated with: (1)the assimilation or release of CO,; (2) the production of sulfuric acid by the oxidation of HzS or thiosulfate; or (3)by the accumulation of organic acids in the medium. These characteristic changes in pH normally associated with the formation of primary products of photosynthesis by purple photosynthetic bacteria are well documented in the literature (Kondrat'eva, 1965). In some cases, the pH changes in the medium are indicative of the utilization rate of organic acids (i.e., acetic and propionic acids) since each mole of organic acid utilized induces a definite change in the pH of the medium (Clayton, 1955). Studies of the mineral requirements of photosynthetic bacteria have established the need for sodium, potassium, calcium, cobalt, magnesium, and iron (Hutner, 1944, 1946). In nature, photosynthetic bacteria are encountered both in freshwater and saline reservoirs, including bodies of water

172

R. H . SHIPMAN, L. T. FAN, AND I. C . KAO

having high salt concentrations. Photosynthetic bacteria isolated from very salty water require 10-15% NaCl in the growth medium (Van Niel, 1931). A concentration of 24% NaCl has been found to be optimal for most purple bacteria originating from various saltwater sources. Purple photosynthetic bacteria isolated from freshwater reservoirs require small amounts of sodium which may be supplied by 0.1-0.2% concentrations of NaCl in the medium (Kondrat’eva, 1965). Potassium requirements of photosynthetic bacteria are on the order of 5.0 mg/liter. Photosynthetic growth may be inhibited when the K:Na ratio exceeds 5, regardless of the absolute concentrations of K and Na in the medium. Purple photosynthetic bacteria have been reported as being very sensitive to calcium deficiency-undergoing agglutination in its absence (Hutner, 1946). The Ca requirements of purple bacteria may be satisfied by the addition of 0.001-0.005% CaClz. Certain photosynthetic bacteria require cobalt in concentrations of 5 pg/lOO ml for the synthesis of cobalamin (BI2) (Kondrat’eva, 1965). Magnesium is required by all photosynthetic bacteria for the biosynthesis of bacteriochlorophyll. Magnesium also participates with various other metals in the activation of several enzyme systems (Van Niel, 1931). The growth of photosynthetic bacteria and C02 fixation may be stimulated by the addition of small quantities of Mn to the media. Quantities of 0.5 pg of Mn per 100 ml may be sufficient for optimum growth; however, higher concentrations of Mn, up to 10 pg/100 ml, have neither stimulatory nor inhibitory effects on growth (Kondrat’eva, 196s). Iron is an indispensable constituent in the medium for the synthesis of iron porphyrins and other iron compounds by purple photosynthetic bacteria (Lascelles, 1953). The important function that cytochrome performs in the metabolism of purple bacteria is well documented. Iron deficiency inhibits the growth of photosynthetic bacteria and markedly suppresses bacteriochlorophyll synthesis (Kamen, 1955).

Ill. Conceptual Design Here, the proposed photosynthetic SCP process, illustrated in Fig. 4,has been designed as a semicontinuousprocess with a mean production capacity of 5 tons of SCP per day. The daily raw material balance data are shown in Table IV. The design is based on the following cultivation parameters: (1) a digester retention time of 24 hours; (2) an operating temperature of 37°C; (3) a pH of 7.0-7.2; and (4) a harvested cellular yield of 10.0 gm of dried cells per liter. The major processing steps include hydrolysis of the wheat bran slurry, solids removal, neutralization, photosynthetic cultivation, recovery, and purification. Major pieces of process equipment and their costs are itemized in Table V.

173

SINGLE-CELL PROTEIN PRODUCTION BY PHOTOSYNTHETIC BACTERIA

Hydrochloric Acid (36%)

1. Hydrolysir - Sterilization

Solids Removal

Bran Wheal Bran Intu8ian

'sunlight/lncandescent

II

Photosynthetic

Solids Recovery

Wheat Bran Residue

Lighting

lnoculatim

Anhydrous Ammonia Yeast Extract

I Supernatant Recycle

H ar ye s t ed ~:/,osynthelic

Centrifugation

Water

FIG. 4. Flow sheet for the photosynthetic single-cell protein process.

In the initial processing step, an aqueous slurry of wheat bran, containing approximately 30% solids, is acidified with HC1(36%). The slurry is injected with steam and held at a temperature of 121°C and a steam pressure of 15 psi for 4 hours. The unhydrolyzed wheat bran residue is separated from the supernatant in a solid-bowl, conveyor-type centrifuge. The wheat bran infusion is discharged into a stainless-steel tank in which neutralization with TABLE IV DAILYRAW MATERIAL BALANCE FOR THE PHOTOSYNTHETIC SINGLE-CELL PROTEIN (SCP) PROCESS Component Input Wheat bran HCI (36%) Anhydrous ammonia Yeast extract Makeup water output Photosynthetic SCP (dry weight) Wheat bran residue (85-90% solids)

Tonslday

15.0 4.7 0.675 0.099 470.0 5.0 6.75

TABLE V MAJOR PROCESS EQUIPMENT FOR PHOTOSYNTHETIC SINGLE-CELL PROTEIN(SCP) PRODUCTION Unit Mixing and storage tanks Mixing and storage tank Storage tank Centrifuge Horizontal transparent polyvinyl chloride (PVC) pipe Centrifuge

Incandescent lamps Boiler unit

No.

2 1 1 1 33

Description

Unit cost

Cost

Type: stainless steel; capacity: 400,000gal Type: carbon steel; capacity: 24,000gal Type: carbon steel; capacity: 12,000gal Type: solid bowl with conveyor discharge; stainless steel; 90 GPM hydraulic capacity Dimensions: 61 cm X 67.1 m; capacity:

$33,o@J

$f%000

$11,200 $8,400 $50,000

$11,200 $8,400 $50,000

$22.501

$163,800

linear ft

4700 gal

1

200 1

Pump

1

Pumps

5

Spray dryer

1

P x

Type: disk bowl with nozzle discharge; stainless steel; includes drive and controls; 90 GPM hydraulic capacity 400 Watts; 115 volts Steam capacity: 30,000Ib/hr; steam pressure: 250 psi Type: radial-flow centrifugal; stainless steel; 10 HP max. capacity: 150 GPM Type: radial-flow Centrifugal; carbon steel; 10 HP max. capacity: 150 GPM Evaporative rate: 4 tons/hr

$32,400

$32,400

$6.00 $52,000

$1,200 $52,000

$5,380

$5,380

$1,680

$8,400

$26,400

$26,400

Total purchased equipment cost:

$425,180

n

E0

TABLE VI COMPOSITION OF WHEAT

Proximate analysis Ash Crude fiber Ether extract N-free extract Protein (N X 6.25)

BRAN"

% Dry basis

6.9 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.2 4.6 .............................. 59.3 .............................. Vitamins 18.0 . . . . . . Amino acids (%) Choline Arginine 1.12 Folic acid Cystine 0.34 Niacin Glycine 1.01 Pantothenic acid Histidine 0.34 Riboflavin Isoleucine 0.67 Thiamine Leucine 1.01 a-Tocopherol Lysine 0.67 Methionine 0.11 Phenylalanine 0.56 Threonine 0.45 Tryptophan 0.34 Tyrosine 0.45 Valine 0.79

}

Minerals Calcium Iron (mdkg) 1,110.0 2.0 235.1 32.6 3.5 8.9 12.1

Magnesium Phosphorus Potassium Sodium Cobalt Copper Manganese

(%)

0.16 0.02 0.62 1.32 1.39 0.07

( d1.10 w

m

13.80 130.00

m

1 E

=Adapted from Jurgens (1973).

176

R. H. SHIPMAN, L. T. FAN, A N D I. C. KAO

anhydrous ammonia occurs. Although wheat bran contains significant amounts of amino acids, vitamins, and minerals (Table VI), yeast extract is added to supplement the infusion medium with vitamins that may have been destroyed during the hydrolysis and heating process. The temperature of the infusion medium is lowered to approximately 40°C at this stage of the process by pumping cooling water from the temperature controller of the photosynthetic cultivation tanks through the cooling jacket of the neutralization tank. Hot water flowing from the cooling jacket is then pumped back to the temperature controller and is used to maintain a temperature of 37°C within the photosynthetic bioreactor. Sterilization of the infusion medium simultaneously occurs during the acid-hydrolysis and heating process, and thus the need for an additional medium-sterilization step is eliminated. During the photosynthetic cultivation phase, wheat bran infusion is pumped from the holding tank at a rate of 5OOO GPH (18,900 Yhour) into a series of horizontal photosynthetic cultivation tanks, 61.0 cm in diameter, constructed from transparent polyvinyl chloride (PVC). Direct sunlight provides illumination for daytime photosynthetic cultivation, and 400-watt incandescent lamps, which may be lighted by solar energy stored during daytime, provide artificial illumination for nighttime operation. The retention time in the transparent PVC cultivation tanks is 24 hours. Continuous inoculation of photosynthetic bacteria is provided by recycling a portion (2%) of the exit stream at a rate of 100 GPH. Photosynthetic bacterial cells are recovered from the exit stream by pumping spent culture media from the recovery tank to a disk bowl-type centrifuge. Harvested bacterial cells are washed with water and the final product is dried in a spray dryer. IV. Economic Analysis The economic feasibility analysis of the photosynthetic SCP process given here is based on the production capacity of the previously described conceptual process. Cost estimates have been compiled from available data on similar items of equipment, cost indexes, and available cost-capacity factors. All estimates have been escalated to the last quarter of 1975 according to the Marshal Stevens Index.

A. CAPITALINVESTMENT The purchase costs for major pieces of photosynthetic SCP processing equipment are included in Table V. The base cost estimates for the stainless steel acid-hydrolysis and neutralization tanks have been taken from Guthrie (1969) and include delivery costs but not installation costs. The process centrifugation costs and their hydraulic capacities have been extrapolated

SINGLE-CELL PROTEIN PRODUCTION BY PHOTOSYNTHETIC BACTERIA

177

from similar items of equipment used for solids removal and dewatering of wastewater sludges (Liptak, 1974). The photosynthetic reactor used in the study is of similar design to a photosynthetic bioreactor constructed of transparent acryl resin (Kobayashiet al., 1971). Since costs for the construction of such reactors are not available, the base cost of the photosynthetic reactor has been estimated from the cost of transparent PVC pipe at $22.50 per linear foot. Costs for steam generation equipment, spray dryers, and additional processing equipment have been obtained from other sources (Perry, 1963; Peters and Timmerhause, 1968; Popper, 1970). Total purchased equipment costs including delivery amount to $425,180. Other direct costs and indirect costs, summarized in Table VII, are assumed to be equivaTABLE VII ESTIMATED CAPITALINVESTMENT Processing equipment Purchased equipment costs' Installation Instrumentation and control' Piping Electricalb Buildings Yard improvements Land Total direct costs

$425,180 98,500 42,000 85,000 24000 66,500 8,000

30,000 $779,180

Indirect cost Engineering and supervisionC Construction expensed Total direct and indirect cost Contractors feese Contingency'

81,800 93,500 $954,480 47,700 95,400

Fixed capital investment (FCI) Working capital0

$1,097,580 121,950

Total capital investment (TCI)

$1,219,530 ~

"Includes delivery costs. 'Includes costs for installation. '@ 10.5% of the direct cost. d @ 12.0% of the direct cost. "@ 5% of the total direct and indirect cost. @ ' 10% of the total direct and indirect cost. "@ 10% of the total capital investment.

178

R. H. SHIPMAN, L.

T.

FAN, AND I. C. KAO

TABLE VIII

ANNUALOPERATING COSTSFOR THE PHOTOSYNTHETIC SINGLE-CELL PROTEIN PROCESS ~~~~~~

~

Direct production costs Wheat bran @ $25.00/ton HCI (36%)@ $37.00/ton Anhydrous ammonia @ $67.50/ton Yeast extract @ $ 2 W t o n NaCl @ $ 25.00/ton Makeup water @ 0.15/1000 gal. Operating labor Supervisory labor Maintenancea Operating suppliesb Fixed charges Depreciation' Taxes and insurance Financin$ Total annual operating cost

$123,750 57,390 15,040 65,340 9,280 5,580 142,560 12,000 32,900 3,300 54,880 27,440 30,490 $579,950

OMaintenance costs = 3% of fixed capital investment (FCI). *Operating supplies = 0.3% of FCI. CAnnualdepreciation of 5% of FCI. dFinancingat one-third of total capital investment at 8% for 10 years.

lent to those of a solid-liquid processing plant. Engineering and supervision costs are 10.5%of the direct costs while construction costs are 12.0% of the total direct costs. The total direct and indirect cost is $954,480. To this figure, an additional 15%to cover contractor's fees and contingency is added resulting in a fixed capital investment (FCI) of $1,097,580. Assuming that the working capital represents 10% of the total capital investment, the total capital investment amounts to $1,219,530.

B. ANNUALOPERATINGCOSTS The annual operating costs, including direct production costs, fixed charges, and plant overhead, have been determined on an annual production basis of 330 days of operation, according to standard procedures outlined by Peters and Timmerhause (1968).A breakdown of the annual operating costs is shown in Table VIII.

SINGLE-CELL PROTEIN PRODUCTION BY PHOTOSYNTHETIC BACTERIA

179

The costs for wheat bran have been estimated at $25.00/ton. The costs for the acid-hydrolysis and neutralization processes have been obtained from costs of similar processes (Liptak, 1974). The remaining raw material costs have been obtained from the Chemical Marketing Reporter. The operating labor requirement for continuous process supervision is based on 3 shifts per day and an average of 1.33 operators per shift. The operating labor costs are based on a rate of $4.5O/man-hour, and a yearly salary of $12,000 is provided for supervisory labor. Maintenance and operating supply costs are based on a percentage of the fixed capital investment. The photosynthetic SCP process is assumed to have an operational expectancy of 20 years with zero salvage value at the end of that time. The annual depreciation, at a yearly rate of 5% of the FCI, amounts to $54,880. Financing is estimated at one-third of the total capital investment at 8% for ten years. The total annual operating cost amounts to $579,950. C. TOTALPRODUCTION COSTAND PROFITABILITY Given the total annual operating cost and a total annual SCP production capacity of 1650 tons, the mean total production cost per ton amounts to $351.48 ($O.lS/lb or $0.39/kg). The profitability of the proposed photosynthetic process may be shown by the gross profit per ton and the gross

Unit Price of Photosynthetic SCP ($/lb)

FIG. 5. Profitability of the proposed photosynthetic process. A0 0, per ton (dollars).

- -A,

Annual (103 dollars);

I80

R. H. SHIPMAN, L. T. FAN, AND I. C. KAO

TABLE IX ESSENTIAL AMINOACID COMPOSITION OF PROTEINS FROM PHOTOSYNTHETIC BACTERIACOMPARED WITH CONVENTIONAL FOODS" Photosynthetic bacterial protein

Soy protein

Amino acid

Egg protein (%)

(%I

(a)

Histidine Isoleucine Leucine Lysine Methionine PhenylaIanine Threonine Tryptophan Valine

2.4 6.6 8.8 6.4 3.1 5.8 5.0 1.6 7.4

2.4 5.4

3.4-3.9 4.1-4.3 7.67.9 5.6-6.0 3.0 4.34.6 2.9-4.4

7.7 6.3 1.3 4.3 3.9 1.4 5.2

-6

6.5-7.0

"Adapted from Shipman (1974a). bNot analyzed.

annual profit for various marketing prices of photosynthetic SCP illustrated in Fig. 5. At a marketing price of $0.30/lb, for example, a gross annual profit of$410,000(i.e., gross profit of$248/ton)may be realized. The photosynthetic SCP process may be economically competitive with current commercial yeast production processes, if the current marketing price of brewer's yeast at $0.45/lb is considered (Anonymous, 1975). One of the most evident advantages of the proposed photosynthetic process is the utilization of photosynthetic SCP for human food supplementation. TABLE X VITAMINCONTENTSOF PHOTOSYNTHETIC BACTERIAAND YEAST"

Vitamin

Photosynthetic bacteria (pg/100 gm dried cells)

Riboflavin (B,) Pyndoxine (Be) Folic acid Cobalamin (BI2) Ascorbic acid (C) Cholecalciferol (D3)

3,600 3,000 2,000 200 20,000 10,Ooob

"Adapted from Kobayashi (1970). bInternationalUnits.

Yeast (pg/IOOgm dried cells) 2,900 2,400 1,700 1.0

-

300,Ooob

SINGLE-CELL PROTElN PRODUCTION BY PHOTOSYNTHETIC BACTERIA

181

Photosynthetic bacterial cells contain approximately 65%protein and significant quantities of the essential amino acids (Table IX). In addition, photosynthetic cells contain relatively large amounts of ascorbic acid, vitamin D, and various B vitamins as shown in Table X. Unlike other SCP processes based on hydrocarbons in which the possibility of substrate toxicities exist, wheat bran offers the advantage of being a readily renewable, toxic-free, natural substrate for the production of edible protein. A reduction in the total production costs may be realized by the possible reclamation and resale of the wheat bran residue as a livestock feed. Processing of the wheat bran residue would involve some form of mechanical or solar drying; however, since the marketing price for such a product is uncertain, these costs have not been considered in the present study. Another potential scheme for the production of photosynthetic SCP involves the cultivation of photosynthetic bacteria in sewage, animal manure, and feedlot wastes. A process described by Kobayashi et al. (1971) involves the cultivation of photosynthetic organisms in municipal sewage sludges and harvesting photosynthetic bacterial cells as a by-product of the purification process. Biomass harvested from photosynthetic cultivation in sewage and animal wastes could be used as a supplement for various livestock feeds (Singh and Anthony, 1968; Coe and Turk, 1973). The proposed process for producing photosynthetic bacteria as a source of edible protein shows definite economic potential. The development of photosynthetic cultivation systems utilizing sewage sludges, livestock manure, and other agricultural waste products should definitely be encouraged. ACKNOWLEDGMENT

This is contribution No. 50-RJof the Department of Chemical Engineering, Kansas Agricultural Experiment Station. Partial support by the Ceres Land Company is appreciated. REFERENCES Anonymous (1975). Chem. Mark. Rep. December 22, 1975. Bose, S. K. (1963). In “Bacterial Photosynthesis” (H. Gest, A. San Pietro, and L. P. Vernon, eds.), p. 501. Antioch Press, Yellow Springs, Ohio. Brock, T. D. (1970). “Biology of Microorganisms,” p. 154. Prentice-Hall, Englewood Cliffs, New Jersey. Clayton, R. K. (1953). Arch. Microbial. 19, 107. Clayton, R. K. (1955). Arch. Microbio2. 22, 195. Coe, W. B., and Turk, M. (1973). In “Symposium: Processing Agricultural and Municipal Wastes,” (G. E. Inglett, ed.), p. 29. AVI Publ., Westport, Connecticut. Cohen-Bazire, G. (1963). In “Bacterial Photosynthesis” (H. Gest, A. San Pietro, and L. P. Vernon, eds.), p. 89. Antioch Press, Yellow Springs, Ohio.

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Cohen-Bazire, G., Sistrom, W. R., and Stanier, R. Y. (1957).J . Cell. Comp. Physiol. 49, 25. Dworkin, M., (1959). Nature, (London) 184, 1891. Eisenberg, M. E., (1953). J. Biol. Chem. 203, 815. Gest, H. (1951). Bactaiol. Reu. 15, 183. Gest, H., and Kamen, M. D. (1949). J. Bacteriol. 58, 239. Gest, H., Kamen, M. D., and Bregoff, H. M. (1950).J. Biol. Chem. 182, 153. Goodwin, T. W. (1955). Annu. Rev. Microbiol. 24, 496. Guthrie, K. M. (1969). Chem. Eng. (N.Y.) 76, 114-142. Heden, C. G . , and Levin, K. (1959).J. Biochem. Microbiol. Technol. Eng. 1, 303. Hirayama, 0. (1968).Agric. Biol. Chem. 32, 34. Hutner, S. H. (1944). Arch. Biochem. 3, 439. Hutner, S. H. (1946).J. Bacteriol. 52, 213. Hutner, S. H. (1950).J. Gen. Microbiol. 4, 286. Hutner, S. H., and Scher, S. (1961). Bacteriol. Proc. 61, 46. Jurgens, M. H. (1973). “Applied Animal Feeding and Nutrition,” p. 55. KendalVHunt Publ., Dubuque, Iowa. Kamen, M. D., (1955). Bacteriol. Reo. 19, 234. Kamen, M. D., and Gest, H. (1949). Science 109, 560. Kak, E., Wassink, E. C., and Dorrestein, R. (1942). Enzymologio 10, 269. Kohayashi, M. (1970). Chem Biol. 8, 604. Kobayashi, M., Matsumoto, M., Nakanishi, H., and Takahashi, E. (1970).J . Sci. Soil Manure (Jpn.) 41, 130. Kohayashi, M., Kobayashi, M., and Nakanishi, H . (1971).J. Fennent. Technol. 49, 817. Kondrat’eva, E. N. (1965). “Photosynthetic Bacteria” (transl. from Russian). Israel Program Sci. Trans]. , Jerusalem. Lascelles, J. (1953). Biochem. J. 55, 1. Lascelles, J. (1960). J. Gen. Microbiol 23, 487. Lascelles, J. (1963). In “Bacterial Photosynthesis” (H. Gest, A. San Pietro, and L. P. Vernon, eds.), p. 37. Antioch Press, Yellow Springs, Ohio. Lascelles, J., and Wertlieb, D. (1971). Biochim. Biophys. Acta 226, 328 (1971). Liptak, B. G., ed. (1974). “Environmental Engineer‘s Handbook,” Vol. I. Chilton Book Co., Radnor, Pennsylvania. Ormerod, J. G., Ormerod, K. S., and Gest, H. (1961). Arch. Biochem. Biophys. 94, 449. Pelzar, M. J., and Reid, R. D. (1965). “Microbiology,” 2nd ed. p. 130. McGraw-Hill, New York. Perry, J. H., ed. (1963).“Chemical Engineer’s Handbook,” 4th ed. McGraw-Hill, New York. Peters, M. S., and Timmerhause, K. D. (1968). “Plant Design and Economics for Chemical Engineers,” 2nd ed. McGraw-Hill, New York. Pfenning, N. (1967). Annu. Reo. Mimobiol. 21, 285. Popper, H., ed. (1970). “Modem Cost-Engineering Techniques” McGraw-Hill, New York. Pratt, D. C., and Frenkel, A. W. (1959). Plant Physiol. 34,333. Rose, A. H. (1968). “Chemical Microbiology,” 2nd ed., pp. 156159. Plenum, New York. Shipman, R. H. (1974a). M.S. Thesis, Kansas State University, Manhattan. Shipman, R. H. (197413). Proc. 4th Annu. Kans. Stateaebr. Biochem. Eng. Symp. p. 26. Shipman, R. H., Kao, I. C., and Fan, L. T. (1975). Biotechnol. Bioeng. 17, 1561. Singh, Y. K., and Anthony, W. G. (1968).J. Anim. Sci. 27, 1136. Sistrom, W. R. (1960).J. Gen. Microbiol. 22, 778. Sistrom, W. R. (1962). J. Gen. Microbiol. 24, 607. Stanier, R. Y. (1961). Bactm’ol. Rev. 25, 1. Stanier, R. Y.,Doudoroff, M. and Adelberg, E. A. (1970). “The Microbial World,” 3rd ed., pp. 559-560. Prentice-Hall, Englewood Cliffs, New Jersey.

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Sybesma, C. (1970).In “Photobiology of Microorganisms” (P. Halldal, ed.), pp. 57-93. Wiley, New York. Thanii, N. C., and Simard, R. E. (1973).J . Water Pollut. Control. Fed. 45, 674. Van Niel, C. B. (1931).Arch. Mimobiol. 3, 1. Van Niel, C. B. (1944). Bacteriol. Rew. 8, 1. Wagenknecht, A. C., and Burris, R. H. (1950). Fed. Proc., Fed. Am. SOC. Erp. Biol. 9, 242. Wai, N. (1971).Proc. Sino-Am. Sci. Coop. Colloq. Ocean Res. 2 , 89. Wai, N. (1972).Bull. Inst. Chem., Acad. Sin. 21.

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Environmental Transformation of Alkylated and Inorganic Forms of Certain Metals JITENDRA SAXENAAND

PHILIPH. HOWARD

Lve Science Division, Syracuse University Research Corporation, Syracuse, New York I. Introduction . . . . . . . . . ..................... 11. Biochemical, Photochemical, and Chemical Tranformation of .......... Metals and Metalloids . . . . . . . . . . A. Valance Changes ................ B. Methylation ... ...................... C. Chelation, Comp dsorption . . . . . . . . . . . . . . 111. B. Biological Transformation in the Soil Environment C. Model Ecosystem and Aquarium Studies (Microcosms) . . IV. Analytical Procedures . . . . . .......... A. Isolation Steps ..................................... B. Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Identification and Speciation ......................... V. Factors Affecting Transformation of Metals . . . . . . . . . . . . . . . . . A. Factors Affecting Methylation of Metals. . . . . . . . . . B. Factors Affecting Degradation of Organometallic

...........

185 186

187 189 192 194 194 197 197 198 198 200 203 205 205 209

VI . Biochemical Pathways and Mechanism for Transformation of

....................................... VII. General Discussion of Various Test Methods . . . . VIII. Correlation between Laboratory and Field Results Monitoring Data ........................... A. Arsenic.. . . . . . . . . .....................

..................................... IX. -Contaminated Areas . . . . . . . . . . . . . . . . . . X. Categorization of Elements . . . . . . . . . . . . . . XI. References . . . . ...........................

210 212 215 215 216 217 219 220 222

I. Introduction

Metals are introduced into the environment &om various sources; these include fuel combustion, incineration, mining and industry, agricultural chemicals, and liquid and solid waste from animals and men. For example, h e 1 combustion for electrical power generation in the United States alone would contribute as much as 300 tons of mercury annually in the environment (Diehl et al., 1972). Annual global emission of nickel from fossil fuels into air has been estimated to be nearly 70,000 tons (Chem. & Eng. News, July 19, 1971) and of arsenic approximately 4000 tons (Ferguson and Gavis, 1971). Metals also exist in one form or the other in the environment as 185

186

JITENDRA S A X E N A A N D PHILIP

n.

HOWARD

natural constituents of the earth’s crust. Elements of major concern today include, but are not limited to: mercury, arsenic, lead, tin, antimony, copper, cadmium, chromium, nickel and vanadium. These exist in the environment in many different chemical forms as air pollutants, as contaminants of water and soil, and as residues in food. Pollution of our environment by mercury and its conversion to the more poisonous methylmercury by microorganisms has caused concern over the effect of such trace metals. The toxic metals in the environment may present a more insidious problem than pollution by organic chemicals because metals, unlike organic chemicals, cannot be degraded to innocuous products, such as carbon dioxide and water. The degradation of organometallic compounds by microorganisms and chemical “weathering’ generally leads to the liberation of toxic elements in addition to other end products. Metals can undergo changes in valance state, be converted into organometallic form, or be mobilized from one environment to another. The interconversions are generally reversible and result in steady-state concentrations of various forms in the environment. Heavy metals have been known to be toxic to humans, animals, and plants. The form in which the metal occurs (e.g., pure metal, inorganic compounds, or organometallic compounds) strongly influences its toxicity and mobility. For example, methylmercury is far more poisonous than inorganic mercury and is excreted very slowly. A small disturbance in the dynamic biological and chemical cycling of these toxic elements can have a considerable impact on their concentrations and distribution in the environment. In view of the toxic nature of many elements, it becomes important to know their fate in the environment. In the case of metals and metalloids, the mechanisms involved in their transport, and transformation from one form to another are extremely important with respect to environmental pollution. This paper reviews information on transformation and fate of certain key metals and their compounds in the environment. The available information on pathways of transformation, experimental methodology used to study these reactions, the suitability of the laboratory techniques for studying environmental transformation of metals and organometallic compounds, and other areas of environmental interest, are discussed.

II. Biochemical, Photochemical, and Chemical Transformation of Metals and Metalloids Chemicals placed in the environment are subjected to chemical alterations and physical processes by sunlight, water, soil, natural inorganic and organic

ENVIRONMENTAL TRANSFORMATlON OF METALS

187

materials, and biological agents and by the combined weathering action of rain, wind, temperature, and humidity. The physical processes, such as adsorption on colloidal substances, volatilization, bioaccumulation or leaching, have a tendency to distribute and dilute or concentrate, but not transform, the contaminants. On the other hand, biological, chemical, and photochemical reactions in the environment result in alteration of the compounds. The interconversions of metals and metal compounds by these reactions could yield products that may be more or less toxic to higher organisms. Although biological transformation of metals and metal compounds might conceivably be accomplished by any living organisms (see Lewis et al., 1966; Evans et al., 1968; Lakso and Peoples, 1975; Wood, 1974), available information indicates that by far microorganisms, especially bacteria and fungi, play a dominant role in catalyzing the modification, activation, or detoxification of compounds of toxic metals and metalloids in the environment. For example, Jernelov and Lann (1973) have noted that the methylation rates of mercury were positively correlated to general microbiological activity. The high order of metabolic activity and versatility, and species diversity in natural communities of microorganisms may provide major advantage to microorganisms over other organisms (see Howard et al., 1975). Also, microorganisms for the most part reproduce more rapidly and have high rates of mutation, thus allowing them to develop enzyme systems over the years that will act upon a variety of substances introduced in the environment. The greater estimated microbial biomass (including algae) over combined animal biomass (Macfadyen, 1963; Dagley, 1972) further substantiates the major role of microorganisms. Organisms of higher trophic levels (e.g., plants, fish, and mammals), on the other hand, are more important to considerations of bioaccumulation and dispersion of metal compounds and their transformation products, thereby affecting their accessibility to bacteria and fungi.

A. VALANCECHANGES Trace metals exist in the environment in several valance forms, and each form may differ in its toxicity. By means of oxidative and reductive reactions, the relative abundance of each species is controlled. For example, mercury can exist in the following three forms (Wood, 1974): H&+ % Hg2+ + H$

The relative amounts of each form will depend on the solubility or the amount of dissociation of the mercuric compound formed and the extent to which metallic mercury enters or leaves the system and by external factors that affect the biosphere. Vaporization of elemental mercury will shift the

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JITENDRA SAXENA AND PHILIP H. HOWARD

reaction to the right, whereas an increase in the concentration of Hg", either due to return fiom the atmosphere or due to addition &om external sources, will shift the equilibrium to the left. Interconversions of the inorganic forms of mercury are catalyzed by microorganisms. For example, bacteria and yeast can volatalize mercury by reducing it from its cationic to its elemental state (Furukawa et al., 1969; Nelson et al., 1973; Schottel et al., 1974; Brunker and Bott, 1974). Alberts et al. (1974) have reported that elemental mercury can also be formed by a chemical mechanism involving interaction of mercuric ions with naturally occurring humic acid. The reverse reaction, i.e., oxidation of metallic mercury to cationic mercury in soils or sediments, occurs under oxidizing conditions (Jernelov, 1969a; Werner, 1967). Microorganisms appear to mediate this reaction, but the extent of their involvement is unclear. Microorganisms are able to reduce the pentavalent form (arsenate) of arsenic to the trivalent form (arsenite) (McBride and Wolfe, 1971). Johnson (1972) obtained evidence for bacterial reduction of arsenate in sea water, which may explain the occurrence of arsenite in the deep ocean. On the other hand, a number of microorganisms are able to oxidize arsenite to less toxic arsenate (Mendal and Mayersak, 1962; Turner, 1954; Turner and Legge, 1954; Armstrong and Harvey, 1951). Johnson and Pilson (1975) presented evidence for slow rates of nonbiological oxidation of arsenite in sea water. A marked increase in the rate of arsenite oxidation upon exposure to direct sunlight suggested a photochemical mechanism. However, since nonsterile water gave overall higher oxidation rates, some oxidation is likely to be microbiologically mediated. Arsenite oxidation in soil has been noted by Quastel and Scholefield (1953). The oxidation was mediated by microorganism in soil as evidenced by the fact that complete inhibition of oxidation occurred when microbial activity was inhibited by sodium azide. The stochiometry of oxygen consumed and arsenite oxidized suggested the reaction NaAsOz

+ HzO + MOz + NaH,AsO,

Alteration of the oxidation state of arsenic catalyzed by the microbial population in activated sludge has been reported by Myers et al. (1973). It was shown that in the presence of oxygen, arsenite was oxidized to arsenate; under anaerobic conditions arsenate was reduced to arsenite, and upon continued anaerobic incubation to even lower oxidation state. Ferguson and Gavis (1971) have suggested that arsenate could be reduced by serving as an electron acceptor in heterotrophic metabolism in the absence of 0%and NO3-. Experimental support for this contention is lacking at the present time.

ENVIRONMENTAL TRANSFORMATION OF METALS

189

Microorganisms have also been implicated in the oxidation and reduction of selenium compounds. Sapozhnikov (1937) observed the oxidation of elemental selenium to selenite by a photosynthetic purple sulfur bacterium. Biological oxidation of selenium in soil has been reported by Geering et al. (1968).The authors noted that some oxidation of selenium also proceeded via nonbiological reactions involving native inorganic material in soil. Microorganisms derived from soil were also able to catalyze the reduction of selenite and selenate to the elemental form (Bautista and Alexander, 1972). The ecological significance of the reduction of selenium compounds is as yet undefined (Alexander, 1974). Bacterial oxidation of trivalent antimony to the pentavalent form has been reported by Lyalikova (1972). The oxidation serves as the sole energy source for the chemosynthetic organism Stibiobacter gen. nov.

B. METHYLATION Biological methylation plays an important role in the transport of toxic metals. Researchers have reported methylation of mercury, arsenic, selenium, tellurium, lead, tin, palladium, gold, and possibly cadmium. Conversion of inorganic forms of metal or metalloids to methylated forms may be employed by microorganisms as a detoxification mechanism.

1 . Mercury Jensen and Jernelov (1969) and Fagerstrom and Jernelov (1971) have reported formation of both monomethyl and dimethyl mercury in lake and river sediments. Bisogni and Lawrence (1973) have pointed out that biological wastewater treatment systems and anaerobic digesters may provide excellent environments for methylation of mercury. The methylation of mercury in many different soils has recently been demonstrated (Beckert et al., 1974; Rogers, 1975). The inability of methyl mercury to accumulate in soil was attributed to dimethylation and volatilization processes (Rogers, 1975). Wood and co-workers (1968)reported that transfer of the methyl group from Co3+ of methylcobalamin to Hg2+ could also be catalyzed nonenzymically under mild reducing conditions. Imura et al. (1971) reported the formation of dimethylmercury from chemical transmethylation from methylcobalamin. Dimethylmercury resulting from microbiological transformation can be released to the atmosphere due to its volatility, where it may be subjected to photolysis. According to Gomer and Noyes (1949), the photolysis of dimethylmercury might proceed as follows: Hg(CW*

2

C H ~+ ' H ~ C H ~

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JITENDRA SAXENA AND PHILIP H. HOWARD

The monomethyl mercuric radical can further undergo decomposition to give rise to metallic mercury and another methyl radical. The methyl radicals probably abstract hydrogen or recombine to form methane or ethane, respectively (Wood, 1974). Spangler and co-workers (1973a,b) have reported evidence for the microbial degradation of methylmercury in lake sediments. It was suggested that organisms responsible for degradation of methylmercury may serve a useful purpose in maintaining the environmental methylmercury concentrations at a minimum. The bacterial species isolated in pure culture caused conversion of methylmercury into volatile elemental mercury (H$) and methane. The cyclic nature of methylmercury formation is also suggested from the studies of Hamdy and Noyes (1975), who noted a continuous breakdown and resynthesis of methylmercury in pure cultures of bacteria. The biochemical, chemical, and photochemical transformations of mercury compounds set up a dynamic system in the environment. The biological cycle for mercury suggested by Wood (1974) is shown in Fig. 1.

HgZ'

Hgz Bacteria

FIG. 1. The biological cycle for mercury (Wood, 1974). Reprinted with permission from Science 183, 1051 (1974). Copyright 1974 by the American Association for the Advancement of Science.

ENVIRONMENTAL TRANSFORMATION OF METALS

191

2 . Arsenic Arsenic, similar to mercury, has also been found to undergo methylation. Microorganisms, particularly bacteria and fungi, have been reported to play active roles in these conversions (McBride and Wolfe, 1971; Challenger, 1945; Cox and Alexander, 1973). Arsenicals are reduced and methylated by microorganisms to give rise to toxic dimethyl and trimethylarsines. Because of their volatility, alkylarsines may appear in the atmospheric environment, where they may be rapidly oxidized. Kearney and Woolson (1973) have reported alteration of organoarsenic compounds via two pathways: (1) an oxidative pathway leading to C-As bond cleavage and (2) a reductive pathway leading to alkylarsine production. The biological cycle of arsenic is shown in Fig. 2.

3. Selenium and Tellurium Selenium and tellurium have been known to be acted on by microorganisms to produce methylated compounds. Fleming and Alexander (1972) isolated a strain of Penicillium from raw sewage which produced dimethylselenide from inorganic selenium compounds. The same organism also catalyzed the conversion of several tellurium compounds to dimethyltelluride. The formation of dimethylselenide from selenite and selenate and an unidentified gas from tellurate by arsenic methylating sewage fungus Candidu humicoh has been reported by Cox and Alexander (1974).The methylation of selenium was later also shown to occur in soil (Alexander, 1974). The conversion of selenium to dimethylselenide and/or dimethyldiselenide and/or an unknown selenium compound by the microorganisms present in lake water-sediment system has recently been noted by Wong et al. (1975). 4. h a d

The methylation of inorganic and organic lead compounds to the volatile and more toxic tetramethyllead by chemical and biological mechanisms has been reported. Wong et al. (1975) noted the microbial formation of tetramethyllead in lake water-sediment system under anaerobic conditions. Addition of inorganic lead nitrate or organic trimethyllead acetate greatly increased the synthesis of tetramethyllead. Whereas conversion of inorganic lead to organic lead was difficult to obtain and perhaps required specific physical, chemical, and biological conditions, the conversion of trimethyl to tetramethyllead proceeded quite readily (Wong et al., 1975). Jarvie et al. (1975) obtained evidence for conversion of trimethyllead to tetramethyllead under anaerobic conditions in the presence of sulfide by a chemical mechanism. The reaction was suggested to proceed via the formation of trialkyllead sulfide.

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JITENDRA SAXENA AND PHILIP H. HOWARD

Air

CH3

I

- As+

HO

CH3

- CH3

- \

- As3 CH3 I - - CH3 + H - CH3 As3 I

Trimethylarsine

- CHJ

Dimethylarsine

Bacteria \

\

OH

CH3

I HO

- As5+11

CH3

I

OH

7As3+ - OH

Bacteria

Bacteria

HO

- As3+11

I OH

7HO -As+

Bacteria

/

I

\

-CH3

II

0

0

0

0

Arsenate

Arrenite

Methylarsenic acid

Dimethylarsinic acid

Sediment

FIG. 2. The biological cycle for arsenic (Wood, 1974). Reprinted with permission from Science 183, 1051 (1974). Copyright 1974 by the American Association for the Advancement of Science.

5 . Other Metals Presumptive evidence for the volatilization of inorganic cadmium by conversion to the methylated species by a Pseudomonas sp. has recently been obtained by Huey et al. (1975). The identification of the volatile cadmium species was based on its ability to transfer a methyl group abiotically in water to Hg+, thereby resulting in the formation of methylmercury. Methylation of other toxic metal ions has not been sufficiently investigated. Wood (1975) has briefly reported the synthesis of dimethyl and trimethyltin, methyl- and dimethylpalladium, and dimethylgold. Evidence for the formation of volatile methylated tin species from Sn4+by a strain of Pseudomonas has also been obtained by Huey et al. (1975). C. CHELATION, COMPLEXATION, AND ADSORPTION Natural and man-made chelates, both organic and inorganic, occur everywhere in the environment at low concentrations. Humic compounds

ENVIRONMENTAL TRANSFORMATION OF METALS

193

(hlvic acid, humic acid, and humin) are among the most widely occurring natural complexing agents. They constitute the bulk of the organic matter in most soils. Their presence has also been noted in sediments and waters of lakes, rivers, and oceans (Rashid and King, 1969; Rashid, 1971; Schnitzer, 1971). Humic substances have an appreciable exchange capacity due primarily to carboxyl and phenolic hydroxyl groups, and can form stable watersoluble and insoluble complexes with metal ions (Rashid, 1971; Schnitzer and Skinner, 1965; Khanna and Stevenson, 1962). Bisogni and Lawrence (1973) have stated that a large fraction of the total mercury in natural water systems exists in sediment as a humate or mineral soil complex. An example of man-made chelates is trisodium nitriloacetic acid, which is contemplated as a possible detergent additive. This could lead to an increase in the concentration of the chelant in water and may play an important role in making metals more soluble and, therefore, accessible, by forming chelates. The anion forms of NTA can react with appropriate metal ions to produce the metal chelate (Thom, 1971): NTA3-

X

Mz+ % NTA - M-

Swisher et al. (1973)have indicated that NTA metal chelates are biodegradable and, therefore, would not be expected to accumulate in the environment. However, the actual effect of metal chelation on the biological cycling of toxic elements in the environment is largely unknown. Metals and their transformation products can also occur in the environment adsorbed on soil and/or suspended particles in air and water. The adsorption of mercury to colloidal particulate matter in natural water systems is a readily observed phenomenon. Hinkel and Learned (1969) reported that the particulate matter in natural waters contained 5-25 times more mercury than the water itself. Rapid adsorption of mercury to alluvium in natural waters has been noted by Dall'Aglio (1971).The adsorption of arsenite by soil has been reported by many researchers (Quastel and Scholefield, 1953; Misra and Benjamin, 1962; Misra and Tiwari, 1963). The amount of arsenite adsorbed was found to increase linearly with the increase in arsenite concentration; a small but constant amount of arsenite, however, appeared to be irreversibly bound to the soil (Quastel and Scholefield, 1953). Other factors that influence arsenite adsorption in soil include the amount of ferric oxide or sesquioxides, amount of calcium or other ions, pH of the soil, and the presence of arsenate (Misra and Tiwari, 1963). Adsorption of the methylated forms of arsenic in various soils has been reported by Dickens and Hiltbold (1967). Adsorption was related generally to the clay content of soil. Selenite may form adsorption complexes with ferric oxide in soil (Geering et al., 1968). Arsine and alkylarsines have a considerable affinity for surfaces; they adsorb strongly to suspended particles in air and water (D. L. Johnson, personal communication).

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JITENDRA SAXENA A N D PHILIP H . HOWARD

Ill. Test Methods for Studying Transformation A number of Merent approaches have been used to study environmental transformation of toxic metals. Four components which are essential to a test procedure are: the metal or metal compound to be investigated, the environmental exposure agents (e.g., microorganisms, light, etc.), exposure medium, and the analytical method. The choice and source of biological agent and environmental conditions used in the test procedure are among the most important fictors that influence transformation of metals and organometallic compounds. The laboratory test procedures cover natural communities of microorganisms, pure culture studies, cell-fkee extract studies, and model ecosystems. In this section the experimental details for the test procedures (e.g., microbiological systems used, incubation conditions, test compound) are described. The analytical procedures used for monitoring biotransformation are described in Section IV. A. BIOLOGICALTRANSFORMATION IN THE AQUATIC ENVIRONMENT

1 . Methylation of Metals a. Mixed-Culture Studies. Biological methylation of mercury compounds by aquatic organisms was first studied by Jensen and Jernelov (1969). They treated bottom sediments from freshwater aquaria with HgCl,; untreated samples and suspensions sterilized by autoclaving served as controls. The formation of dimethylmercury from mercury or from monomethylmercury was studied using homogenates of rotten fish (Xiphophorus maculatus) as the biological material. The conversion was studied under anaerobic conditions, which were obtained by flushing the flasks with nitrogen. A lake water sediment system, supplemented with nutrient broth and glucose to stimulate microbial growth, has been used by Wong et al. (1975) to study methylation of lead. The authors found that the oxygen consumption accompanied with growth was sufficient to generate anaerobic conditions. Pond water supplemented with nutrients was used as a biomethylation system for arsenite by Braman (1975). He found that bottom sediment or anaerobic conditions were not needed for methylation. This led him to conclude that methanobacterium were not the sole source of environmental biomethylation. Langley (1973) used a system containing river water sediment and overlaying water (collected simultaneouslywith the help of Jenkins sampler) to which gold fish had been added to allow for possible concentration of the methylmercury generated. The increase in the methylmercury content of fish above the background was taken as a measure of methylmercury generation. The author assumes that the methylmercury formed in the sediment will be quantitatively removed by the fish, and that no methylation

ENVIRONMENTAL TRANSFORMATION OF METALS

195

of mercury will occur in the fish itself. Since neither of these points have been proved yet, the results obtained with a methylmercury assay system such as above should be viewed with caution. Bisogni and Lawrence (1973) constructed microbial reactors for studying mercury transformations; the reactors could be operated under anaerobic (low redox potential) and aerobic (high redox potential) conditions and were equipped with a dimethylmercury trap and a mercury scrubber system. In either mode, the units were operated on a semicontinuous basis. Inorganic mercury in the form of mercuric ion was introduced with the feed solution consisting of carbonaceous-nitrogenous media, nutrient salts, buffer salts, and tap water. b. Pure-Culture Studies-lntact Cells and Cell-Free Extracts. Tonomura et al. (1972) have studied the formation of methylmercury by an anaerobic bacterium (Clostridium cochlearium) isolated from the soil. The organism was incubated anaerobially (gas phase, nitrogen) in a medium containing organic nutrients, cysteine, mercuric chloride, and cobalamin, and the methylmercury formed was assayed. Vonk and Sijpesteijn (1973) studied the methylation of mercury during aerobic growth of several bacterial and fungal species which are commonly found in water and soil; sublethal concentration of mercuric ions were used in their experiments. A few reports are available in the literature in which researchers have utilized the mercury-resistant bacteria in transformation studies with mercury and its compounds. For example, Hamdy and Noyes (1975) used H$+-resistant Enterobacter aerogenes isolated from river sediment. The culture chamber was connected to a series of traps designed to scrub organomercury compounds as well as elemental mercury. McBride and Wolfe (1971) studied the biological formation of alkylarsines utilizing pure cultures of a methanogenic bacterium (Methanobacterium strain M.O.H.). This bacterium was chosen since the extracts of this bacterium were earlier shown to catalyze the formation of methylmercury from inorganic mercury (Wood et al., 1968). In order to assay for the formation of alkylarsine, whole cells of the bacterium were incubated with sodium arsenate in a gas atmosphere of H,-C02. Fleming and Alexander (1972) studied the formation of dimethylselenide and dimethyltelluride by a strain of Penicillium isolated from raw sewage. The fungus was isolated by plating on a medium containing inorganic salts, organic carbon source, and N+,SeO,. The ability of the isolated fungus to form volatile products (dimethylselenium and dimethyltellurium) was investigated by inoculating the medium (defined medium or autoclaved municipal sewage) containing inorganic selenium or tellurium compounds with the washed spore suspension of the fungus. For many metals, methylation reactions have been studied with the cellfree extracts of microorganisms. Wood et al. (1968) have studied the synthe-

196

JITENDRA SAXENA A N D PHILIP H. HOWARD

sis of methylmercury compounds using the extracts of methanogenic bacterium (Methanobacterium strain M.O.H.) grown on hydrogen and carbon dioxide (80:ZO). The reaction mixture for the assay of the formation of methylmercury and dimethylmercury contained crude extracts, adenosine triphosphate as energy source, H$+, and methylcobalamin. The gas phase was hydrogen which served as the source of electrons. Similar conditions were used by McBride and Wolfe (1971) to study the formation of dimethylarsine fi-om sodium arsenate. A reaction flask which was heated in a boiling water bath prior to incubation was run parallel to the experimental flask to determine the extent of chemical methylation of arsenic. c. Field Studies. Jacobs and Keeney (1974) studied methylmercury formation and Hg loss from mercury-treated river sediments during in situ equilibration. Bulk sediments were collected from river sites, treated with mercuric chloride or phenylmercuric acetate and returned to the river with untreated controls. After equilibration with the river environment for various intervals, samples were removed for analysis. Kania et al. (1973)have investigated the h t e of mercury in artificial stream channels 300 feet long, 2 feet wide, and 1 foot deep lined with a polyvinyl chloride film. Washed builders sand was distributed into the channels to a uniform depth of 2 inches. The flow of water was set to provide 25 gallons per minute into each channel. The average retention time in the stream was 2 hours. The channels were allowed to seed naturally except that mosquito fish, Gambusiu affznis, were introduced a month before mercury was added. Various components of the biotic community were periodically sampled and analyzed for mercury.

2 . Degradation of Organornetallic Compounds Spangler et al. (1973a) have studied the bacterial degradation of methylmercury in lake sediment. They incubated a mercury-enriched sedimentwater mixture under aerobic conditions to permit the formation of methylmercury; an aliquot from this flask was used to inoculate the medium containing methylmercury in tryptic soy broth to study methylmercury degradation. Uninoculated controls containing methylmercury were run simultaneously. By direct isolation and isolation from enrichment cultures, these researchers obtained pure cultures of aerobic and anaerobic microorganisms capable of degrading methylmercury (Spangler et al., 197313). Biodegradability of a range of NTA metal chelates in river water has been investigated by Swisher et al. (1973). River water was allowed to settle for 2 weeks and then decanted before use. After the addition of NTA to the water, selected metals were added separately as chlorides. Another sample was set up with NTA and no added metal. Biodegradability was assessed by analyzing for NTA at various intervals.

ENVIRONMENTAL TRANSFORMATION OF METALS

B.

BlOLOGICAL TRANSFORMATION IN THE

197

SOIL ENVIRONMENT

The degradation work in the soil environment has largely been restricted to organoarsenical pesticides and some mercurials. Researchers have used many different types of soil in these studies. The soil types vary from sand (low in organic matter content, usually less than 1%)to muck soil or peaty soil (high in organic matter content, usually higher than 50%).The organic matter in soil affects the microbial activity, and may be responsible for adsorption and chelation of metals. The decomposition of methanearsonate (MSMA) was studied by Dickens and Hiltbold (1967)in several different soils of varying organic matter content, which had been moistened to field capacity. Herbicide decomposition was also investigated in the presence of added decomposable organic matter. For studying persistence of cacodylic acid, Woolson and Kearney (1973)brought the soils to 75%field capacity and the incubation was carried out under aerobic as well as anaerobic conditions. Mercury methylation in the terrestrial environment has been studied by enriching soil samples with Hg2+ and then analyzing soil and/or the atmosphere above the soil for methylated mercury compounds (Beckert et al., 1974; Rogers, 1975). Von Endt et al. (1968) used soil-enrichment technique and isolated a fungus, several actinomycetes and several bacteria capable of metabolizing methanearsonate. Solidified universal salt solution (Kearney et al., 1964) containing methanearsonate was inoculated with the isolates of soil microorganisms, and degradation was studied. A few field studies dealing with the transformation of metals in soil have been reported. Johnson and Braman (1974)enriched a soil plot of nearly 0.05 m3 with Hg2+ solution, and placed bottomless bottles over the plot. Samples of air were periodically drawn for analysis by pumping from the tops of the bottles. Braman (1975) used a similar technique to study the transformation of arsenic compounds in soil. To demonstrate methylation of mercury under field conditions, Beckert et al. (1974) enriched small agricultural plots with radiolabelled H$+. Plots were watered daily and soil samples were collected for organic mercury analysis at periodic intervals. C. MODEL ECOSYSTEM AND AQUARIUM STUDIES

(MICROCOSMS) A model ecosystem is a laboratory simulation of a dynamic biological environment characteristic of the natural ecosystem. By varying the number of the food chain elements, both simple and very complex laboratory model ecosystems have been devised to study transformation of toxic metals and other environmental perturbants.

198

JIPENDRA SAXENA AND PHILIP H . HOWARD

Bahr and Ball (1969), in their studies on arsenic transformation, used separate aquariums for groups of similar organisms as well as a complete ecosystem. Aquariums containing sand, pond mud, or gravel and water with plants, fish, or invertebrates, each in separate aquariums, were used to study the fate of arsenicals in the absence of interference by other &tors. A complete microcosm containing all the above food chain elements was also simultaneously used by these researchers. Metcalf and co-workers (1971; Lu et al., 1975) used a terrestrial-aquatic ecosystem, involving a food web of both herbivorous and carnivorous organisms, to study the environmental fate of cadmium and lead. The terrestrial portion of the ecosystem consisted of washed sand and soil mixture molded into a sloping surface. Application of the metal was made directly to the terrestrial phase to simulate an application to a crop and provide for transport from land to a typical lake water situation. In another experiment, sewage sludge served as the source of heavy metal. Isensee et al. (1973)have used a modified version of Metcalf's model ecosystem to study the distribution and fate of alkylarsenicals. In the modified system, the terrestrial phase of the ecosystem of Metcalfwas omitted, but a small soil inoculum was added to treated and control (untreated) tanks. [14C]-Cacodylicacid was added directly to the tanks while [14C]-dimethylarsinewas first absorbed to the soil and then added to the aquarium. In view of the bct that cacodylic acid is used predominantly in controlling pests of cotton, Schuth et al. (1974) reexamined the fate of cacodylic acid in a model ecosystem which contained bottom-feeding organisms (cat fish and cray fish-indigenous to cottonproducing areas) and duckweed (Lemna minor L), daphnids, and snails. IV. Analytical Procedures The accuracy of the experimental results from metal transformation studies depends to a great extent upon the analytical method employed. Transformation of heavy metals, like other environmental contaminants, is generally studied by following the formation of the metabolites and products. The volatility of the several intermediate metal compounds and their tendency to adsorb on particles and surface may sometimes present a problem in accuracy. The analytical procedure employed for studying metal transformation usually involves following three steps.

A. ISOLATION STEPS Prior to analysis, the metal and its transformation products have to be extracted from the reaction medium and then concentrated. The choice of extractant is, perhaps, one of the critical steps in any analysis. Volatile

ENVIRONMENTAL TRANSFORMATION OF METALS

199

metabolites of heavy metals are usually first trapped in a suitable mixture and then recovered by extraction with organic solvents. HgBr2-KBr solutions are generally employed as the trapping solution. This is particularly suited for trapping mercury vapors or organomercurials (dimethylmercury) (Spangler et al., 1973a,b). A Hg(N03)2-HN03 trapping solution is equally effective in trapping, but is not widely used owing to low extraction efficiency for organomercurials (Spangler et d.,1973b). For extraction of organic mercury, the benzene-cysteine extraction technique developed by Westoo (1967) is frequently used (Fang, 1973; Bisogni and Lawrence, 1973; Vonk and Sijpesteijn, 1973). Inorganic mercury is not extracted into the organic solvent phase and, therefore, provides no interference. The recovery in benzene-cysteine extraction technique is 98 & 3%. For measurement of dimethylmercury, the gas samples containing dimethylmercury are passed through a solution of mercuric chloride in 2 N HC1 prior to subjecting them to benzene-cysteine extraction. For extraction of mercury from fish, sulfuric acid:nitric acid mixtures have been used. Methylmercury and inorganic mercury have been collected from natural waters with the help of chelating resins (Law, 1971). The collected mercury compounds are eluted with a slightly acidic solution of thiourea, and the resin is reused. Chelating resins frequently have also been used to collect gold and platinum metals (Koster and Schmuckler, 1967; Green and Law, 1970; Green et al., 1970). Volatile alkylarsines have been trapped by oxidation to nonvolatile acids by treatment with nitric acid kept in ethanol-ice bath (McBride and Wolfe, 1971). Other researchers have used silver diethyldithiocarbamate-pyridine solution for trapping alkylarsines (Powers et al., 1959; Sachs et al., 1971). A second method based on the property of alkylarsines to react with the redrubber stopper used to seal the reaction vessel has also been used (McBride and Wolfe, 1971). Braman (1975) reported trapping alkylarsines from air samples in a column containing glass microbeads coated with metallic silver. Volatile selenium compounds have been collected by flushing the gaseous phase of the culture flasks into a stainless-steel column containing Porapak 0s (Cox and Alexander, 1974). Low-temperature collection of dimethylselenide and dimethyldiselenide on a column packed with OV-1 on Chromosorb W has been used by Chau et al. (1975). Volatilization by conversion to arsine has frequently been practiced as a method of separation of arsenicals from background impurities. In this process As (5+) is first reduced to As (3+)with stannous chloride and/or KI, which is further reduced to arsine with zinc in acid (Madsen, 1971; Fernandez and Manning, 1971). Some researchers have used titanium chloride for the preliminary reduction and magnesium in acid for generating arsine (Pollock and West, 1973). Braman and co-workers (1972; Braman and Foreback, 1973) have used sodium borohydride to reduce arsenic compounds to arsine.

200

JITENDRA SAXENA AND PHILIP

n. HOWARD

B. ANALYSIS The following analytical procedures have been used for detection of metal transformation products.

1. Gas Chromatography The method is used widely for determining trace amounts of volatile metabolites of heavy metals which are thermally stable (Fleming and Alexander, 1972; Jensen and Jernelov, 1969; Spangler et al., 1973a,b; Vonk and Sijpesteijn, 1973). Methylated forms are analyzed by gas chromatographic detection of CH3MX or CH3MCH3(where M = metal, X = halogen) using an electron capture detector. Dimethyl intermediates may be first converted to monomethyl derivates prior to injection in the gas chromatograph in order to increase the sensitivity of the electron capture detector. Gas chromatographyhas also been used for analysis of trace metal via their conversion to thermally stable volatile derivatives. Arsenic has been converted to trichloride (Vranti-Piscou et al., 1971), trifluoride (Juvet and Fisher, 1965), trimethylsilyl derivative (Butts and Rainey, 1971), and more recently to triphenylarsine (Schwedt and Russel, 1972; Talmi and Bostick, 1975); antimony to triphenylstibine (Talmi and Norvell, 1975); selenium to 5-nitrobenzoselenadiazole (Talmi and Andren, 1975); and certain other metals to trifluoroacetylacetonate derivative (Ross et al., 1965; Bayer et al., 1971, Lisk, 1974). Of the various GC systems used for trace metal analysis through synthesis of volatile derivatives, the most recently applied is the GC interfaced with a microwave emission spectrometric detector described by Talmi and co-workers (Talmi and Bostick, 1975).

2. Use of Labeled Compounds. One of the simpler ways to study the transformation of heavy metals is by the use of radioactive elements, or organometallic compounds labeled in the element or in the carbon. Researchers have used 14C-labeledcompounds for studying the fate of arsenicals in soil and in model ecosystems (Von Endt et al., 1968; Dickens and Hiltbold, 1967; Woolson and Kearney, 1973; Isensee et al., 1973). 74As-labeledcompounds have been utilized by McBride and Wolfe (1971)and Bahr and Ball (1969). In studies concerning the degradation of organomercurials, z03Hgor 14C-labeledcompounds have been used (Fang, 1973; Furukawa et d.,1969; Nelson et al., 1973). Radioactive metabolites formed are separated by thin-layer chromatography and may be identified by cochromatography. Preparative chromatography can be used to obtain small quantities of material for further identification of unknown metabolites. The radiolabeled metabolites can be more easily detected and identified. Furthermore, if the label is in the metal, a mass balance of the metal and transformation products is possible.

ENVIRONMENTAL TRANSFORMATION OF METALS

20 1

3. Atomic Spectroscopy In conventional flame atomic absorption spectrometry, the specific element is burned in the flame and the energy absorbed at a specific wavelength is measured. The conventional flame emission or absorption spectrometry is not widely used primarily because of the availability of the techniques that afford superior sensitivity. For example, flameless or cold vapor method is both sensitive and rapid and has been used in heavy metal analysis by Iskandar et al. (1972), Jacobs and Keeney (1974), Chu et al. (1972), Bisogni and Lawrence (1973), and others. The technique involves chemical reduction of test metal to the elemental form, its volatilization into a long-pathlength absorption tube, and measurement of the absorption at appropriate wavelengths. Chemical agents, such as benzene, toluene, xylene, chloride, interfere with the method and can produce positive absorbance peaks. In atomic fluorescence spectrometry, the metal or metal compound is atomized by flame or nonflame cell, illuminated from a source of excitation, and the resulting fluorescence radiation is measured. Atomic-fluorescence has less frequently been used in environmental studies. West (1974) has shown by theoretical treatment that in the case of mercury, atomic fluorescence using cold vapor technique is more sensitive and offers considerably less interference from nonspecific impurities than the atomic absorption technique. A modified cold-vapor mercury-fluorescence detector having increased sensitivity has recently been described by Thompson and Godden (1975). A number of reports have dealt with the application of emission spectrometry in environmental analysis and transformation studies. Flame emission is, however, not sufficiently sensitive owing to inadequate excitation energies provided by the flame. To obtain the required sensitivities, more efficient excitation sources, e.g., induction-coupled plasma and direct current-discharge plasma have been used. In plasma emission spectroscopy, the metal or metal compound is introduced into a helium or argon plasma to yield free electronically excited metal atoms; their emission intensity is measured by standard photometric detection equipment. The technique has been used to determine trace amounts of volatile arsenic compounds (Lichte and Skogerboe, 1972b; Braman and Foreback, 1973), mercury (Braman and Johnson, 1974; April and Hume, 1970; Lichte and Skogerboe, 1972a), and antimony (Talmi and Norvell, 1975). 4 . Spectrophotometric Procedure

Although not as sensitive as some of the recently available techniques for trace metal analysis, spectrophotometric techniques are fairly commonly used because of low cost and simplicity. The diphenylthiocarbazone

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JITENDRA SAXENA AND PHILIP H. HOWARD

(dithizone)method is probably the most widely used colorimetric method for detection of trace levels of mercury. In this method, dithizone is allowed to react with H&+ or H e + in acid solution to form a colored complex, which is extracted with chloroform or carbon tetrachloride (Sandell, 1959; Snell and Snell, 1949). The absorption of the complex is measured at 490 nm or alternatively the decrease in dithizone absorbance at 610 nm is measured. The procedure can be used to determine 0.5 to 50 ppm of mercury. Presence of copper, silver, gold, etc., interferes with the determination. Preliminary treatment to ensure decomposition of organic material is required in order to determine organic-bound mercury by this method. The silver-diethyldithiocarbamate (AgDDC) colorimetric method has been used for determination of arsenic (Powers e t al., 1959; Dickens and Hiltbold, 1967). In this method arsenate is first reduced to arsenite, which is then allowed to react with hydrogen to form gaseous arsine. Arsine is trapped in silver diethyldithiocarbamate-pyridine solution and the stable red complex formed is measured at 540 nm. The lower limit of detection for this method is 2 ppb (Braman and Foreback, 1973). The method can be used for determination of arsenic in organic arsenicals. However, the carbonarsenic bond should be first ruptured by digestion with nitric acid-sulfuric acid oxidation procedure (Sachs et al., 1971). Arsenic has also been determined by reaction with acidified molybdate; the arsenomolybdate complex upon reduction produces a blue complex (Portmann and Riley, 1964; Johnson and Pilson, 1972).

5. Neutron Activation This procedure can be used for determination of several elements with great sensitivity (Robertson and Carpenter, 1974). The technique involves exposing a sample to a source of neutrons to produce a radioactive nuclide of the element (Lyons, 1964). For mercury, for example, irradiation results in two radioactive nuclides, lS7Hgand '03Hg. Although the method is extremely sensitive, it has rarely been used in environmental transformation studies. Its use has so far been restricted to monitoring for trace levels of metals in environmental samples. The major disadvantage of this technique is that it requires special irradiation facilities and data handling. 6. X-Ra y Fluorescence

In this method low-energy photons are used to excite the characteristic X-ray energy of the element (Wallace et al., 1971). The X-rays emitted are then sorted and measured using a solid-state Ge(Li)or Si detector coupled to a multichannel analyzer. Because of the low sensitivity of this technique, preconcentration of the sample is usually required. In one reported method (Marcie, 1967), the element is reacted with ammonium pyrollidine

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203

dithiocarbamate prior to detection by X-ray fluorescence. Watanabe et al. (1972)determined arsenic by precipitation with diethyldithiocarbamate and subjecting the precipitate directly to X-ray fluorescence.

7. Polarographic Methods This technique is more suitable as a supplementary and reference method, since its sensitivity is not high enough for use in analysis of environmental samples (Wallace et al., 1971). The method can be useful for monitoring changes in the oxidation state of arsenic (Myers et al., 1973; Whitnack and Brophy, 1969). 8 . Limits of Detection of Various Methods The limits of detection of the well known instrumental methods which have been used in studying transformation of metals are summarized in Table I. C. IDENTIFICATIONAND SPECIATION Differentiation of the various chemical forms of toxic metal is an integral part of any environmental transformation study. Thin-layer chromatography is one of the commonly used techniques for separation and identification of mercury compounds. Most frequently researchers have used silica gel and alumina thin-layer plates (Westoo, 1969;Tatton and Wagstaf€e, 1969; Hamdy and Noyes, 1975). Johnson and Vickers (1970) have used thin-layer plates to separate various organic mercurials and inorganic mercury. In several studies organomercurials have been reacted with dithizone to form dithizonate complexes prior to separation by thin-layer chromatography (Beckert et al., 1974). Von Endt et al. (1968) have used thin-layer plates coated with silica gel G (calcium sulfate binder) to separate various inorganic and organic arsenicals. The compounds were identified by cochromatography. A few researchers have separated the arsenicals by electrophoresis on cellulose thin-layer plates (McBride and Wolfe, 1971). More recently, arsenic compounds have been separated by their reduction to corresponding arsines, which are accumulated in cold trap and selectively volatilized in the order of their boiling point (Braman and Foreback, 1973). The disadvantage of speciation following reduction of arsenicals to correspondingarsine is that it will fail to distinguish between certain types of organoarsenic compounds-for example, between dimethylarsine and dimethylarsinic acid. Gas chromatography has been used for separation of arsines (Talmi and Feldman, 1975; Talmi and Bostick, 1975) and of volatile alkyllead and selenium compounds (Chau et al., 1975). Separation of atmospheric mercury species has been obtained by passing the samples of air

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JITENDRA SAXENA A N D PHILIP H. HOWARD

TABLE I INSTRUMENTAL LIMITSOF DETECTION IN TRACEMETAL ANALYSIS~

Method

Gas chromatography Electron capture Helium ionization Hydrogen atmosphere FID Mass spectrometry Electron impact

Sensitivity (9)

10-13 10-12

10-12 10-12

Spark source 10-13 Ion probe 10-15 10-11 GC/MS/computer Atomic absorption spectroscopy Flame Flameless Atomic emission spectroscopy

10-9 10-12

X-Ray fluorescence

10-7

Neutron activation analysis Wet chemical methods Polarography

(ppm-ppb) 10-*-10-"

Application

Electrophilic organics, e.g., CH,HgCl All gases Highly specific to metalloorganic compounds Detects all elements and most inorganic and organic compounds

Over 60 elements detectable with different sources

High sensitivity (lo-''via inductively coupled and direct current discharge plasmas Used for elements with atomic numbers above 11 Responds to all elements Methods available for most metals Detects most metallic elements and compounds

"Karasek (1975), and Karasek and h u b (1974). 'Talmi and Feldman (1975), Johnson and Braman (1975), and Braman and Johnson (1974).

through a connected series of tubes containing selective absorbers for various species-a glass fiber filter to retain particulate mercury, Chromosorb W, siliconized with SE 30 methyl silicone for H$+, Chromosorb W, treated with sodium hydroxide for methylmercury, silvered glass beads for elemental mercury, and gold-coated glass beads for dimethylmercury (Braman and Johnson, 1974; Johnson and Braman, 1974). This speciation device has been used by Rogers (1975) to study environmental transformation of mercury in soil. An ion-exchange procedure involving the use of the reagent isothiocyanatopentaaquochromium for differentiating inorganic and organic mercury ions has been reported by Baltisberger and Knudson (1975). Determination of mercury in water before and after photodestruction of dissolved organic carbon by ultraviolet irradiation has been used to measure organic mercury in natural waters (Fitzgerald and Lyons, 1973). Combined gas chromatography-mass spectrometry has been extensively used for detection of metabolites (Fleming and Alexander, 1972; Jensen and

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205

Jernelov, 1969). The technique has an almost universal ability to separate components and provide a mass spectrum for identification as well as detection. Overall fiagmentation patterns of the authentic methylated form of the trace metal can be compared with that of unknown compounds for identification. In certain cases, however, absorption of the chemical on the large surface area in G U M S systems and thermal decomposition may greatly reduce the applicability of this technique.

V. Factors Affecting Transformation of Metals The factors that affect transformation of metals are numerous and have varied from test to test and from laboratory test to natural conditions. A discussion of these factors is important to understand how and to what extent they are responsible for variations in the test results and consequently to the evaluation of the experimental methodology.

A. FACTORS AFFECTINGMETHYLATIONOF METALS Factors that influence the methylation of metals are generally the same as those which &ect other microbiological transformations. These include the concentration of the metal, number and species of microorganisms present and their growth rates, acclimation, adsorption and chelation of the metal, presence of other chemicals including supplemental nutrients, and physical parameters, such as pH, temperature, and redox potential of the test medium.

1. Concentration of the Metal Jensen and Jernelov (1969)studied the formation of methylmercury under aerobic conditions as a function of inorganic mercury concentrations in the lake sediment. Their findings indicated that methylmercury production increased with increasing inorganic mercury dosage up to 1OO p g per gram of sediment. A further increase in inorganic mercury caused a sharp decrease in the formation of methylmercury, probably due to the inhibition of the methylating microorganisms. In the river sediment-water systems in which gold fish were used to concentrate methylmercury generated in the sediment, maximum methylation rate was observed at a mercury concentration of 3 mditer (Langley, 1973). An increase in methylmercury formation with increasing inorganic mercury concentrations has also been observed under anaerobic conditions (Bishop and Kirsch, 1972; Bisogni and Lawrence, 1973). Fleming and Alexander (1972) studied the formation of dimethylselenide from inorganic selenium, and they found that in the medium containing 10 pg of of selenite per milliliter, as much as 13-24% of the added

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JITENDRA SAXENA A N D PHILIP H. HOWARD

selenite was converted to dimethylselenide, whereas at 10-fold higher selenite concentration, less than 2% of the selenium was recovered in the dimethylated form. 2. Microbial Activity

Perhaps one of the most important factors that affects the results of a metal transformation study is the type of microorganisms present and the number of each type. The source of microorganisms in the environmental transformation studies has been river and lake sediments, sewage, activated sludge, river water, soil, etc. Many of these sources have been examined for their bacterial composition and have been found to be extremely diEerent from each other. For example, activated sludge from varied sources was found to contain 72 different species classified in 14 genera (McKinney and Wetchlein, 1955). Hoadley and McCoy (1965)have reported isolation of 11species of bacteria from the lakes and streams. Methylation of metals has been demonstrated to be catalyzed by anaerobic as well as aerobic bacteria (Tonomura et al., 1972; Vonk and Sijpesteijn, 1973; McBride and Wolfe, 1971), and fungi (Fleming and Alexander, 1972; Cox and Alexander, 1973; Vonk and Sijpesteijn, 1973). Since all five organisms investigated by Vonk and Sijpesteijn (1973) were able to methylate mercury, these researchers concluded that a slight capacity to methylate mercury may be a rather common property of aerobic bacteria. Whether anaerobic or aerobic methylation plays a dominant role in methylation of metals under natural conditions is unclear. Since fungi generally do not contribute significantly to the microbial activity in lake sediments, their role in methylation may be more important in soil. In addition to the type of microorganisms, microbial growth rates have also been found to influence methylation rates of metals. Bisogni and Lawrence (1973) have reported that with doubling of net specific anaerobic growth rates (from 1/24 per day to 1/12 per day) the net specific methylation rate increased by a hctor of approximately 3. A similar doubling of the aerobic growth rate caused approximately a %fold increase in the methylation rates. 3 . Microbial Acclimation

Frequently there is a lag between the exposure of toxic metal to the microbial agents and the beginning of transformation. This lag is attributed to the need for “acclimation” which refers to a variety of processes which take place during the lag period, such as enzyme induction, development of tolerance, selection of a species of microorganisms, etc. Cox and Alexander (1973) noted that cells of C. humfcola grown in the presence of arsenate produced alkylarsine more rapidly than cells proliferating in media without

ENVIRONMENTAL TRANSFORMATION OF METALS

207

arsenate. These observations suggest the need for induction of enzymes for alkylarsine synthesis. Although the fungus could also catalyze the methylation of selenium, the findings indicated that the enzymes catalyzing the methylations of arsenic and selenium were not induced simultaneously (Cox and Alexander, 1974). It has been noted that certain microorganisms can develop tolerance to rather high mercury concentrations which is not lost upon subsequent growth on mercury-free media (Rissanen and Miettinen, 1972). Plasmidmediated resistance to toxic metals has been observed in enteric bacilli (Summers and Silver, 1972). Furukawa and Tonomura (1972a,b) have noted that Hg-volatilizing enzyme can be induced in a Pseudomanas strain by growing the cells in the presence of a number of mercurials. The relationship between resistance to mercury and the mobilization and transformation of mercury is unclear at the present time. It has been proposed that mercury volatilization and production of methylmercury may be a means of resistance and detoxification against mercurials (Hamdy and Hoyes, 1975; Wood, 1975). Colwell and co-workers (1975; Sayler et al., 1975) studied the role of mercury-resistant bacteria in the amplificationof mercury in the aquatic food chain and found a 200-fold increase in mercury accumulation in oysters dosed with mercury-resistant, mercury-metabolizing bacteria compared with oysters held in the absence of the bacteria. 4 . Adsorption and Chelation of Metals The adsorption of elemental contaminants to inorganic and organic constituents of water and soil systems is well documented (Dall’Aglio, 1971; Hinkel and Learned, 1969; LagenverfF, 1972). Both adsorption and complexation of the metal ion may influence the methylation process by making a particular metal ion unavailable for methylation. It is apparent, however, that adsorption and chelation by sediment does not completely prevent methylation of mercury because many successful methylation studies have been performed in the presence of sediment (Jensen and Jernelov, 1969; Olson and Cooper, 1974).

5 . Presence of Other Chemicals The presence of a number of chemicals has been shown to influence the methylation of metal ions. The effect is generally observed via one of the following mechanisms: the chemical may influence the metabolic activity of the test organisms; the chemical may react with the metal ion, thereby rendering it unavailable for methylation; sometimes the chemical (for example, other metals) may be preferentially methylated. Fagerstrom and Jernelov (1971)were able to show that methylmercury production is dependent on the biochemical availability of inorganic mercury. These investigators

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n. HOWARD

observed that sulfide was extremely inhibitory to the methylation of mercury. Sulfide serves as a binding agent by converting the soluble mercuric ions to insoluble HgS. To obtain the same yield of methylated mercury from mercuric sulfide as from divalent inorganic mercury, the concentration of mercury had to be nearly 103 times higher (Fagerstrom and Jernelov, 1971). Sulfide is formed in anaerobic benthic regions of the environment from sulfate by sulfate-reducing bacteria. Tonomura et al. (1972) confirmed the inhibitory effect of sulfide by studying the methylation of mercury by a culture of C . cochkarium in the presence of sulite-reducing bacteria. These researchers observed very small amounts of methylmercury formation in the combined culture compared with that produced in the control. Fleming and Alexander (1972), while studying the alkylation of selenite, observed that dimethylselenide formation increased with increasing sulfate concentrations. In Candida humicola phosphate was able to inhibit the synthesis of trimethylarsine from arsenite, arsenate, and monomethylarsenate but not from dimethylarsinate (Cox and Alexander 1973). High antimonate, selenate, selenite, and tellurate concentrations also depressed the rate of conversion of arsenate to trimethylarsine, but nitrate was without effect (Cox and Alexander, 1974). The decrease in trimethylarsine production by selenium and tellurium was suggested to be due to their ability to complex with trimethylarsine. The presence of biodegradable organic matter in the reaction medium has generally been shown to enhance the rates of methylation. Bishop and Kirsch (1972) observed a stimulation of methylation rates in anaerobic systems when organic nutrients, e.g., glucose-glutamic acid and acetate, were added to the medium. Langley (1973) showed that methylation rates increased with increase in the organic sediment index, a measure of the organic carbon and nitrogen in the sediment sample. Wood et al. (1968) have suggested that nutrient enrichment of methylation systems (for example, by the addition of sewage) would increase methylation rates by increasing the numbers of bacteria capable of synthesizing alkylcobalamin (a methyl donar and cofactor for methylation). Increased production of methylmercury in the presence of methylcobalamin has been reported by Hamdy and Noyes (1975). Addition of homocysteine, a compound known to stimulate methylmercury synthesis in Neurospwa (Landner, 1971), was found to decrease the formation of methylmercury in bacterial cultures (Hamdy and Noyes, 1975).

-

6. Physical Parameters, such as p H , Temperature, and Redox Potential of the Test Medium

The pH of the methylating system could affect the rates of methylation of inorganic metal either by affecting the microbial enzyme system responsible for this transformation, by affecting the responsible organisms, or by affect-

ENVIRONMENTAL TRANSFORMATION OF METALS

209

ing the distribution and availability of the proper species of metal ion for methylation. Jernelov et al. (1972) have reported that the pH optimum for methylation of mercury either under laboratory or natural conditions is 4.5. A negative correlation between the levels of methylmercury in fish (presumably an indicator of microbiological methylation of mercury) and pH has been noted by a number of researchers (Olsson, 1969; Jernelov, 1972). This has been attributed to a changeover from the formation of mono- to dimethylmercury, which might evaporate when formed (Fagerstrom and Jernelov, 1972; Jernelov, 1969b). Several investigators (Langley, 1971; Bishop and Kirsch, 1972) have reported that the methylation process was dependent on the temperature of the reaction medium. Bisogni and Lawrence (1973) found that temperature (range 10"-30°C) does not significantly affect the aerobic or anerobic methylation reaction if a constant growth rate of microorganisms is maintained. These authors have suggested that the effect of temperature on methylation observed by Langley (1971), and Bishop and Kirsch (1972) may most likely be due to temperature-related changes in the microbial growth rate, which also affected the methylation process. A relationship between the conversion of mercury compounds and redox potential has been studied by Tonomura et al. (1972). They found that methylmercury is formed by Clostridium cochlearium at about +50 mV. If the redox potential is lowered to -200 mV, sulfate-reducing bacteria become predominant and produce large amounts of sulfide. Mercuric ions combine with sulfide to form insoluble mercury sulfide, which is not available for methylation. No methylation was observed under aerobic conditions, redox potential approximately 300 mV. These observations suggest that factors other than mere exclusion of oxygen (e.g., redox potential) may be important in anaerobic methylation of metals.

+

B. FACTORS AFFECTINGDEGRADATION OF ORGANOMETALLIC COMPOUNDS Only a few reports are available concerning the effect of various environmental and nutritional parameters on the transformation of organometallic compounds. The biodegradation mechanisms of organometallic compounds and purely organic compounds are somewhat similar except that degradation of organometallic compounds may give rise to a metal in addition to other chemical metabolites and end products. In view of the similarity between the biodegradation mechanisms of organometallic compounds and that of other organic chemicals, it appears likely that important variables pertinent to their biodegradation will be similar. These may include types of microorganisms, mineral salt composition, test chemical concentrations, supplemen-

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n. HOWARD

tary nutrients, and physical parameters such as pH, temperature, light. For example, the stimulation of the breakdown of monosodium methylarsonate (MSMA) due to the presence of supplemental organic carbon source was reported by Von Endt et d. (1968). These researchers observed that soil isolates released much greater amounts of 14C02from [14C]-MSMA when yeast extract was added to the culture medium. Woolson and Kearney (1973) have reported that soil which had received cacodylic acid previously and presumably had an adapted microbial population, metabolized cacodylic acid more readily than fresh soil. These findings suggested that microbial adaptation could significantly affect the biodegradation of chemical compounds in the environment. VI. Biochemical Pathways and Mechanisms for Transformation of Metals Information concerning the pathway and mechanism of metal transformation has been derived largely from pure culture and cell-free extract studies. Although very reproducible, the disadvantages associated with these studies include (1)failure to account for transformation catalyzed by a combined action of many microorganisms or by synergistic action, and (2) the general remoteness of the conditions from natural processes in soil or water. An additional problem due to the absence of permeability barrier is that the intermediates that are formed by the cell-free extracts may never appear outside the cell. Although the pathways or the mechanisms established in pure cultures of microorganisms and/or in cell-free extracts may not be easily extrapolated to natural conditions, Alexander (1972) has suggested that such studies could serve as useful guides for what to look for under natural conditions. Biochemical pathways and transformation mechanisms are known for some metals. Methylmercury has been reported to be formed by both enzymic and nonenzymic reactions. According to Wood, cobalamin plays a central role in the synthesis of organometallic compounds in both enzymic and nonenzymic systems (Wood et al., 1968, 1972; Desimone et al., 1973). In aerobic microorganisms and facultative anaerobes, the mechanism of methylmercury synthesis involves electrophilic attack on the CH3 group of the methylcobalamin-methionine synthetase complex leading to the formation of methylmercury and the aquocobalamin-enzyme complex (Wood et al., 1972). The coenzyme is reduced by FADH2 and remethylated by CH3 transfer from N-methyltetrahydrofolate. In strict anaerobes, such as Methanobacillus, methane synthetase is associated with metalalkyl formation. In view of the fact that low redox potential (> -400 mV) is required for

ENVIRONMENTAL TRANSFORMATION OF METALS

21 1

growth of these anaerobes, any inorganic mercury salts added to the methane synthetase enzyme system should be reduced to HgO which, after methyl-radical addition may give rise to alkylmercury compounds (Wood et al., 1972). The methylation process involving methylcobalamin has been demonstrated in the cell-free extracts of a methanogenic bacteria (Wood et al., 1968).In Neurospora crassa, a fungus in which cobalamin is not known to be involved in metabolism, the methyl group is transferred to the mercury atom, which is complexed on homocysteine (Landner, 1971). Thus, methylation of mercury by this mechanism may be visualized as “incorrect” synthesis of methionine. A mechanism for nonenzymic methylation of mercury which involves transfer of the alkyl group from alkylcobalamin to mercuric ion to form dimethylmercury as the first product, has been suggested by Imura et al. (1971). Dimethylmercury then reacts with mercuric ion to give monometh ylmercury . The enzymic reduction of inorganic divalent mercury to metallic mercury has been shown to be mediated by electron transfer agents-NADH (Wood, 1974), NADPH, FADHP and a c-type cytochrome (Furukawa and Tonomura, 1972a,b). Involvement of cellular electron andlor energy transfer reactions in the reduction of H 2 + is also suggested from the inhibition of mercury volatilization by respiratory poisons, observed by Summers and Silver (1972). The mechanism of alkylation of arsenic is unclear at the present time. Studies have indicated that methylcobalamin is a methyl donor in biomethylation of arsenic (McBride and Wolfe, 1971). The biochemical pathway leading to formation of methylated forms of arsenic has been suggested to involve first the reduction of arsenate (5+) to arsenite (3+), which is methylated to form methylarsonic acid (arsenic, 3+); the latter compound is reductively methylated for form dimethylarsinic acid (arsenic, 1+)which is finally reduced to dimethylarsine (arsenic, 3-) (Fig. 3). The synthesis of monomethyltin has been shown to occur by a reaction involving oxidative addition to methylcobalamin (Wood, 1975). The process involves oxidative methylation of the stannous ion by reaction with methylcobalamin (Co3+)to give rise to methyl-stannic ion coupled with the formation of reduced cobalamin (Co’). Alkylation of metal ions in the environment could also occur by the process of transmethylation. For example, methyliodide, which is known to be synthesized biologically in the marine environment, can methylate a number of metal ions (Wood, 1975). Also, tin has been reported to transfer its methyl group abiotically in water to H$+, resulting in the formation of methylmercury (Huey et al., 1975).

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JITENDRA SAXENA AND PHILIP H. HOWARD

II

II

II

0

0 Arrenite

A rsen ate

0 Methylarsonic acid CH3-B1 Z

2eJBlZr CH3 CH3

I

I Asp3

- CH3

I

HO - AS - CH3

I1 0

H

Dimethylarsinic acid ( cacodylic acid ) CH3-B,

= Methylcob(%+)alamin

B12r = Cob(Z+)alarnin

FIG. 3. Pathway of methylarsine synthesis from inorganic and methylated arsenicals in Methonobacterium sp.

VII. General Discussion of Various Test Methods Toxic heavy metals can enter the environment either in organometallic, inorganic, or elemental form. Organometallic compounds, such as phenylmercuric acetate, upon degradation yield inorganic heavy metals which may be more or less toxic than the organic compound forms. The resulting metals may be environmentally transformed via the routes previously shown in the biological cycle for each metal. The fate of organometallic compounds has generally been determined by methods similar to those employed for determination of environmental persistence of an organic compound. An inherent problem in studying the fate of organometallic compounds by most laboratory test methods will be the accumulation of toxic metal with degradation. The resulting metal may inhibit further breakdown of organometallic compounds. In contrast, in natural aquatic environments, the inorganic toxic metal formed is continuously removed by dilution and/or volatilization. The possibility of methylation-demethylation interconversion of metallic compounds sets up a dynamic system of reversible reactions that results in steady-state concentrations of various metallic and methylated forms in the environment. The disturbances produced in the steady-state concentrations

ENVIRONMENTAL TRANSFORMATION OF METALS

213

caused by the introduction of metal into the natural ecosystem as a result of man’s activities will affect the natural equilibrium, which, in turn, will affect the concentrations of toxic intermediates. In assessing the environmental fate of toxic metals, therefore, what needs to be answered is not only whether a particular metal can be methylated, but also whether the kinetics of the process will allow significant levels of the methylated form to build up. The test methods used thus far have succeeded in answering only part of the question, e.g., whether a particular metal can be methylated. The techniques used for these studies have included microbial reactors, shake culture studies with pure and mixed cultures of microorganisms, and microecosystems. The aerobic and anaerobic microbial reactions constructed by Bisogni and Lawrence (1973) appear to be more suitable for investigational use. Bisogni and Lawrence have used these reactors for studying the effect of various environmental conditions on microbial methylation of mercury, where a careful control of incubation conditions is desirable. More commonly used are simpler tests, i.e., batch type in which investigators have incubated pure or mixed cultures of microorganisms with inorganic forms of a metal under aerobic and/or anaerobic conditions and have identified the methylated product formed (Vonk and Sijpesteijn, 1973; Jensen and Jernelov, 1969; Fleming and Alexander, 1972; McBride and Wolfe, 1971). Mixed cultures may sometimes give erroneous results since they may be expected to contain microorganisms responsible for both methylation and demethylation of the test heavy metal. Depending upon the conditions chosen for the test, their activities could be affected and may influence the results of the test. Natural communities, however, may be preferred over pure cultures, since it is unlikely that any single microorganism can methylate all the metals. The use of aquatic microecosystems has become an eRective tool for studying biotransformation of metals (Isensee et al., 1973; Schuth et al., 1974; Lu et al., 1975). A microecosystem allows the investigator to examine the interconversions of metallic compounds in a somewhat dynamic system. Since metal transformation as well as transport and bioaccumulation of the transformation products are expected to take place in the model ecosystem owing to the presence of a complete food chain, a kinetic study of the metal transformation is also feasible. The information obtained could perhaps be more easily extrapolated to the natural environment to assess whether biological transformation of any metal would result in accumulation of toxic intermediates in significant concentrations in the environment. There can, however, always be doubt that the kinetic rate of each transformation reaction is the same in the model ecosystem as in the environment. Since the accumulation of substantial quantities of any toxic intermediate is so much dependent

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JITENDRA SAXENA AND PHILIP H . HOWARD

on the rate of each reaction, a change in the kinetic rate of one single reaction in the model ecosystem could yield results different from those in the actual environment. Numerous reports have become available from which it is clear that mercury can be methylated either by enzymic (microbial) or chemical mechanisms (Jensen and Jernelov, 1969; Wood et aZ., 1968; Imura et ul., 1971; Bertilisson and Neujahr, 1971). Whether these reactions occur to a significant extent in the environment has yet to be determined. Spangler et ul. (1973b)have stated, “the inability to find even traces of methylmercury in most sediments taken from areas highly polluted with inorganic mercury leads one to question whether significant methylation occurs in sediment under environmental conditions or whether the turnover rate of any methylmercury formed is such that significant concentrations do not accumulate.’’ These investigators (Spangler et al., 1973b) have shown that a large number of microorganisms occurring in the lake sediment possess the ability to aerobically degrade methylmercury. These species may be important in suppressing the methylmercury content of the sediment. Similarly, species of microorganisms that degrade methylated forms of arsenic have also been found to be present in the environment (Von Endt et aZ., 1968). Although many researchers have failed to detect significant concentrations of methylmercury in lake sediments, it has generally been assumed that the methylmercury found in fish is the result of formation before intake beginning with methylation of inorganic mercury in sediments. A few studies have been conducted to determine whether methylation of mercury and mercury compounds occurs in fish. Ukita and Imura (cited in Kojima and Fujita, 1973) reported that yellow fin tuna liver homogenates were able to methylate mercury. Other fish or mammalian liver homogenates tested failed to catalyze mercury methylation. Jernelov (1972) reported that microorganisms predominant in fish slime and fish intestine have the ability to methylate mercury, but found no evidence of mercury methylation in the fish itself. Wood (1973) has pointed out that measurement of steady-state concentrations need not reflect the rate of synthesis of methylmercury. He further stated that the rate of synthesis of methylmercury does not have to be very rapid in sediments for fish to accumulate dangerous levels of it; when the rate at which methylmercury is produced, then released into the water and taken up by fish, exceeds the rate of metabolism of methylmercury in fish, then methylmercury will accumulate in fish. Therefore, what should be measured, according to Wood (1973), is the concentration of total mercury in the sediment and the rate of methylmercury uptake in fish. However, such measurements still would not distinguish whether methylmercury was taken up by or formed in the fish.

ENVIRONMENTAL TRANSFORMATION OF METALS

215

VIII. Correlation between Laboratory and Field Results and Monitoring Data Laboratory studies that model environmental reactions of metals and metal compounds have little utility if they are not comparable to reactions in the natural environment. Generally, laboratory techniques have attempted to retain as many important factors of the normal environment as possible. These factors are, however, so numerous, and their influence so interwoven in the case of metals and metal compounds, that any laboratory test method may be unable to account for all the parameters. In spite of these shortcomings, suitable laboratory transformation studies have provided some information that can be extrapolated to some degree to the natural conditions. The ultimate evaluation of a technique for studying environmental transformation reactions is a comparison of its results to field studies and environmental monitoring data.

A. ARSENIC The monitoring data of Braman and co-workers (Braman and Foreback, 1973; Johnson and Braman, 1975) on the methylated forms of arsenic in the environment can provide some insight into the type of transformation reactions that are of environmental significance. Braman and Foreback (1973) have reported that dimethylarsinic acid was the major form of arsenic in the environment. Methylarsonic acid, although found, was present in much smaller concentrations. Laboratory studies have shown that microorganisms can methylate arsenic to alkylated arsines and that both dimethylarsinic acid and methylarsonic acid may be intermediates in the arsenic methylation sequence (McBride and Wolfe, 1971). The presence of alkylarsenic (primarily trimethylarsine) in atmospheric samples has been shown by Johnson and Braman (1975). In view of the fact that dimethylarsinic acid may be resistant to oxidation, and if it is reduced by microorganisms to dimethylarsine it will be readily oxidized back to dimethylarsinic acid, this form of arsenic may predominate in the environment. The actual situation is more complex than this, since both dimethylarsinic acid and methylarsonic acid are added in the environment in the form of pesticides and, therefore, one will expect to find these forms in the environment. The estimated United States production for arsenic pesticides indicates that methylarsonic acid is produced in much larger quantity than dimethylarsinic acid. For example, in 1971, 70 million pounds of disodium and monosodium methylarsonate were produced, whereas only 2 million pounds of sodium cacodylate were produced (U. S. Environmental Protection

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Agency, 1972). If no transformation of these compounds occurred in the environment, one would expect to find methylarsonic acid as the major form of arsenic in the environment. The fact that dimethylarsinic acid was the predominant form as shown by the studies of Braman and Foreback (1973), can be interpreted to mean that methylation of methylarsonic acid was occurring in the environment and was responsible for the accumulation of dimethylarsinic acid. McBride and Wolfe (1971) were able to show transformation of methylarsonic acid to dimethylarsinic acid in their laboratory studies with cell-free extracts of Methanobacterium. Field studies had demonstrated the conversion of arsenicals to alkylarsines (Braman, 1975), which upon oxidation will also yield dimethylarsinic acid, the persistent form of arsenic. B. MERCURY It has been estimated that 70% of the mercury consumed in the United States is lost to the environment (Saha, 1972). Jernelov (1969a)has reported that the main types of mercury discharged in the environment are: inorganic divalent mercury, metallic mercury, phenylmercury, and methylmercury. Alkylmercury compounds are used predominantly for seed dressing,and this usage accounts for less than 1%of the total mercury consumption (calculated &om the data in U.S. Environmental Protection Agency, 1973; U.S. Tariff Commission, 1973). Other major sources of mercury in the environment are industrial effluents, consumer use and misuse and disposal of products containing mercury; losses of mercury from these sources appear to be largely in the form of metallic and inorganic divalent (e.g., from chloralkali industries), and phenylmercury (e.g., from paper and pulp industry). If no transformation of mercury compounds was occurring in the environment, one would expect to find less than 1%of the mercury in the form of methylmercury. The analysis of the atmospheric samples for various species of mercury revealed that 14-19% of the total atmospheric mercury was in the form of methylmercury (Table 11). The statistics of total mercury and methylmercury in a lake or estuary showed that roughly 1-10% of the total mercury present in the aquatic environment may be in the form of methylmercury (Jernelov and Lann, 1973).An increase in the methylmercury concentration relative to the total mercury introduced in the environment would suggest interconversion of mercury compounds in nature. Conversion of inorganic and phenylmercury compounds to alkylmercury has been demonstrated in numerous laboratory studies (Jensen and Jernelov, 1969; Fagerstorm and Jernelov, 1971; Jernelov and Rudling, cited in Jernelov, 1969a; and others) and is supported by the field studies (Johnson and Braman, 1974, Rogers, 1975). However, in many instances there are certain quantitative differences be-

217

ENVIRONMENTAL TRANSFORMATION OF METALS

TABLE I1 MERCURY SPECIESCONCENTRATION IN ENVIRONMENTAL SAMPLESO Percentage of total mercury in

Mercury species Particulate mercury Inorganic divalent mercury Metallic mercury Dimethylmercury Monomethylmercury

Atmosphere near ground 2-6 19

60

<1-1 14-19

Lake or estuary sediment

t I

Lake or estuary water

Biota

t I

t I

90-99

99

1-10

1-10

il

90-99

‘Johnson and Braman (1974) and Jernelov and Lann (1973).

tween the laboratory studies and the monitoring data. For example, during incubation of soil with Hg2+ in the laboratory, Rogers (1975) noted that dimethylmercury was the predominant species, followed by elemental mercury, ionic mercury, and then methylmercury. The order of predominance of the mercury species is not in agreement with the monitoring data of Johnson and Braman (1974), who found that in the atmospheric samples the predominant species was HgO followed by ionic mercury, methylmercury, and then dimethylmercury.

IX. Restoration of Metal-Contaminated Areas Nearly 4 million pounds of mercury are lost to the environment annually (calculated from the data in U.S. Environmental Protection Agency, 1973; Saha, 1972). It has been estimated that ~ 0 . 1 % of the total mercury present in the aquatic ecosystems will be converted to methylmercury annually (Jernelov and Lann, 1973). This suggests that even if the use of mercury were to stop today, there would still be enough mercury in the environment to continue to release methylmercury for a considerably long time. The efforts to restore mercury-contaminated bodies of water and reduce health hazards must, therefore, be oriented toward slowing down or inhibiting the synthesis of methylmercury from the mercury already present in the environment. A number of theoretically possible methods have been suggested for this purpose (Wood, 1972; Jernelov and Lann, 1973; Dean et al., 1971; D’Itri, 1971).These include: (1) dredging heavily polluted areas, (2) covering the sediment of heavily polluted areas to reduce the rate of methylmercury formation and its release into water, (3) making mercury unavailable for methylation by converting it to mercury sulfide, or binding to inorganic

218

JITENDRA SAXENA AND PHILIP H. HOWARD

material such as silica minerals, (4) improving water quality to reduce microbial population, (5) increasing pH to facilitate the synthesis of volatile dimethylmercury over monomethylmercury, and (6) inhibition of microbial methylmercury formation by addition of metabolic inhibitors. Some laboratory experiments directed toward testing the feasibility of the proposed restoration methods have been described in the literature Uernelov and Lann, 1973; Langley, 1973; Lindstron, 1968; Lann, 1970). The efficiency of the measures in reducing mercury contamination in fish as revealed from laboratory studies is shown in Table 111. Dredging is not successful because it has a tendency to resuspend mercury and distribute inorganic mercury over a wider area (Wood, 1972). Of the other methods tested, binding of the mercury with sulfide or silica mineral proved to be highly efficientin reducing mercury methylation. Small-scale field tests have been carried out by Jernelov and Lann (1973) to evaluate the feasibility of these methods in the field. The results of these studies are, however, inconclusive. It has been observed that chlorinated hydrocarbons irreversibly inhibit methane synthetase and methionine synthetase, the two enzyme systems generally implicated in methylmercury synthesis (Wood et al., 1968; Penley

RESULTS OF

THE

TABLE 111 LABORATORY INVESTIGATION OF THE RESTORATION METHODS FOR MERCURY-CONTAMINATED WATER Reduction in CH3Hg in fish over control

Method tested 1. Dredging 2. Substances added to cause the formation of mercuric sulfide Glucose (to achieve anaerobiosis) Sz-

3.

4. 5. 6.

FeS FeSz Covering mercury contaminated sediment with sand Combination of mercury binding and covering with silica mirierd Raising the pH from 6.7 to 7.1 by CaC03 addition Sediment covered with fluorspor tailings

("/.I

Reference

Levels increasedover control

Wood (1972) Jemelov and Lann (1973)

75 95

84 60 53 >99.9

Lann (1970) Lindstrom (1968)

81

Jernelov and Lann (1973)

82

Langley (1973)

219

ENVIRONMENTAL TRANSFORMATION OF METALS

et al., 1970). Interestingly enough, in locations where sediments were polluted with both inorganic mercury and chlorinated hydrocarbons, fish were found to accumulate very little methylmercury. The problem with chlorinated hydrocarbon application for control of mercury methylation is that under these conditions, chlorinated hydrocarbons accumulate in fish (Wood, 1971; Wood et al., 1972).

X. Categorization of Elements By examining the current knowledge of the physical and chemical properties of a toxic element, it is possible to make certain predictions as to how some of these materials may behave in the environment. Such information could be helpful in deciding which of the toxic metals should be monitored in the environment. Wood (1974) has classified elements on the basis of their toxicity and their relative availability for environmental transformation, as determined by their solubility characteristics and concentrations at which they occur in the environment naturally. According to this classification, toxic elements can be considered to be (1)noncritical, (2) toxic and relatively accessible, or (3) toxic but very insoluble and very rare. The elements fit in these categories as shown in Table IV. The elements classified as very toxic and relatively accessible should be of major concern since they have the highest potential for environmental hazard. The relative mobility of these elements in the environment, as well as their toxicity, is somewhat dependent on their ability to undergo methylation. Wood (1974) has predicted that tin, palladium, platinum, gold, and

TABLE IV CATEGORIZATION OF ELEMENTSFROM THE STANDPOINT OF ENVIRONMENTAL HAZARDS"

Noncritical

Very toxic and relatively accessible

Toxic but very insoluble or very rare ~~~

Na

K Mg Ca H 0 N

C P Fe S C1 Br

F Li

Rb Sr A1 Si

Be Co Ni Cu Zn Sn

As Se

TI Pd Ag Cd Pt

Au Hg Te Pb

Sb

Ti Hf Zr W Nb

Bi

Ta Re

Ga

La 0s

Rh Ir Ru Ba

~~~~~

"From Wood (1974). Reprinted with permission from Science 183, 1049 (1974). Copyright 1974 by the American Association for the Advancement of Science.

220

JITENDRA SAXENA AND PHILIP H . HOWARD

thallium will be methylated in the environment, but that lead, cadmium, and zinc will not be methylated. This prediction is based on the fact that the alkylmetals of lead, cadmium, and zinc are not stable in aqueous systems and that methylcobalamin does not transfer methyl groups to these elements. Recently, Wood (1975) has shown methylation reactions in case of tin, palladium, and gold. Contrary to Woods (1974) prediction, however, researchers have also been able to show methylation of lead (Wong et al., 1975); also presumptive evidence for the formation of methylated cadmium species has recently been presented by Huey et al. (1975). Wood (1974) in these predictions does not take into account the possibility that even if certain alkylmetals are unstable in aqueous systems, in the cell microenvironment they may be sufficiently stable or perhaps remain enzyme bound to serve as methyl donors for the formation of another and more stable methylated compound. It is interesting to note in this context that Huey et d. (1975) have proposed a transmethylation reaction between CH3-Cd species to H 8 + . The authors stated that although protolysis in the aqueous environment would suppress accumulation of methylcadmium species, the present information does not disallow kinetically competitive transmethylation from CH3-Cd species to H$+. No experimental evidence is available at the present time to support or dispute the occurrence of such transmethylation reaction. Even if it can be predicted which metals can be methylated, it still needs to be determined whether methylation will occur in the natural environment, and if so whether it will result in accumulation of substantial quantities of the methylated form in the environment. The methylation process in the environment is considerably more complex than in laboratory studies, and prediction of environmental methylation rates is extremely difficult. For example, the kinetics of degradation of an alkylmetal [as shown by Spangler et al. (1973a,b) for methylmercury].will be important in determining the net quantity of the methylated form in the environment. No attempts have so f;?r been made to formulate a methylation model that will take into account the kinetics of all the processes occurring in the environment.

XI. Summary and Conclusions Understanding the behavior and transformation of metals and metal compounds is an important parameter in the overall evaluation of their potential environmental hazards. The study of metal and metal compound transformations in the environment requires a variety of different considerations than for organic chemicals, since the metal portion of the compound cannot be converted to innocuous end products, as with the organics (CO,, H,O). Also, many of the reactions are reversible and thus the kinetics become very important to the

ENVIRONMENTAL TRANSFORMATION OF METALS

22 1

cycling of the metal through the environment. A variety of processes may take place, such as degradation of organometallic forms to inorganic forms and valence changes, methylation, or chelation of the metal. For studying the transformation of toxic metals, both mixed and pure cultures and model ecosystems have been used. However, in most cases these techniques only answer the qualitative aspects of metal transformations (e.g., whether a metal will be methylated). Qualitative information is important for organic compounds, since the processes are generally irreversible. However, with metals, reversible reactions, e.g., methylation-demethylation, oxidationreduction, can take place, so that the reaction rates are extremely important. These reaction rates will vary from one test method to another. Thus, although laboratory techniques are important for determining organometallic degradation and the possibility of metal methylation and/or valence change, field studies should be used to determine the kinetics of metal transformations. Setting priorities for environmental research on metals and organometallic compounds is an extremely complex process. A number of parameters can assist in the decision-making process. The quantity released into the environment and the toxicity of the material provides some indication of the environmental hazards involved, and thus the degree of testing needed. Physical and chemical properties of a metal, such as solubility, stability of the methylated form, may give some idea of the possible behavior in the environment, and thus be helpful in deciding the type of testing needed and the experimental procedure. During this review a number of areas were identified where more information is needed to h l l y assess the environmental behavior and hazards from metals. The kinetics and mechanisms of various metal transformation reactions in the environment have not been adequately studied. Also important to understand is the actual role of different biological systems in metal transformation. For example, at present it is normally assumed but not proved that microorganisms methylate metals that are taken up by higher food chain organisms. Additional work is needed to elucidate the role of nonbiological processes (photochemical and chemical) in metal transformation in the environment. More quantitative comparisons between laboratory and field studies and monitoring information concerning various forms of metal in the environment are necessary. And finally, more effort needs to be directed to find ways of predicting the transformation reactions that a metal may undergo in the natural environment.

ACKNOWLEDGMENTS The authors wish to thank Dr. D. L. Johnson for valuable assistance in the preparation ofthis review. Supported in part by Environmental Protection Agency Contract 68-41-2210.

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Rissanen, K., and Miettinen, J. K. (1972). In “Mercury Contamination in Man and His Environment,” STUDOC/10/137. IAEA, Vienna. Robertson, D. E., and Carpenter, R. (1974). “Neutron Activation Techniques for the Measurement of Trace Metals in Environmental Samples,” NAS-NS-3114. Prepared by Subcommittee on Radio-chemistry, U.S. Atomic Energy Commission, Washington, D.C. Rogers, R. D. (1975). Proc. Int. Conf. Heavy Metals Enoiron., 1975 Paper C-218. Ross, W. D., Sievers, R. E., and Wheeler, G. (1965).Anal. Chem. 37, 5 9 M 0 0 . Sachs, R. M., Michael, J. T., Anastasio, F. B., and Wells, W. A. (1971).Weed Sci. 19,41%416. Saha, J. G. (1972). Residue Rev. 42, 103-163. Sandell, E. B. (1959). “Colorimetric Determination of Metals.” Wiley (Interscience), New York. Sapozhnikov, D. I. (1937). Mikrobiologiya 6, 643444. Sayler, G. S., Nelson, J. D., and Colwell, R. R. (1975). Appl. Microbiol. 30, 91-96. Schnitzer, M. (1971). In “Organic Compounds in Aquatic Environment” (S. J. Faust, ed.), 5th Rudolf Res. Conf., pp. 297315. Dekker, New York. Schnitzer, M., and Skinner, S. I. M. (1965). Soil Sci. 99, 27%284. Schottel, V., Mandal, A,, Clark, D., Simon, S., and Hedges, R. W. (1974). Nature (London) 251, 335-337. Schuth, C. K., Isensee, A. R., Woolson, E . A., and Kearney, P. C. (1974). J . Agric. Food Chem. 22, 999-1003. Schwedt, G., and Russell, H. A. (1972). Chromatographia 5, 242-245. Snell, F. D., and Snell, C. T. (1949). “Colorimetric Method of Analysis,” 3rd ed., Vol. 11. Van Nostrand-Reinhold, Princeton, New Jersey. Spangler, W. J., Spigarelli, J. L., Rose, J. M . , and Miller, H. M. (1973a). Science 180, 192-193. Spangler, W. J., Spigarelli, J. L., Rose, J. M., Flippin, R. S., and Miller, H. H. (1973b).Appl. Microbiol. 25, 488-493. Summers, A. O., and Silver, S. (1972).J. Bacteriol. 112, 1228-1236. Swisher, R. D., Taulli, T. A,, and Malee, E. J. (1973). In “Trace Metals and Metal-Organic Interactions in Natural Waters” (P. C. Singer, ed.), pp. 237-263. Ann Arbor Sci. Publ., Ann Arbor, Michigan. Talmi, Y., and Andren, A. W. (1975). Anal. Chem. 46, 2122-2126. Talmi, Y., and Bostick, D. T. (1975). J. Chromatogr. Sci. 13, 231-237. Talmi, Y.,and Feldman, C. (1975). In “Arsenical Pesticides” (E. A. Woolson, ed.), pp. 13-35. Am. Chem. SOC.,Washington, D.C. Talmi, Y., and Norvell, V. E. (1975). Anal. Chem. 47, 151CL1516. Tatton, J. 0. G., and WagstaEe, P. H. (1969).J . Chromatogr. 44, 284-289. Thom, N. S. (1971). Water Res. 5, 391-399. Thompson, K. E., and Godden, R. G. (1975). Analyst 100, 544-548. Tonomura, K., Furukawa, K., and Yamada, M. (1972). In “Environmental Toxicology of Pesticides” (F. Matsumura, G. M. Boush, and T. Misato, eds.), pp. 115-133. Academic Press, New York. Turner, A. W. (1954). Aust. J . Biol. Sci. 7 , 452A78. Turner, A. W., and Legge, J. W. (1954). Aust. J. Biol. Sci. 7 , 479-495. U. S. Environmental Protection Agency (1972). “The Pollution Potential in Pesticide Manuhcturing,” Pestic. Study Ser. 5, Tech. Rep. TS-00-72-04. U.S. Environmental Protection Agency (1973). “National Disposal Site Candidate Waste Stream Constituent Profile Report-Mercury, Arsenic, Chromium, and Cadmium Compounds,” Vol. VI, TRW Syst. Group, Rep. No. 21485-6013-RU-00. U. S . Tariff Commission. (1973). “Synthetic Organic Chemicals, United States Production and Sales 1971,” T.C. Publ. No. 614. U.S. Tariff Comm., Washington, D.C. Von Endt, D. W., Kearney, P. C., and Kaufinan, D. D. (1968).J. Agric. Food Chem. 16,17-20.

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Vonk, J. W., and Sijpesteijn, A. K . (1973). Antonie oan Leeuwenhoek 39, 505513. Vranti-Piscou, D., Kontoyannakos, J., and Parissakis, G. (1971). J. Chromtogr. Sci. 9, 499501. Wallace, R. A., Fulkerson, W., Shultz, W., and Lyon, W. S. (1971). “Mercury in the Environment, the Human Element,” NSF-EP-1. Oak Ridge Natl. Lab., Oak Ridge, Tennessee. Watanabe, H., Berman, S., and Russel, D. S. (1972). Tahnta 19, 1362-1375. Werner, J. (1967).So. Kern. Anal. Lung, 1967 (cited in Jernelov, 1969a). West, C. D. (1974). Anal. Chern. 46, 797-799. Westoo, G. (1967). Acta Chem. Scand. 21, 17W1800. Westoo, G . (1969). I n “Chemical Fallout” (M. W. Miller and G. Berg, eds.), pp. 7596. Thomas, Springfield, Illinois. Whitnack, G. C., and Brophy, R. G. (1969). Anal. Chirn. Acta 48, 123-127. Wong, P. T. S., Chau, Y. K., and Luxon, P. L. (1975). Nature (London) 253, 26%264. Wood, J. M. (1971). Ado. Enoiron. Sci. 2, 39-55. Wood, J. M. (1972). Environment 14, 33-39. Wood, J. M. (1973). Rev. I n t . Oceanogr. Med. 31-32, 7-16. Wood, J. M. (1974). Science 183, 104S10.52. Wood, J. M. (1975). Absh., Int. Conf. Heaoy Metals Enoiron., 1975 Paper A-5. Wood, J. M., Kennedy, F. S., and Rosen, C. G. (1968). Nature (London) 220, 173-175. Wood, J. M., Penley, M. W., and Desimone, R. E. (1972).1. A. E. A . , Tech. Rep. Ser. No. 137, pp. 49-65. Woolson, E. A., and Kearney, P. C. (1973). Enoiron. Sci. Technol. 7 , 47-50.

Bacterial Neuraminidase and Altered Immunological Behavior of Treated Mamma1ian CelIs PRASANTA K. RAY Chittaranjan National Cancer Research Centre, Calcutta, India I. Introduction ........................ .......... 11. Neuraminidase-the Receptor-Destroyin A. Occurrence in Bacteria and Viruses . . . . B. Neuraminidase in Animal Tissue, Cells, and Biological Fluids ........................... C. Neuraminidase and Its Relationship D. Substrate of the Enzyme-Sialic Acid ................. E. Neuraminidase Mechanism of Action and Substrate Specificity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F. Cell-Surface Sialic Acid and Malignancy . . . . . G. Sialolipids .............................. 111. Sialic Acid and Its Relationship to the Antigenicity of the Cell .................................... Surface . . . . IV. Increased Immunogenicity of Neuraminidase-Treated Cells . . A. Treated Normal Cells . . . . . . ......... B. Treated Embryonic Cells ............................ C. Treated Tumor Cells ...................... V. Regression of Established e Tumor . . . . . . . . . . . . . . Regression of Growing Tumors Caused by Neuraminidase-Treated Syngeneic Tumor Cells . . . V1. How Do Neuraminidase-Treated Tumor Cells React in the Host to Establish Specific Antitumor Immunity?. ........... A. Facilitated Contact between Neuraminidase-Treated and Untreated Cells ............................... B. Facilitation of Other Interactions Involving Killer an Target Cells . . . . . . ................. C. What is the Source ccurring Antibody? D. Nature of Neuraminidase-Exposed Antigenic Specificities E. Is There a Shift from Sialoglycolipid Synthesis to Sialoglycoprotein Synthesis during Malignant ............. Transformation? . . . . . . . . F. Why the Naturally Occu Anticarbohydrate Antibody .......................... VII. A Probable Mechanism by Which Neuraminidase-Treated tumor Immunity . . . . . Tumor Cells Give Rise to

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

227 228 229 229 231 231 233 234 236 237 243 243 244 244 246 246 249 249 250 254 255

258 259 259 260 261

I. Introduction A great deal of emphasis has been placed for the last few years on investigations of cell-surface properties. Especially in the field of immunobiology, 227

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cell-surface research has drawn the attention of a number of laboratories because of its tremendous importance in various types of immune reactions. Transplantation and tumor immunologists were involved in investigations relating to the cell surface because both the rejection of a transplanted graft and also the rejection and/or enhancement of a tumor depend very much on immune reactions involving target and effector cell surfaces. Investigators have spent considerable time trying to control the induced immune reactions that follow the graft of a foreign tissue or a tumor, with the objective of either having the transplanted tissue survive for a considerable time or destroying the tumor graft. Obvious procedures involved include the use of immunosuppressive drugs, various chemotherapeutic agents, radiation, etc. One of the significant and recent alternative approaches has been the modification of cell-surface antigenic specificities either directly or indirectly so as to make a transplanted tissue less antigenic and a tumor more antigenic or immunogenic. However, in this review I shall not discuss modification of a transplanted tissue to make it acceptable to the host or survive longer; discussions of the modification of normal and tumor cell surfaces to make them more immunogenic will be given priority. The unique tool that investigators found to be very useful was the enzyme-induced modification of the cell surface, since the substrates of many of these enzymes were normally expected to be present on the complex cell-membrane “glycocalyx” structure. However, these investigations have been centered around only a few enzymes. Of these, the most important are P-glucosidase (Gesner and Ginsberg, 1964), P-galactosidase (Gesner and Ginsberg, 1964), 1-asparaginase (Cappizzi et al., 1970; Bernard and Boiron, 1970), trypsin (Woodruff and Gesner, 1968; Billingham and Sparrow, 1955), and neuraminidase (Sanford, 1967; Currie and Bagshawe, 1968a,b; Currie et al., 1968; Woodruff and Gesner, 1967; Weiss et al., 1968; Forrester, et al., 1962; Gesner and Thomas, 1966; Dalmasso and MiillerEberhard, 1964). Because of limitations of space, I shall concentrate on the enzyme neuraminidase in this review. This review will deal mainly with the occurrence of neuraminidase in biological fluids and cells and its role in various pathogenic processes, and various functions of the substrate of the enzyme, i.e., sialic acid on the mammalian cell surface, antigenicity and immunogenicity of various cell types treated with neuraminidase, and the phenomenon of tumor regression induced by neuraminidase. II. Neuraminidase-The

Receptor-Destroying Enzyme

The discovery of neuraminidase has opened a wide area of biochemical and immunological research. Only a few enzymes in the history of enzyme

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research have created such an impact in a wide variety of scientific investigations. The enzyme, first detected in a virus (Hirst, 1941, 1942), was considered to be of tremendous importance as it was at that time generally believed that viruses were devoid of enzymes. Both Hirst (1941, 1942) and McClelland and Hare (1941) showed that chicken red blood cells were agglutinated by influenza virus particles. Hirst’s assumption that an enzyme present in influenza virus was responsible for the agglutination of red blood cells was later supported by Burnet et al. (1946). They termed the enzyme receptor-destroying enzyme (RDE) since the filtrates from Vibrio comma (V. cholerae) and Clostridium perfringens (C. welchii) destroyed the receptor sites for influenza viruses on the surhce of human erythrocytes. Subsequently, Ada and French (1959a,b) purified RDE and identified it as neuraminidase. Neuraminidase is an enzyme that falls in the category of “glycosidases.” It is also known as sialidase, since it splits off the terminal sialic acid residues (a %carbon sugar) from the nonreducing end of disaccharides, oligosaccharides, and polysaccharides (Gottschalk, 1957).

A. OCCURRENCE IN BACTERLA AND VIRUSES Neuraminidase is present in a number of bacteria and viruses (Table I). The various bacterial neuraminidases are generally found in their culture medium, and in certain cases it has been observed as a part of the cell, e.g., Corynebacterium diphtheriae neuraminidase. Ada and French (1959a,b) have shown that V. cholerae neuraminidase is an inducible enzyme and can be induced by sialic acid and N-acetylmannosamine. The protozoan Trichomonas foetus has also been shown to contain neuraminidase (Romanowska and Watkins, 1963). Most of the organisms when infecting their hosts usually focus on the respiratory or intestinal tracts, the lining of which contain mucinous substances susceptible to neuraminidase action (Burnet, 1948; Marion et al., 1953). Thus neuraminidase occurring in these bacteria and viral particles may have a role in the establishment of infections. It is possible that neuraminidase in the bacteria or viruses may bind with the substrate on the host cell and facilitate contact between the invading and attacked cell types, thus helping in the infective processes. B. NEURAMINIDASEIN ANIMAL TISSUE,CELLS,AND

BIOLOGICALFLUIDS Apart &om the wide availability of neuraminidase in intact bacteria and viruses, it has been found in a variety of animal tissues, cells, and biological fluids. It has been detected in saliva (Perlitsh and Glickman, 1967), rabbit

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K. RAY

TABLE I NEUIUMINIDASE IN VARIOUS BACTERIA AND VIRUSES

class

Name of the organism

Vibrio cholerae

Bacteria

Closhidium welchii Pneumococcus type I1 Diphtheroid bacilli Closhidium perfringens Clostridium septicum Bacteroides species Corynebacterium zenes Pasteurella multocida Corynebacterium diphtheriae

Mycoplama gallisepticum Streptococcal species Diplococcus pneumoniae Streptococcus sanguinis

Viruses

Corynebacterium hemolyticum Corynebacterium pyogenes Fusobacteriurn polymwphusm Trichomonas foetus Corynebacteria, mycobacteria, and nocardias kctobacillus bi$dus Pseudomonas group Measle virus Myxoviruses Fowl plague viruses Influenza virus A and B

Influenza virus Mumps virus Newcastle disease virus Parainfluenza

References Burnet et al. (1946), Gottschalk (1956), Heimer and Meyer (1956), Faillard (1956) Bohm et al. (1957) Heirner and Meyer (1956) Burnet (1951) Popenol and Drew (1957), Cuatrecases (1971) Gandalla et al. (1968) Muller (1970a) Muller (1971a) Muller (1971b) Jagleski (1969), Blurnberg and Warren (1961),Moriyamaand Barksdale (1967) Roberts (1967) Hyano (1967), Hyano and Tanaka (1969) Heimer and Meyer (1956), Kelly (1967) Muller (1974), Hyano and Tanaka (1969) Muller (1973b) Muller (1973h) Muller (1973a) Muller (1972) Arden (1972) Shilo (1957) Shilo (1957) Howe (1970), Howe et al. (1969) Drzeniek (1966) Drzeniek et al. (1966) Gottschalk and Perry (1951), Kuhn and Brossrner (1956), Gottschalk (1956, 1957) Hirst (1972) Ada and Stone (1950) Seto et al. (1959), Vasilera (1968) Sekol et al. (1961)

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23 1

kidney (Kuratowska, 1967),human sputum (Rolla, 1966),rat liver and kidney (Mahadevan et al., 1967), rat brain (Carubelli, 1968), thrombocytes and erythrocytes of cattle (Gielen et al., 1969), pig brain (Tettamanti and Zambotti, 1968), human intestinal mucosa (Ghosh et al., 1968), rat liver and Ehrlich ascites tumor cells (Horvat and Touster, 1968),bovine brain (Gielen and Harprecht, 1969), pig kidney (Tuppy and Palese, 1968), salivary glands of rats and human saliva (Nijar et al., 1970), mammalian sperm (Srivastavaet al., 1970), human brain (Ohman et al., 1970), rat mammary glands (Tulsiani and Carubelli, 1971), L cells (Glick et al., 1971),ganglioside (Ohman, 1971), HeLa cells (Carubelli and Griffin, 1970), transformed cells (Schenogrund, 1973), hepatic plasma membranes (Visser, 1973), etc. It should be noted that various types of both prokaryotic as well as eukaryotic cells and tissues contain neuraminidase. The presence of the enzyme in serum and various biological fluids may be due to the presence of neuraminidase-containing microorganisms in those fluids, which might have released the enzyme in the environment. It is possible that tissue injury caused by microorganisms may also lead in some cases to the release of lysosomal neuraminidase. Availability of this enzyme in a large number of tissues and biological fluids naturally raises the question: What are the functional necessities of this enzyme in those tissues, and why is it found in appreciable quantities in various biological fluids? AND ITS RELATIONSHIP TO C. NEURAMINIDASE PATHOGENICITY

The occurrence of neuraminidase in a broad spectrum of bacteria and viruses raises the question of why so many organisms contain this enzyme. The most obvious question is: Does neuraminidase have any role in bacteriaand virus-induced pathogenesis? At least in those cases where the organisms contain neuraminidase, it seems to be a possibility. Reports from various laboratories are in agreement with this view. These reports are summarized in Table 11. In view of these observations, and also of the fact that an increased level of sialic acid is available in various body fluids during various pathological conditions, it is reasonable to suggest that neuraminidase may play a key role in the process of pathogenesis by various bacteria and viruses. It remains to be established, however, that all the organisms that contain this enzyme use it as a means of invading the otherwise protected environment of tissues and cells. D. SUBSTRATE OF

THE

ENZYME-SIALICACID

The substrate for neuraminidase is widely distributed in animal tissues, secretions, and excretions. It is commonly known as sialic acid, a 9-carbon

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TABLE I1 NEURAMINIDASE AND ITS RELATIONSHIP WITH VARIOUS DISEASES Disease

References

Influenza Pneumococcal infections Gas gangrene caused by Clostridium perfingens Abscess caused by Bacteroides fi-agilis Bacterial meningitis Pneumonia Various other microbial pathogenicity Tdchomonas foetus infections Experimental meningitis

Krizanova (1967) Muller (1969) Muller (1970b) Muller (197Oc) O’Toole (1971) Fischer (1971) Vertievliuv (1973) Muller (1972) Carruthers (1973)

amino sugar, and it is found in a number of forms, e.g., N-acetylneuraminic acid, N-glycolylneuraminic acid, and 7-O-acetylneuraminic acid (Blix et al. , 1957). The N-acetyl and N-glycolyl derivatives of neuraminic acid have been observed in the cell periphery (Carubelli and Griffin 1968). N-Acetylneuraminic acid is an integral part of urine mucoprotein, bovine submaxillary gland mucoprotein, serum mucoproteins, and mucoproteins present in human erythrocyte stroma (Faillard, 1957; Bohm et al., 1957; Klenk and Lempfrid, 1957). Sialic acids are also present in such mucoproteins as meconium, ovarian cyst fluids, and human cervical mucus (Odin, 1955a,b,c). Porcine submaxillary gland mucoprotein also contains sialic acid (Blix et al., 1955, 1956). In 1950, Ada and Stone reported that the RDE from V. c o m m could reduce the cell-sudice negative charge of human erythrocytes, and they suggested that the released material contained acidic groups. In 1958, Klenk reported that acylated neuraminic acids might be largely responsible for the negative charges on erythrocytes. Later, Cook and Jacobson (1968) showed that reduction of cell-surface negative charge after neuraminidase treatment was associated with the liberation offi-ee sialic acid moieties from the cells. It should be pointed out, however, that sialic acids in mucins or glycoproteins are susceptible to neuraminidase hydrolysis. Sialic acids in glycolipids are resistant to neuraminidase digestion (Klenk, 1958; Weinstein et al., 1970). The presence of sialic acid on the surface of cells was demonstrated to be released by neuraminidase action (Weiss, 1961). Two different methods are normally used to have the direct estimation of neuraminidase-releasable

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sialic acids on the cell surface: (1) electrokinetic methods in which neuraminidase treatment is associated with the loss of cell surface net negative charge measured in a cell electrophoresis apparatus; (2) direct estimation of sialic acid in the supernatants of cells treated with the enzyme (Winzler, 1970; Weiss, 1969; Warren, 1959; Aminoff, 1961; Ray and Simmons, 1972, 1973a). A number of important factors have to be taken into consideration when measuring the sialic acid from cell surfaces. Sialic acid on the cell surface is not a constant factor available in equal quantities all the time. Its concentration may vary at various phases of the cell cycle (Mayhew, 1966; Brent and Forrester, 1967). It has been shown that neuraminidase-susceptible anionic sites and the cell-surface negative charge increases at the G2 phase of the cell cycle. However, contradictory reports are also available (Shank and Burki, 1971). Glick and associates (1971) showed an increase in the total sialic acid content on the surface of KB cells at about the time of mitosis. However, Warren and Glick (1968)stated that on cultured mouse fibroblasts the rates of synthesis of surface membranes were approximately similar in growing and nongrowing cells. E. NEURAMINIDASEMECHANISM OF ACTION AND SUBSTRATE SPECIFICITY As described earlier, Ada and French (1959a) purified RDE and identified it as neuraminidase, the enzyme which hydrolyzes the a-glycosidic bond joining the keto groups of sialic acid to a sugar or sugar derivative (Gottschalk, 1957). This enzyme is now known as Vibrio comma neuraminidase EC 3.2.1.18) which cleaves the 2.3-, 2,4-, 2,6- and 2,S-cll-glycosidic linkages between terminal N-acetylneuraminic acids and mucopolysaccharides (Drzeniek, 1967; Drzeniek and Gaube, 1970). It is important to note that all four types of linkages are cleaved by Vibrio cholerae neuraminidase (Drzeniek, 1967; Drzeniek and Gaube, 1970)and by Clostridium perfringens neuraminidase (Cassidy, et al., 1965). Viral neuraminidases, however, show significant differences in their ability to split various a-ketosidic linkages. Under well-defined conditions, fowl plague virus cleaves only 2,3 linkages, but Newcastle disease virus hydrolyzes 2,3and 2,s-a-ketosidic linkages. Thus, fowl plague virus neuraminidase fails to remove a sialic acid from disialyllactose, whereas Newcastle disease virus removes both the sialic acid residues (Gottschalk and Bhargava, 1971). It appears that bacterial and viral neuraminidases perhaps can be used as a probe for the elucidation of structural linkages between sialic acids and the carbohydrate prosthetic groups of the glycoproteins. Thus, the functional

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importance of various a-glycosidic linkages between sialic acid and carbohydrate moieties can be evaluated. Although careful experimental analysis is needed to find out the differential capabilities of bacterial and viral neuraminidases, some information is available to this effect. At a fixed period of incubation, bacterial and viral neuraminidases do show marked differences in their ability to split sialic acid moieties from normal and malignant cell surfaces (Ray et ul., 1970, 1972; Ray and Simmons, 1971b, 1972, 1973a). This differential response of their splitting capacities is also associated with the differences in properties of bacterial or viral neuraminidase-treated cells (Ray and Simmons, 1972, 1973a; Ray et ul., 1972; Ray and Sundaram, 1975a,b). Bacterial enzyme-treated mouse lymphoid cells become extremely sensitive to antibody- and complementmediated cytolysis (Ray et al., 1970, 1972; Ray and Simmons, 1972). Influenza virus neuraminidase is devoid of this property (Ray et ul., 1970; Ray and Simmons, 1972). Vibrio choZerue neuraminidase (VCN) is capable of making even cells of autologous origin susceptible to autologous immune destruction (Ray et al., 1970, 1971, 1972; Ray and Sundaram, 1975a,b), but influenza virus neuraminidase (IVN) is incapable in this regard (Ray and Sundaram, 1975b). Using VCN and IVN, it has been described that VCNreleasable sialic acids are present in larger amounts in both normal and various malignant cells (Ray and Simmons, 1973a); this suggests the predominance of the VCN-susceptible sialic acids in both normal and malignant cell types.

F. CELL-SURFACE SIALICACID AND MALIGNANCY The exact relationship with the sialic acid content in the cell periphery and the malignant transformation of growth is not known. How is sialic acid concentration increased on the cell surface of a malignant cell and why? Is it related to protection of the cell against an otherwise hostile immune environment? Is it a part of the process of malignant transformation of cells that genetic information is there for the increased synthesis of sialic acid on the cell surface of malignant cells? Does sialic acid help the malignant cell to escape the immune surveillance of the host? All these questions remain unanswered or partially answered. Pioneering experiments of Ambrose and associates (1956; Ambrose, 1966, 1967) showed that cell-surface negative charge was increased in stilbestrolinduced hamster kidney tumor cells and on dimethylaminoazobenzeneinduced rat hepatomas in comparison to what was present on the normal cells. In cultured hamster fibroblasts converted by polyoma virus, two distinct types of cells were observed: One was not distinguishable from the original cell type, and the other showed increase in sialic acid content. De-

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fen& and Gasic (1963) showed that polyoma-induced conversion of embryonic hamster BHK cells was associated with the increase in a dense, pericellular, neuraminidase-susceptible, Hale-positive acid mucopolysaccharide. Fuhrmann (1965) reported that there were no diEerences in the electrophoretic mobilities between proliferating normal cells and an ascites hepatoma. Both were higher than for the normal liver cells. Fuhrman (1965) suggested that malignancy was associated with an increase in the sialic acid-mediated cell-surface charge density. This hypothesis, however, did not receive support from other laboratories (Cook and Jacobson, 1968; Weiss and Sinks, 1970; Weiss and Hauschka, 1970). Vassar and colleagues (1967) observed that mesenchymal tumor cells had higher negative charges than carcinoma cells. More charges were lost from mesenchymal tumor cells when treated with neuraminidase than were carcinoma cells treated with neuraminidase. Vassar et al. also reported that normal and malignant epithelial cells did not have significantly different surface charge densities. Smith and Welborg (1972) reported that the progression to a more virulent form of the rat ascites hepatoma AS-30 D was accompanied by a substantial decrease in the neuraminidase-susceptible cell-surface sialic acid. Decreased amounts of sialic acids following transformation in vitro of strains of fibroblasts were also reported (Ohta et al., 1968). Grimes (1970) showed that simian virus 40 (SV40)-transformedcells have only 60% as much sialic acid as “normal” 3T3 cells. After transformations with SV40 virus, BALB/c cells lose 65% of their sialic acids. This reduced level of sialic acid was associated with reduction in the activity of sialyltransferase-the enzyme that catalyzes the transfer of sialic acids during sialoglycoprotein synthesis. Thus both increased and decreased levels of sialic acids were seen in various types of tumors after neuraminidase treatment; this was also substantiated in recent experiments (Ray and Simmons, 1973a). It is not possible to draw definitive conclusions regarding the relationship between sialic acid content in the cell membrane sialoglycoprotein and malignancy. Furthermore, there exist considerable discrepancies with respect to estimation of free sialic acid as released by neuraminidase hydrolysis from the intact cells and the estimates based on changes in electrophoretic mobilities after the incubation of cells with neuraminidase (Wallach and Perez-Esandi, 1964). A wide range of variability of neuraminidase-susceptible sialic acid residues has been observed by Ray and Simmons (1973a). Some tumors contain high concentration of cell-surface sialic acid, and some contain amounts somewhat similar to those present on their normal counterparts (Ray and Simmons, 1973a). Two important factors have to be taken into consideration when estimating sialic acid by colorimetric methods: (1)there exist signifi-

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cant differences in cell size among various types of cells; (2)there ought to be a variation in the content of sialic acid in cells at various stages of the mitotic cycle. It appears from the above studies that normally surfaces of malignant cell contain more sialic acid than do various types of normal cells, and the occurrence of 2,6-a-glycosidic linkages between sialic acid and other carbohydrates in the polysaccharide chain of the glycoprotein membrane complex is higher in both normal and malignant cells (Ray and Simmons, 1973a). The possible discrepancies existing between the chemical analysis of cellsurface sialic acid and the interpretation of the data based on the electrophoretic mobility studies have been discussed in detail earlier (Weiss, 1969; Mehrishi, 1972). In general, it appears that cell-surface sialic acid can be detected only by direct chemical estimation from the membrane complex. As the cell periphery is a three-dimensional structure, one cannot expect that the sialic acid molecules will be available at the same plane all the time. Their location might very well be at different depths and contours of the complex membrane structure. The farther away an ionized molecule is, the less will be its projected contribution at the plane of zeta potential; therefore, electrophoretic mobility cannot precisely quantitate threedimensional charge density and distribution.

G . SIALOLIPIDS Cell membrane glycolipid has been found also to contain sialic acid (Sweeley and Dawson, 1969). Hakomori and Murakami (1968) reported that polyoma virus-transformed BHK2l cells have less N-acetylneuraminyl lactosylceramide than do the corresponding untransformed cells. In a more definitive study, Hakomori et al. (1971) reported that Rous sarcoma virusinfected chicken embryo fibroblasts contain less sialoglycolipids than do the noninfected cells. The levels of some of these sialoglycolipids, which disappear on or after malignant transformation, increase in cells exhibiting contact inhibition of growth with increased amounts of cell contact (Hakomori, 1970). Later it was shown that in polyoma virus-infected cells of NIL-BE and BHK there is a consistent fall in both the sialoglycolipid content and the enzyme UDP-galactose: Lactosylceramide a-galactosyltransferase activity (Kijimoto and Hakomori, 1971). Weinstein et al. (1970), indicated signifcant variation in the type and amount of sialolipids present on various types of cell membrane. The isolated surface membranes contain six times as much sialic acid per milligram of protein as do whole L cells. Weinstein et al. said that monosialogangliosides are restricted to intracellular membranes. Therefore, analyses of whole cells appear not to give the exact sialoglycolipid content of the cell membrane.

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It is very interesting to note that, in malignant cells, while sialoglycoprotein is increasing, sialoglycolipid content is decreasing. Is malignancy a process where there is a shift or by-pass toward the synthesis of more sialoglycoproteins rather than sialoglycolipids?Or is it a process where the sialic acid pool in the body is shifted from sialoglycolipid synthesis to sialoglycoprotein synthesis?This shifting may have something to do with the availability of the particular enzyme involved in the transfer of sialic acid to the specific acceptor molecule. Whether, during malignant transformation, the synthesis of specific sialyltransferase is increased or decreased is a matter for investigation. The precise biological role of the relative increase in the terminal sialoglycoproteins in various malignant cells is a subject of speculation. Apffel and Peters (1970) suggested that one of the biological roles of the sialoglycoproteins may be the repression of antigenic determinants. It is known that sialoglycoproteins are very poor immunogens (Meyers and Deutsch, 1955; Schultze and Heremans, 1966; Silverberg et al., 1955). Removal of sialic acid molecules from orosomucoid (Peters, 1963; Winzler, 1965) and from both normal (Lundgren and Simmons, 1971; Simmons et al., 1970, 1971a,b) and malignant cells (Sanford, 1967; Bagshawe and Currie, 1968;Currie and Bagshawe, 1969; Simmons et al., 1971c,d 1972; Ray and Sundaram, 1974a,b, 197% Ray et al., 1974, 1975, 1976) increases their immunogenicity. Among many other properties associated with the neuraminidase-treated cells, which will be discussed later, exposition of newer antigenic specificities appears to have tremendous implications in immunobiological research. 111. Siaiic Acid and Its Relationship to the Antigenicity of the Cell Surface Sanford (1967), working with TA-3 tumor, reported that these tumor cells showed reduced transplantability if they had been previously treated with the enzyme neuraminidase. Currie and Bagshawe (1968a,b) working with Landschutz ascites tumors in mice, also found reduced transplantability of this tumor when previously treated with neuraminidase. The explanations put forward by both Sanford (1967)and Currie and Bagshawe (1968a,b)were that the reduced transplantability of those tumors after neuraminidase treatment was due to the “unmasking” of histocompatibility antigens on the tumor cells. The possibility of exposition of histocompatibility antigens by neuraminidase treatment was later investigated by Ray and Simmons in a series of investigations (Ray et al., 1970; Simmons et al., 1970, 1971a,b; Ray and Simmons, 1971a). Using specific anti-H-2 antisera, it was possible to show that neuraminidase-treated mouse lymph node cells did not absorb

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more anti-H-2 antibody compared to the untreated cells (Ray et al., 1970; Ray and Simmons, 1971a). Subsequently, these findings were confirmed in a number of independent laboratories and in several systems (Sanford and Codington, 1971a; Shimada and Nathenson, 1971; Hauschka et al., 1971; Schlesinger and Gottesfeld, 1971; Rosenberg and Rosentine, 1972; Reisner and Amos, 1972). Sanford and Codington (1971a) observed that TA-3 tumor cells did not absorb more anti-H-2, -3, -4, -11, and -28 nonspecific antibody after VCN treatment. Shimada and Nathenson (1971) reported that sialic acid was not a constituent part of H-2 alloantigens. Hauschka et al. (1971) could not detect any difference in H-2 antigenic titers of TA-3 tumor cells before and after neuraminidase treatment. Schlesinger and Gottesfeld (1971) also showed that neuraminidase treatment of lymphoid cells had no consistent effect on the absorption of H-2 or 8 antibodies by the treated cells. In HLA system, Rosenberg and Rosentine (1972) and Reisner and Amos (1972) independently reported that human lymphocytes treated with neuraminidase’ did not show increased availability of HLA antigens. Thus, the explanations put forward by Sanford (1967) and Currie and Bagshawe (1968a,b) that the decreased transplantability of neuraminidase-treated tumor was due to the “unmasking’ of histocompatibility antigens on the tumor were found to be inconsistent by several laboratories. A number of reports are available in the literature regarding the increased susceptibilityof neuraminidase-treated cells to the cytolytic action of specific alloantibody and complement. The pioneering report by Ray and colleagues (Ray et al., 1970, 1972; Ray and Simmons, 1971a)that neuraminidase-treated mouse lymph node cells were increasingly sensitive to the cytolytic action of alloantibody and complement stimulated further interest in these areas of investigation. Increased immunolysis of neuraminidase-treated cells was also reported from other laboratories (Lauf, et al., 1971; Schlesinger and Gottesfeld, 1971). Increased cytolysis of mouse spleen cells in the presence of a number of H-2 antisera and guinea pig serum complement was reported by Schlesinger and Gottesfeld (1971). Ray et al. (1970, 1972; Ray and Simmons, 1971a) showed that neuraminidase-treated mouse lymph node cells were more susceptible to the cytolytic action of human serum, rabbit serum, and guinea pig serum where alloantibody was not added. Various complement inhibitors were shown to abrogate the lytic activity of rabbit serum against neuraminidase-treated mouse lymph node cells (Ray et al., 1970, 1972). Thus it appears that neuraminidase-treated cells are sensitive to complement-mediated cytolysis whatever may be the mechanism of com‘Unless otherwise mentioned, the word neuraminidase stands for Vibrio chokrae neuraminidase in these discussions.

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plement activation (Ray et al., 1970, 1971, 1972). Schlesinger and Gottesfeld (1971)observed that neuraminidase treatment of murine spleen cells results in an increased ability of these cells to absorb cytotoxic factors in the guinea pig serum. Dalmasso and Muller-Eberhard (1964) showed increased complement binding on neuraminidase-treated red blood cells. They said that reduction of mutually repellent negative charges from the surface of cells by neuraminidase treatment facilitated the binding between cells and negatively charged complement molecules. However, investigations on the effects of neuraminidase on the absorption of polyanions did not support such simplistic explanations (Julliano and Mayhew, 1972). Ray and colleagues (1970, 1972, 1973; Ray and Simmons, 1971a)described that neuraminidase-treated mouse lymphoid cells become susceptible to cytolysis by alloantibody and complement and also by normal human, guinea pig, and rabbit sera when no antibody was added from an exogenous source. Further, autologous rabbit lymph node cells previously treated with neuraminidase become susceptible to lysis by autochthonous rabbit serum components (Ray et al., 1970, 1971, 1972, 1975a,b). The lytic factor in xenogeneic sera (Ray et al., 1970, 1972; Ray and Simmons, 197313) could be absorbed out by neuraminidase-treated mouse cells, but not by untreated cells. Later, Schlesinger and Amos (1971) and Schlesinger and Gottesfeld (1971) reported an increased capacity of neuraminidase-treated cells to absorb the cytolytic component of guinea pig serum for mouse thymocytes. Most recently, Rosenberg and Rosentine (1972), and Reisner and Amos (1972) have independently reported that human lymphocytes treated with neuraminidase were capable of absorbing antibodies from both xenogeneic (Rosenberg and Rosentine, 1972) and allogeneic sera (Reisner and Amos, 1972),which react primarily with neuraminidase-treated cells and less well, or not at all, with normal cells. It has been suggested (Reisner and Amos, 1972)that the human leukocyte antigens exposed by neuraminidase were not related to HLA determinants and that the antigens might be panreactive, in that both neuraminidase-treated monkey and human erythrocytes could absorb them. This interpretation might also help explain the phenomenon of lysis of neuraminidase-treated cells by autologous serum (Ray et al., 1970, 1971, 1972; Ray and Sundaram, 1975a,b; Rosenberg and Rosentine, 1972) since autologous sera have been shown to contain antibodies against self cellular antigens that are not normally available for reaction but become exposed only after neuraminidase treatment (Ray and Sundaram, 1975a,b: Rosenberg and Rosentine, 1972). The practical significance of the presence of these types of molecules in normal sera has not yet been properly evaluated. This type of phenomenon may have some correlation with the immune clearance of defective, aged, mutated, and/or malignant cells from the body if these situations are some-

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how associated with the exposition of those normally hidden or crippled antigens. The mechanism through which various types of defective cells are cleared from the system may follow, at least in some cases, procedures described above. Destruction of some vitally important cell types after infections with neuraminidase-containing bacteria, viruses, etc., may follow the mechanism described above. Invading bacteria and viruses can destroy those cells, resulting in the severe loss of blood cells and cells capable of effecting immune function. A question remains whether “infectious hemolytic anemia” has any relationship to this type of mechanism. It is possible that cells that become defective as a result of aging processes, due to mutation following transformation of a genetic trait by oncogenic agents, may very well be cleared from the system by a similar mechanism if the changes are associated with the exposure of crippled antigens on them. However, these speculations, though having some scientific basis, need detailed investigations to warrant acceptance of the claim. At the same time, it should be mentioned that the above propositions are in conformity with what has been descriped in connection with neuraminidase-induced destruction of cell types by even autologous immune components. Thus it appears that sialic acid may play a big role in conferring structural rigidity and stability on the cell (Ray and Chatterjee, 1975b). Sialic acid, however, may serve many valuable functions, being an integral part of the cell membrane “glycocalyx” structure. Removal of sialic acid is associated with a variety of altered behaviors of the cell surface. The most drastic is the cytolysis of cells by the immune components, as described earlier. Among the other properties associated with the neuraminidase treatment of cells are the following: reduction of negative charges of the cell surface (Ambrose, 1966), alteration of migratory pattern of treated cells (Woodruff and Gesner, 1969), enhancement of the adhesive property of cells (Ray and Chatterjee, 1975a), decreased pinocytic behavior of the treated cell surface (Chatterjee and Ray, 1975), increased phagocytosis of the treated cells (Lee, 1968a,b; Weiss et al., 1966; Schmidtke et al., 1973; Schmidtke and Simmons, 1975), altered viral (Baylor, 1964)or mycoplasma (Gesner and Thomas, 1966) induced hemagglutination, inhibition of cell aggregation (Kemp, 1968), increased deformability of the treated cells (Weiss, 1965a,b), altered amino acid transport across the treated cell membrane (Brown and Michael, 1969), increased stimulation of responding cells by treated stimulatory cells (Lindahl-Kissling and Peterson, 1969; Lundgren and Simmons, 1971; Han, 1972, 1973; Etheredge et al., 1972), enhancement of delayed skin hypersensitivity (Han, 1974), accentuation of human host blood lymphocytic transformation cultured with neuraminidase-treated cancer cells (Watkins et aZ., 1971, 1974), increased rosette-forming ability even of treated autologous red blood cells with autologous T cells (Baxley et al., 1973),

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and last, but not least in importance, the increased immunogenicity of neuraminidase-treated normal, fetal, and tumor cells. These will be discussed in a separate section later. In view of the above discussion relating to the mechanism of immune clearance of defective, damaged, aged, or malignant cells, it may be appropriate to speculate that, if the cell-surface configuration is subjected to reorientation such that antigenic moieties like those exposed by neuraminidase are exposed as a result of any physiological disturbance in the body, these cells might become susceptible to immune clearance by the mechanism described earlier. It may be that the body maintains such an effective tool to balance the dynamic stability of cell population. It is possible that, at a very early stage of malignant transformation, the transformed cells may have exposed antigens on the surface and so are subjected to immune clearance. Who knows whether the concept that cells are being transformed continuously and are immunolyzed by the body by some such active immune mechanism has any relevance with the mechanism described above? It remains to be established that these continuously produced transformed cells simply await a situation where the hostile immune environment of the host’s body is found to be immunologically weak; e.g., if the host is immunosuppressed either as a result of nutritional deficiency; following an infectious disease; or artificially, owing to the incorporation of a high dose of immunosuppressive drugs, as in the case of transplant patients, or to exposure to radiation. In any such situation, the transformed cells may not be cleared effectively from the system because proper immune activity is lacking. The result is an accumulation of large masses of transformed cells able to grow effectively. When this process continues for some time, these cells may induce a protective mechanism covering the exposed antigen on their cell surface. One of these mechanisms may involve triggering of sialic acid synthesis so that sialic acid molecules are found to be universally related, directly or indirectly, to tumor-specific antigenic moieties. Exposure of these antigens would make a cell susceptible to immune destruction, as we shall see in subsequent sections of this review. The mechanism described supports the concept of continuous generation of transformed cells and the concept of immune surveillance and its relationship to the development of malignancy. This gives a logical explanation, on the basis of immune clearance of cells, with respect to the escape of transformed cells from immune attack. It is not necessary, however, that the patient’s immune system should continuously be suppressed or depressed. But perhaps it is essential that, during the initial events of the growth of transformed cells, particularly during the process of establishment, the depressed immune reactivity in the host helps establishment of the tumor. It has been seen that an animal otherwise resistant to a particular type of tumor

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becomes susceptible to that tumor if it is in an immunosuppressed condition (P. K. Ray, V. S. Thakur, and K. Sundaram, unpublished observation). In the tussle of two entirely opposite concepts of immune functions in the host during malignant growth, perhaps it can be said that immune functions in the host are not necessarily in a depressed state during all the time of tumor growth. On the contrary, it is perhaps true that immunosuppression facilitates tumor development. It should be kept in mind that the phenomenon of spontaneous development of tumors and that of the serially transplanted tumors would certainly differ insofar as the immune status of the host are concerned. Once the tumor is established, immune function may be otherwise normal, but it is possible that it cannot react against the tumor itself. The reason lies perhaps in the architecture of the tumor itself, not in the immune system. After the tumor grows to a certain extent, it may release some cytotoxin or other factors, which may in turn enter the circulation to destroy or inactivate potentially reactive immunocytes to effect immunosuppression. However, although the scope of this review is too limited to permit of discussing this matter in detail, a speculative discussion is meaningfbl at least to initiate interest in further research. Future investigation in this area would certainly bring more definitive and clearer understanding. It is becoming more and more evident that neuraminidase treatment increases accessibility to some antigens on the cell surface, the specificities of which are normally not available for reaction. Human leukemic lymphocytes treated with neuraminidase had increased amounts of heteroantigens on their surfaces as compared to untreated cells (Kassulke et al., 1971). Rosenberg and Rosentine (1972) showed that most human serum samples contained antibodies which, in the presence of complement, can cytolyze even autologous and allogeneic lymphocytes. Grothaus et al. (1971) reported that the susceptibility of human lymphocytes to antisera directed against HLA and other antigens is increased after neuraminidase treatment. Neuraminidase treatment apparently exposes ABH (Saber et al., 1965) and Forssmann (Drzeniek et al., 1966)antigens. Ray and colleagues (1970, 1971, 1972; Ray and Simmons, 1972; 1975a,b)earlier reported that neuraminidase treatment of murine lymphoid cells is associated with the exposure of newer antigenic specificities against which antibody molecules are present in autologous (Ray et al., 1970, 1971, 1972, 1975a,b; Ray and Simmons, 1972), allogeneic (Ray et al., 1970, 1972a,b), and xenogeneic (Ray and Simmons, 1972; Ray et al., 1972) sera. By specific antibody absorption experiments, these antibodies could be absorbed out, using VCN-treated cells for absorption (Ray and Simmons, 1973b; Ray and Sundaram, 1975a,b). From the above discussion, it is evident that removal of sialic acid moieties by bacterial neuraminidase leads to the availability of a variety of antigens on various cell types; this in turn is responsible for altered behaviors.

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IV. Increased lmmunogenicity of Neuraminidase-TreatedCells

A. TREATEDNORMAL CELLS From the immunological point of view, one of the most important properties of neuraminidase is its ability to increase the immunogenicity of the treated cells. In skin graft experiments, Simmons and colleagues (1970, 1971b) have shown that recipients of C3H cells treated with neuraminidase rejected the C3H skin grafts more rapidly than the recipients of cells treated with inactivated enzyme. It was also demonstrated that recipients of neuraminidasetreated CBA cells rendered the C3H recipients far more immune to subsequent CBA skin grafts than did the infusion of cells treated with inactivated neuraminidase. To prove the specificity of reaction, these authors gave inoculations of CBA cells treated with active or inactive neuraminidase to C3H recipients and subsequently grafted the CBA and C57 skin. There was, however, accelerated rejection of CBA skin grafts but not of the C57 grafts, thus showing that the immunogenicity of those cells was increased by neuraminidase treatment and that the host could be specifically sensitized against the infused inoculum. In a beautiful dissection of the system, Lundgren and Simmons (1971) showed that neuraminidase treatment of the stimulatory cells was associated with an increase in its immunogenic capability such that the treated cells could stimulate the responding cells much more than the untreated ones in human mixed-lymphocyte culture. This was reported to be true in both allogeneic and autologous cell-culture systems (Lundgren and Simmons, 1971; Etheredge et al., 1972). These authors did not observe any effect produced by treating the responding lymphocytes with neuraminidase. Han (1972) reported that stimulating cells treated with the enzyme and inactivated by X-irradiation could produce increased stimulatory capacity, thus substantiating the observation made by Lundgren and Simmons (1971). Further, Han (1972) observed a similar response after treating the responding lymphocytes with neuraminidase in a one-way, human, mixed-lymphocyte reaction. He observed that in vitro blastogenic response of sensitized human lymphocytes to specific antigens, e.g., purified protein derivative, varidase, monilia, and mumps, and to weak antigens like concanavalin A and pokeweed mitogen was greatly enhanced by neuraminidase treatment; the enzyme had no effect on strong antigen like phytohemagglutinin (Han, 1973). Delayed skin hypersensitivity was enhanced when the skin-testing antigen was given together with neuraminidase (Han, 1974). Thus, from these studies it appears that neuraminidase treatment is associated with more immunogenic capability of the treated cells, even if autologous by

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nature, and the presence of neuraminidase along with the antigen results in heightened immune reactivity in the recipient against the corresponding antigen.

B. TREATED EMBRYONIC CELLS Simmons and colleagues (1971a) reported that C3H embryos removed from a 7-day pregnancy-sensitized recipient mouse to C3H skin grafts when a neuraminidase-treated embryo was used for sensitization. Thus neuraminidase could increase the immunogenicity of fetal cells. Increase in the immunogenicity after neuraminidase treatment, therefore, does not seem to be restricted to certain types of cells, It has been discussed earlier that neuraminidase treatment increases the immunogenicity of normal cells (Simmons et al., 1970, 1971b; Lundgren and Simmons, 1971; Etheredge et al., 1972; Han, 1972).

C. TREATED TUMORCELLS Sanford (1967)originally demonstrated that the transplantability of ascites TA-3 tumors was greatly reduced if these cells were previously treated with neuraminidase. Subsequently Currie and Bagshawe (1968a,b) showed that Landschutz ascites tumor, L 1210 leukemia (Bagshawe and Currie, 1968), and 3-methylcholanthrene-induced tumor (Currie and Bagshawe, 1969) which had been pretreated with neuraminidase and injected intraperitoneally failed to kill normally susceptible hosts. Lindenmann and Klein (1967) obtained similar results using Ehrlich ascites tumors. Simmonset al. (1971c,d, 1972) and Ray and Sundaram (1974a) observed that neuraminidase-treated 3-methylcholanthrene-inducedfibrosarcoma grew less well in C3H mice compared with the untreated tumor, thus confirming the earlier findings of Currie and Bagshawe (1969). Sethi and Brandis (1973) further confirmed that L 1210cells showed reduced transplantabilityif inoculated in susceptible mice after neuraminidase treatment. Ray and Sundaram (197413, 1975a,b,c) and Ray et al. (1974)reported that a dimethylbenzodithionaphthene (DBDN)induced fibrosarcoma showed reduced transplantability if pretreated with neuraminidase. Thus, although a large number of reports show that neuraminidase treatment of tumor cells reduces the transplantability of the tumor in a susceptible host, contradictory reports are also available (Ruhenstroth-Bauer, et ul., 1962). Hauschka et al. (1971) used low cell densities during enzyme incubation and got significant reduction in the viability of the cells. They explained their findings of reduced transplantability of neuraminidase-treated tumor

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cells as due to the reduction in tumor-cell viability after neuraminidase treatment. It is well known that at low concentrations of cellular suspensions during incubation (e.g., 3 x lo4 cells/ml), and in the absence of adequate amounts of protective proteins in the media, cell survival rates are significantly less than at greater cell densities (e.g., 106 to 107/ml). In careful experiments it was recently shown by Ray et al. (1975, 1976) and Ray and Sundaram (197%) that neuraminidase impaired the transplantability of a DBDN fibrosarcomain Swiss mice. These authors (Ray et al., 1975,1976; Ray and Sundaram 1974a,b, 1975c) did the enzyme incubation at a higher concentration of cells, washed the cells after incubation with a large excess of medium, measured the viability by the dye-exclusion method, and adjusted the final viable cell concentrations to have identical numbers of viable cells in both treated and sham-treated controls. Serial dilutions were done to have lower cellular concentrations just before inoculation. Ray et al. (1975, 1976) injected the same number of treated and untreated viable tumors and still observed that neuraminidase-treated cells showed impaired transplantability. Therefore, Hauschka’s supposition of reduction of viability during enzyme incubation, although it may be true at lower cellular concentrations, as the only cause of reduced transplantability of neuraminidase-treated tumor does not seem to be valid. A number of reports available in the literature also showed that neuraminidase was not cytotoxic to various types of cells (Currie and Bagshawe, 1968a,b, 1969; Woodruff and Gesner, 1969; Sanford and Codington, 1971a,b;Rayet al., 1970,1972,1974,1975,1976;Ray and Simmons, 1971a,b, 1973a,b). Human lymphocytes treated with neuraminidase responded normally to both phytohemagglutinin and allogeneic stimulation (Lundgren and Simmons, 1971). Bone marrow incubated with neuraminidase was fully capable of reconstituting mice treated with lethal doses of cyclophosphamide and mounting a graft-versus-host reaction (Stutman, 1970). The ability of neuraminidase-treated tumor cells to grow, and to kill immunosuppressed recipients (Simmons et al., 1971b; Ray et al., 1974; Ray and Sundaram, 1974a,b, 1975a,b), strongly argues that neuraminidase-treated tumor cells have a normal ability to proliferate into tumor masses in recipients with reduced immune capacity. Neuraminidase-treated methylcholanthreneinduced fibrosarcoma grew readily in sublethally irradiated mice (Currie and Bagshawe, 1969), and in goat antimouse lymphocyte serum and cyclophosphamide-treated mice (Simmons et al., 1971d). Normal growth of neuraminidase-treated L 1210 leukemia was observed in cyclophosphamide-treated mice (Sethi and Brandis, 1973). High doses of neuraminidase-treated DBDN-induced fibrosarcoma grew even in normal individuals (Ray et al., 1975, 1976). Lower doses of treated tumor that did not show tumor development in normal animals showed tumor development in

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hydrocortisone-treated, and also in sublethally X-irradiated, mice (Ray et al., 197513). All these observations strongly suggest that neuraminidase treatment does not eliminate the tumorigenic ability of the tumor cells, and so the suppression of the growth of neuraminidase-treated tumor is immunologically mediated. Neuraminidase-induced loss of transplantability of tumors had been ascribed by some workers to the loss of oncogenicity of the treated tumor (Bekesi et al., 1972). However, on the basis of the above information, the observations of Bekesi and colleagues (1972) do not appear to be valid. It is extremely interesting that inoculation of neuraminidase-treated tumor can give rise to tumor-specific immunity. This has been verified in a number of laboratories and in a variety of tumor systems (Sanford, 1967; Currie and Bagshawe, 1968a,b, 1969; Currie et al., 1968; Simmons et al., 1971c,d; Ray and Sundaram, 1974a,b, 1975c; Ray et al., 1974, 1975, 1976; Sethi and Brandis, 1973; Bekesi et al., 1972). Obvious attempts therefore involved the use of neuraminidase-treated tumor for the therapy of a growing established tumor. This is discussed in the following section.

V. Regression of Established Solid-Tissue Tumor b3GRESSION OF GROWING TUMORS CAUSED BY NEURAMINIDASE-TREATED SYNCENEICTUMOR CELLS

In elegant experiments, Simmons et al. (1971c,d) showed for the first time that a solid-tissue tumor could be made to regress completely after inoculation of neuraminidase-treated tumor of syngeneic origin. The regression was shown to be immunospecific in that a particular type of tumor could be made to regress using neuraminidase-treated tumor of the same type (Simmons et al., 1971~). An inoculum containing 20,000 normal MC-42 fibrosarcoma cells was inoculated into C3H/HeJ mice. When the tumors were less than 1.0 cm in diameter, neuraminidase-treated MC-42 tumor cells were injected in those animals. Animals which received inocula containing neuraminidasetreated tumor strongly inhibited the growth of tumors that were already firmly established and growing rapidly. All the tumor-bearing animals receiving therapy showed temporary cessation of tumor growth, and many tumors regressed and disappeared (Simmons et al., 1971~).All animals whose tumors regressed, survived for an indefinite period. Some animals showed temporary cessation of tumor growth followed by regrowth leading to death of the animal. No tumor greater than 1.0 cm in diameter totally regressed. Injections of intratumor neuraminidase (Simmons et al., 1972) could stop the growth of injected tumors in general, and a number of tumors totally regressed. In animals bearing bilateral immunologically identical

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tumors, injection of one tumor with neuraminidase induced the regression not only of the injected tumors, but also of the contralateral tumors. When the bilateral tumors were of different types, injections of one did not lead to regression of the other, but the animals were still capable of rejecting the injected tumor while dying of the immunodistinct untreated tumor (Simmons et al., 1972). Ray and colleagues extended their work in the case of a DBDN fibrosarcoma transplantable in Swiss mice and made similar observations (Ray and Sundaram, 1974b, 1975c; Ray et al., 1974, 1975, 1976). Further, they (P. K. Ray, M. Seshadri, and K. Sundaram, unpublished observation) could show significant growth inhibition of rat ascites Yoshida sarcoma following inoculation of neuraminidase-treated and mitomycin C-inactivated syngeneic Yoshida sarcoma cells. Control animals died within about 2 weeks, but animals receiving therapy survived more than 100 days in some cases. This work has been extended further in dogs with spontaneous mammary tumor (Sedlack, et al., 1975).Sedlack et al. presented their data based on 35 dogs and showed that in 13 out of 15 dogs having spontaneous mammary tumors there was significant tumor regression following the inoculation of mitomycin plus neuraminidase-treated tumor cells (2x 10' cells per animal). However, the application of 1x lo8 mitomycin- and neuraminidase-treated cells resulted in an accelerated tumor progression in all 8 dogs treated. In the case of responsive groups, they could detect similar response with both benign and malignant tumors, no matter whether their initial tumor volumes were 1 cm3, 2 cm3, or more. These results further corroborate the initial observation by Simmons et a2. (1971c,d, 1972) that an established tumor can be made to regress completely following an inoculation of neuraminidase and mitomycin C-treated syngeneic tumor. This has been substantiated further by the finding that mice having leukemia L 1210 show prolongation of survival following methotrexate treatment, but with no cures. With a single inoculation of neuraminidase-treated L 1210 cells, much greater extension of survival occurred; indeed 40% of the animals were cured (Holland, 1975). Holland suggested that with a better experimental drug, capable of causing 20% cures by itself, 8O-W% of animals could be cured following a timed inoculation of neuraminidase-treated tumor along with the drug. As discussed earlier, tumor treated only with neuraminidase, although it cannot induce a tumor if inoculated at a smaller dose, retains its tumorigenic properties (Ray et al., 1975, 1976). Higher doses of tumor treated with neuraminidase alone produce tumor development even in normal animals (Ray et al., 1975, 1976). Lower doses of treated tumor, which cannot induce tumor development in normal animals, do produce tumors in immunosuppressed animals (Ray et al., 1976). Thus, it appears that there exists a potential risk in inoculating large doses of tumor cells treated only with neuraminidase in immunotherapy as a fresh tumor may develop as well.

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Furthermore, cancer patients are normally in an immunosuppressed condition. This fact has to be taken into proper consideration before one uses tumor cells treated only with neuraminidase in immunotherapy. Math6 et al. (1972) reported the deleterious effects of neuraminidase-treated tumor which they used for immunotherapy of AKR leukemia. In their attempt, they used high doses of tumor cells treated only with neuraminidase. Thus indiscriminate use of neuraminidase-treated tumor cells for immunotherapeutic purposes may give rise to harmful effects rather than to any beneficial results. &yet al. (1974,1975,1976;Ray and Sundaram, 197%) described a method for suppressing completely the tumorigenic ability of neuraminidase-treated tumor without any substantial loss of its immunogenic properties. They observed that their “tumor vaccine” preparation was highly effective in immunizing animals against the corresponding tumors (Ray et al., 1974, 1975, 1976; Ray and Sundaram, 1975~).The potential benefits of this type of vaccine for the immunotherapy of cancer was also suggested (Ray et al., 1974, 1975, 1976; Ray and Sundaram, 1975c) to be as follows: 1. There is no danger of inducing fresh tumor growth by the vaccine preparation as such. 2. Tumor vaccine containing large numbers of tumor cells can be inoculated at one time, and this is necessary sometimes to build up effective immunity. 3. A syngeneic tumor or a portion of the same tumor to be treated can serve as the source of tumor cells for the vaccine preparation; this would eliminate the chance of any graft-versus-host reaction. 4. There will be no inhibition on the part of the patient to accept such tumor vaccine, if he(she) knows that the vaccine is developed from his(her) own tumor cells modified in vitro. 5. There is no ethical inhibition such as might exist against injection of viable tumor cells, as the vaccine preparation does not induce any fresh tumor growth. 6. Once prepared, the vaccine can be stored for a considerable length of time at low temperature (-ZOOC) without substantial loss of potency, though freshly prepared vaccine is always more reactive. The important parameters of this vaccine therapy as suggested by Ray and colleagues (1974, 1975, 1976; Ray and Sundaram, 197%) were (a) the number of inoculations of the vaccine and the number of cells present in it; (b) the immunological status of the individual receiving therapy; (c) the total number of tumor cells present in the tumor mass to be treated; (d) the level of blocking antibody at the beginning and during the time of treatment. Using a immunotherapeutic regimen involving this type of tumor vaccine, Ray and colleagues (P. K. Ray, unpublished observation) observed that immunization of AKR mice, which normally developed spontaneous lymphatic

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leukemia in about 80%of animals after a certain age, could grossly inhibit the appearance of leukemia in the immunized animals. The vaccine-injected animals survived longer than did untreated controls. Further, the above protocol of treatment was found to be useful in the immunization of female C3H mice, which normally developed mammary adenocarcinoma at a particular phase of their lives. Immunization of these mice with a syngeneic tumor vaccine could inhibit the appearance of mammary tumors. Immunized animals survived longer than did untreated controls (P. K. Ray, unpublished observation). From all these observations it appears that recurrences of tumor even in the case of a human cancer like leukemia, might be checked by the use of an immunization protocol based on the neuraminidase-treated tumor vaccine. It may also be possible to check the secondary growth of tumors after the primary tumors are taken care of by other means, e.g., chemotherapy, radiotherapy, surgical amputation, etc. Results obtained in the author’s laboratory are extremely interesting and offer many possibilities for future use, at least in a limited sense, for the immunotherapy of cancer in man. However, extensive laboratory investigations and clinical trials are needed to determine the applicability and usefulness of this method in cancer control and treatment.

VI. How Do Neuraminidase-TreatedTumor Cells React in the Host to Establish Specific Antitumor Immunity? The question is still not very much understood. Only scattered results are available. Before any probable speculative propositions are made, let us survey the available information in the literature. A. FACILITATED CONTACTBETWEEN NEURAMINIDASE-TREATED AND UNTREATED CELLS

It is now well established that all mammalian cells have a net negative charge on their surfaces. Because of like charges on the surfice of mammalian cells, electrostatic repulsion is seen when two cells come closer. A comprehensive account of the physical background leading to interactions of cells was given by Weiss and Harlos (1971). They suggested that reductions in cell-surface negativity brought about by neuraminidase treatment are likely to produce cell-to-cell interactions by direct mechanisms. Kolodny (1972) was unable to demonstrate the effect of neuraminidase on the attachment of cells to culture dishes. McQuiddy and Lillian (1971) working with neural retina cells from chick embryos cultured with neuraminidase could not demonstrate an effect on the size or number of cell aggregates. Adhesion or nonadhesion of two cell

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lines to glass could not be correlated with either net surface negativity or the contributions of sialic acid to this parameter (Weiss, 1968). But observations contrary to these are also available. Cormack (1970) noted that incubation of Walker 256 carcinoma cells with neuraminidase reduced the rate of attachment of the treated cells to mesothelial membrane. Kemp (1968) observed that the mutual reaggregation of embryonic-chick muscle cells was inhibited by prior treatment with neuraminidase. Ray and Chatterjee (1975a) showed increased lysolecithin-induced cell adhesion and/or aggregation in Amoeba proteus. Thus it is not known why in certain systems neuraminidase treatment gives rise to increased cell adhesion, whereas in others it becomes ineffective. Concentration and also distribution of sialic acid in the cell periphery appear to be important parameters that determine the surface-related characteristics-a complicated system that needs detailed investigation.

B. FACILITATION OF OTHERINTERACTIONS INVOLVING KILLER AND TARGET CELLS The precise immune mechanism by means of which the target cells are ultimately killed is not known. Target cells may be killed directly by immune lymphocytes or macrophages that have been made specifically cytotoxic by a factor produced by thymocytes or immune lymphocytes (Grant et al., 1972). Feldman and Basten (1972) suggested that sensitized T cells interact with antigen to produce a fictor causing antigen to be concentrated on macrophages, which in turn pass on information to B cells; this would lead to the production of humoral antibodies. Apparently, the contact between T cells and antigen seems to be extremely important. Prominent among other cell types involved in immune reactions are macrophages. Granger and Weiser (1964, 1966) reported the contact destruction of tumor cells. The in uitro induction and release of a cell toxin by immune mouse peritoneal macrophages was reported by Kramer and Granger (1972). The role of macrophages in processing the antigen had been emphasized by Fishman and Adler (1963). Further, it was reported that transformation of lymphocytes may be caused by macrophages previously sensitized by an antigen (Seiger and Oppenheim, 1970). However, in an attempt to find out whether neilraminidase treatment of target cells affects the target and effector cell interaction, Weiss and Cudney (1971) showed that neuraminidase treatment of p 815 mastocytoma target cells did not increase susceptibility to the attack by sensitized spleen cells. When cells are incubated with neuraminidase, some enzyme may be absorbed to the cell surface (Rebrishi, 1972). A strong suggestion that neuraminidase, when used in high concentrations, may be absorbed to the cell surface comes &om Weiss (1973), who reported a progressive loss of net

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surface negativity over a enzyme concentration ranging from 0.001 to 10 units per lo6cells. Weiss pointed out that absorbed neuraminidase might be released ultimately to modify a cell that came in contact, i.e., lymphocytes, macrophages, etc., and thus might modify the interactions. This is a proposition that must be very carefully investigated, particularly when there is a great possibility of a cis and/or allosteric effect when any molecule is added to the membrane or stripped off. The cell membrane is fluid and continuously in dynamic movement. It is not a rigid structure. So an induced effect in some part of the surface structure might be associated with remote or distant modification. In addition to what may be absorbed onto the cell surface by neuraminidase incubation of cells, neuraminidase is as well available in lysosomes of various cell types, e.g., rat mammary glands (Tulsiani and Carubelli, 1971), rat liver and kidney (Mahadevan et al., 1967), Ehrlich ascites tumor cells (Horvat and Toustar, 1968), etc. Lysosomal activation is possible by a variety of substances including antisera (Weiss and Dingle, 1964; Fell and Weiss, 1965). The liberated neuraminidases may modify the surfaces of the cells releasing them by an autolytic process (Weiss, 1967). These neuraminidases may alter certain cell contact reactions (Weiss, 1965b). Lysosomal enzyems may also attack the surface of adjacent cells that are not themselves the source of the enzymes (Butherworth, 1970). Whether or not lysosomal enzymes other than neuraminidase, at least in certain instances, do play a role in exerting the overall effect in uiuo is a matter for future investigations. Recently, Brandis et. al. (1974) reported that vitamin A alcohol may exert an effect similar to that of neuraminidase on L 1210 leukemia cell surface. They observed a reduction in negative charges and increase in positive charges after vitamin A treatment of the cell surface. They proposed that vitamin A might have released the lysosomal neuraminidase, which in turn processed the cell surface so that there was a reduction in cell-surface negativity after vitamin A treatment. Thus, whether or not lysosomal activation by a variety of agents may exert an effect similar to that of neuraminidase both in vivo and in uitro is very difficult to say at this moment. These results may, however, bring support, in an indirect way, of the neuraminidase effect on cell membranes and the altered properties associated with these phenomena. In mixed-lymphocyte cultures, Lundgren and Simmons (1971) showed that the stimulatory activity of target cells was very much increased if the cells were treated previously with neuraminidase. The stimulation index was measured by the increased ability of the responding cells to incorporate [3H]thymidine. However, when the responding cells were treated in neuraminidase, no difference was observed between the untreated and neuraminidase-treated responding cells. It was of extreme interest that,

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even in an autologous system, these authors observed significant stimulation using neuraminidase-treated autologous stimulatory cells. However, Han (1972, 1973), while confirmed the findings of Lundgren and Simmons (1971) that neuraminidase treatment of stimulatory cells could show increased stimulation in the responding cells, observed similar effects if the responding cells or both were treated with neuraminidase. Weiss and Cudney (1971) showed enhanced killing of mastocytoma cells in vitro by a neuraminidase-treated, sensitized spleen-cell population. Thus, either the target or effector cells or both, after neuraminidase treatment, could sensitize the other cell types. Neuraminidase as such exerts a variety of effects on lymphocytes, macrophages, etc. A significant loss in surface negativity of neuraminidasetreated lymphoid cells (Mayhew and Weiss, 1968; Bennett et al., 1969; Weiss and Sinks, 1970) and circulating blast cells from patients with acute and chronic lymphocytic leukemia (Weiss and Sinks, 1970) was reported. Ray et aZ., (1970, 1972) and Ray and Simmons (1971a, 1972) reported increased immunolysis of mouse lymphoid cells by autologous, allogeneic (Ray et al., 1970, 1971, 1972), and xenogeneic sera (Ray et al., 1972; Ray and Simmons, 1973b). Human lymphocytes treated with neuraminidase are also more susceptible to lysis by antisera directed against HLA, 8, and TL antibodies than are corresponding controls (Grothaus et al., 1971). Sensitized human lymphocytes if treated with the enzyme show increased sensitivity to concanavalin A, pokeweed mitogen, and various other soluble antigens used for skin testing (Han, 1974). While the lymphocyte surface properties are altered by neuraminidase treatment, other cell types also show changed behavior after neuraminidase treatment. Human circulating monocytes showed a significant reduction in surface charge density (Weiss et al., 1966), and this was also associated with an increased cell deformability. Macrophages from peritoneal exudates of mice also exhibited mean reductions of approximately 19%of their mobilities after incubation with neuraminidase (Mayhew and Weiss, 1968). Murine sarcoma 37 and Ehrlich ascites tumor cells also showed an increased deformability associated with loss of surface negativity after neuraminidase treatment (Weiss, 1965a,b). Now the question is whether neuraminidase-induced changes in either target cells or the antigen-processing cells can lead to enhanced immunogenicity of target cells. Interactions of the tumor cells and macrophages often result in destruction and/or phagocytosis of the tumor cells (Bennett et al., 1964). It is interesting that neuraminidase-treated macrophages showed increased ability to phagocytose negatively charged particles in vitro (Weiss et al., 1968). On the other hand, the degree of ingestion of opsonized erythrocytes by peritoneal macrophages was greatly increased if the erythrocytes were pretreated with

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neuraminidase (Lee, 1968a,b; Schmidtke et al., 1973; Schmidtke and Simmons, 1975). On the other hand, adherence of opsonized bacteria to mouse macrophages treated with neuraminidase is greatly increased compared to the untreated macrophages. Thus here again we see that neuraminidase treatment of either the target cells or the Ag-processing cells may facilitate the contact between these cell types leading to the establishment of strong immunity. There are various reports that neuraminidase-treated cells bind naturally occurring antibodies (Ray et al., 1970,1971,1972; Ray and Sundaram, 1975a,b; Rosenberg and Rosentine, 1972; Rosenberg and Schwarz, 1974; Rosentine and Plocinik, 1974). These antibody or opsonin-coated cells may be increasingly phagocytosed and may stimulate the efferent limb of host cell-mediated immune response. However, apart from this effect, neuraminidase treatment is associated with a number of other changes in the properties of treated cells. Nordling and Mayhew (1966) showed that during incubation neuraminidase might be internalized and in the process might react with intracellular organelles. Thus, in addition to the alteration of surface properties, one may expect some modifications of the intracellular characteristics to occur also. Neuraminidase treatment can affect potassium transport across cell membranes (Click and Githens, 1965). In human erythrocytes, a reversible cation exchange behavior of sialoglycopeptides has been demonstrated by Ohkuma and Furuhata (1970). Neuraminidase treatment affects the amino acid transport in HeLa cells (Brown and Michael, 1969). Vaheri et al. (1972)reported that neuraminidase in low concentrations stimulated division and sugar uptake in density-inhibited cell cultures. Although these altered biochemical behaviors of neuraminidase-treated cells apparently do not seem to influence the increased immunogenic ability of the treated cells, there are a few other observations that may be of some importance in immune reactions. Burnet and Anderson (1947) reported that normal human sera could induce agglutination of human RBCs treated with neuraminidase. Kassulke et al. (1971) demonstrated that treatment with neuraminidase increased the A and H antigens present on human leukemic leukocytes. Grothaus et al. (1971) reported that human HLA antisera showed increased lysis for neuraminidase-treated cells. Rosenberg and Rosentine (1972) showed that virtually all normal human sera contain lytic antibodies directed against autologous and allogeneic cell-membrane determinants exposed by neuraminidase treatment. In rodent systems, Ray and colleagues originally reported that neuraminidase-treated murine lymphoid cells were extremely sensitive to lysis by alloantisera and complement (Ray et al., 1970, 1971, 1972; Ray and Simmons, 1971a). Later, Schlesinger and Amos (1971) reported that neuraminidase-treated mouse spleen cells were increasingly

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cytolyzed by allogeneic H-2 antisera. Hughes et al. (1972) and Sanford and Codington (1971b) showed that normal C3H serum contained factors cytotoxic for neuraminidase-treated TA-3 cells from strain A mice. Rosenberg and Schwarz (1974) recently reported that normal sera from C3H/HeJ mice were cytotoxic to both autologous and allogeneic cells treated with neuraminidase, but had no effect on untreated cells. Sera from CBA/J, AKWJ, A/J, BALBkJ, and DBA/2J mice were decreasingly cytotoxic for neuraminidase-treated cells, and serum from C57BWSJ mice contained no cytotoxic activity. This toxicity was strictly complement dependent and was assumed to be due to an antibody. It was stable at 56°C for 30 minutes but was inactivated at 80°C for 30 minutes. Neuraminidase-treated lymphocytes absorbed this cytotoxic substance from C3H/HeJ serum, but untreated lymphocytes did not. Cells from strains containing the natural antibodies contained as much of the “hidden” membrane component as cells from strains lacking the antibody. Ray et al. (1970, 1971, 1972) and Ray and Simmons (1973b) demonstrated that mouse lymphoid cells were increasingly cytolyzed by guinea pig serum, rabbit serum, and human serum where no anti-H-2 antibody was added. Later these authors demonstrated that the cytotoxic hctor was specific and could bind with neuraminidase-exposed antigenic determinants, but not with previously available cell-surface antigenic specificities (Ray and Simmons, 1973b). These factors could be absorbed out by neuraminidasetreated cells, but not by untreated cells. These molecules as such could not cytolyze neuraminidase-treated cells and needed the complement to effect cytolysis. These molecules were having antibody-like activity (Ray and Simmons, 1973b). Thus various normal sera contained naturally occurring antibody molecules against neuraminidase-exposedantigenic determinants. Ray and Sundaram (1975a,b)also observed that, even in autologous serum, antibody against self cellular antigens exposed after neuraminidase treatment was available. Both we (Ray et al., 1970, 1971, 1972; Ray and Sundaram, 1975a,b), Rosenberg and Rosentine (1972), and Rosenberg and Schwarz (1974) showed that the antibody was of the IgM type. The biological significance of this type of antibody is not known, but several possibilities regarding the role of this type of naturally occurring antibody will be discussed in the next section. C. WHAT IS THE SOURCEOF THE NATURALLY OCCURRINGANTIBODY? The mammalian cell membrane is a complex of lipid, protein, and carbohydrate in general. Sialic acid, a 9-carbon sugar, is usually situated at the terminal positions at or near the nonreducing ends of the carbohydrate

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chains of glycoproteins and glycolipids in the plasma membrane (Winzler, 1970) and the binding is through 2,3-, 2,6-, and 2,8-a-glycosidic bonds with the next carbohydrate molecule in the polysaccharide chain (Drzeniek, 1966, 1967; Drzeniek and Gaube, 1970). Neuraminidase hydrolyzes the a-glycosidic bonds between neuraminic acid and the next sugar in the chains (Gottschalk and Bhargava, 1971), though there exist differences in their ability to hydrolyze different types of bonds between sialic acids and carbohydrates among neuraminidase obtained &om various sources (Ray and Simmons, 1973a). D. NATUREOF NEURAMINIDASE-EXPOSED ANTIGENIC SPECIFICITIES Nicolson (1973) showed that neuraminidase treatment of erythrocytes, lymphoma, and normal and virus-transformed fibroblast cell membranes unmasks p-D-galactose-like sites on the membranes. Rosentine and Plocinik (1974) showed that the exposed antigen is a carbohydrate. In carbohydrate inhibition studies, these authors showed that a family of oligosaccharides containing D-g&CtOSe at the nonreducing end of the chain and linked by p-1,4 or p-1,3 linkage to the next sugar can inhibit the human lymphocytotoxic antibody reaction with neuraminidase-treated LTC or PBL. They suggested that the naturally occurring antibody molecules against neuraminidase-exposed carbohydrate antigens might be of a heterogeneous nature, but to a varying extent all the p-galactosyl compounds inhibit all sera regardless of the target test cell. The natural cytotoxic antibody present in guinea pig serum for neuraminidase-exposed antigens on murine tumor cells was weakly inhibited by glycoproteins containing terminal P-galactosyl residues (Huges et al., 1973). Thus the naturally occurring antibody molecules appear to be directed against oligosaccharides containing p-galactosyl nonreducing end residues. There is ample evidence that mammalian cell membrane oligosaccharides contain this type of molecules. The GalP-1,4 Glc NAcP-1,3 Gal/3-1,4 Glc sequence, by itself, or as NANAa-2,3 Gal p-1,4 Glc NAc, is found in membranes of human red cells, white cells, spleen, muscle, brain, and nerve (Koscielak et aZ., 1973; Li et al., 1973; Siddiqui and Hakomori, 1973; Wherrett, 1973). The Gal-Glc NAc sequence with galactose being either p-1,3 or p-1,4 linked is found in human red cell membrane glycoproteins (Winzler, 1972). The Gal &1,3 Gal NAc sequence, especially as its sialyl derivative, is found in human gangliosides (Hakomori, 1970) and in membrane glycoproteins of human red cells (Presant and Kornfeld, 1972). Uhlenbruck et al. (1969) showed that Gal /3-1,3 Gal NAc inhibits the agglutination of neuraminidase-treated human group 0 erythrocytes by Arachis hypogaea

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lectin and the T agglutinin and is part of the T receptor originally described by Hubener (1925; Thompson, 1927; Friedenreich, 1930). Thus the compounds most inhibitory for these antibodies in normal sera are found as natural components of cell membrane oligosaccharides, usually in a sialated form. The widespread occurrence of this naturally occumng antibody molecule in various normal sera raises the question of how this antibody is formed and what is the biological significance of these molecules? One of the most probable explanations may be as follows. We are continuously being exposed to myriads of microbes, and the microbial antigens are mostly polysaccharide in nature. The body raises antibodies against these polysaccharide antigens and thus may protect it from future infections due to various microbes. It may also use it against any cell having exposed antigens so as to destroy those defective cells. Now absence of sialic acid on the cell membrane oligosaccharides may be the result of enzymic cleavage or failure of synthesis or both. Thus by any of these processes the hidden antigens may be exposed. It is very much possible that these endogenously occurring antigens may be exposed on even autologous cells during the life time of an individual so that they become autoantigenic. Further, neuraminidases are present in many microorganisms, as described earlier. Therefore, after infection with microbes, neuraminidase may be released in the system and the hidden or crippled cell-surface antigens may be unmasked on normal cells of the host, making them susceptible to destruction. Neuraminidase is a component of mammalian cell lysozymes. It is sometimes possible that activation of lysosome may release the lysosomal neuraminidase, which would in turn process the cells, rendering them susceptible to Iysis by autoantibody. Thus both infection by neuraminidase-containing microorganisms and release of endogenous enzymes, as in tissue injury may result in cells being stripped of their sialic acid and thus becoming autoantigenic. Thus the antibody already available in the serum and other body fluids may have an important role in removing damaged or defective cells from the system. These antibodies may have a role in eliminating the continuously generated transformed cells (if any) and may play a vital role in the immune surveillance system of the host, at least where unmasking similar antigens may be a part of the process. It is known that the body is continuously exposed to environmental infusions or infections. Further, many biochemical changes occur constantly within the body. Any imbalance in the dynamic equilibrium may result in disease symptoms-malignancy, of course, is a part of it. One school of opinion holds that altered or malignant cells are continuously being generated and are removed because of the active immune surveillance system.

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But it is not known exactly how these cells are destroyed by the immune components of the body. The mechanism described above in relation to the immune destruction of neuraminidase-treated autochthonous cells by autologous antibody may provide a logical explanation of this phenomenon, at least in those instances where the possibility exists of a similar exposure of hidden or crippled cell-surface antigens, as we normally see in the case of neuraminidase treatment of cells. Incomplete synthesis of oligosaccharide chains on autologous cell membrane glycoproteins and glycolipids could generate or make available this antigen also. Hakomori and Murakami found incomplete glycolipid oligosaccharide chains in malignantly transformed hamster tissue culture cells (Hakomori and Murakami, 1968). The malignant cells had low levels of the glycolipid, hematoside (NANA a-2,3 Gal p-1,4 Glc ceramide) and high levels of its precursor, lactosyl ceramide (Gal /3-1,4 Glc ceramide) as compared to untransformed cells. Either the malignant cells were unable to add neuraminic acid to the nascent oligosaccharide chain, or neuraminidase removed NANA faster than it could be added. Brady et al. (1973) provided evidence for a decrease in glycolipid glycosyltransferase activity in malignantly transformed murine tissue culture cells. They demonstrated no difference in neuraminidase activity, suggesting that the incomplete glycolipids on the transformed cells were the result of impaired synthesis. Keenan and Morre (1973) demonstrated decreased amounts of glycolipid sialyltransferase activity in dimethylbenzanthracene-induced rat mammary carcinoma. This resulted in an accumulation of the ganglioside GMI in tumor tissue. The nonreducing terminal disaccharide of GM1 is GalP-1,3 Gal NAc. Keenan and Morre’s observation is in agreement with Hakomori’s general hypothesis (1971) that a fundamental aspect of malignant transformation is an inability to complete a cell-surface oligosaccharide chain. However, if this is so, this defect would make all transformed cells antigenic to their hosts and might provoke antibody response, which in turn would destroy the cell if it was not destroyed by already available antibody in the serum (where the naturally occurring antibody may serve as part of the immune surveillance system). Thus continuously generated malignantly transformed cells might be cleared from the system by means of this mechanism. If it is possible to isolate transformed cells in uiuo during the transformation process and test that it is actually through this mechanism that these cells are destroyed, at least in certain instances, and if ultimately no malignancy is detectable in that host, then it would be conclusively established that these antibody molecules are functioning as part of the immune surveillance system against transformed cells. If this is so, one would expect an increased amount of neuraminidase-revealed antigen on untreated tumor cells during the initial phases of transformation. Over and above this, one would predict a higher

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incidence of cancer in persons not having this naturally occurring antibody. Until the fact is firmly established, it remains a speculation. In a preliminary investigation, G. N. Rosentine (personal communication, 1975)described the naturally occurring antibody levels in normals and compared them to levels in patients with newly diagnosed malignancies, patients with immunodeficiency syndromes, some of whom subsequently developed cancer, and in normal and affected members of multiple malignancy families. However, no correlation between antibody level and the presence of malignancy was established. In normal individuals antibody level was high in childhood and declined with advancing age. Although G. N. Rosentine’s observation (personal communication, 1975) does not prove or disprove that there is a higher incidence of cancer in persons having a very low level of this naturally occurring autoantibody, it does indicate that there is no readily discernable relationship between antibody level and malignancy in patients with many of the common types of cancer. A large prospective study with proper experimental subjects is needed in order to obtain definitive information. Cell-surface glycoproteins have an oligosaccharide sequence similar to that of glycolipids, but there is no clear-cut information regarding their modification during malignant transformation. Warren et al. (1973) reported increased sialic acid content in membrane glycoproteins after transformation. Ray and Simmons (1973a) also observed a very large amount of neuraminidase-releasablesialic acid on different types of malignant-cell surfaces. Compared to various types of normal cells, sialic acid on the malignant cell surface is generally h r greater (Ray and Simmons, 1973a). Further, it was reported that glycolipid on transformed cells contains low levels of sialic acid (Hakomori and Murakami, 1968) and that the sialic acid attached to glycolipid is neuraminidase resistant (Brady et al., 1973). E. Is THEREA SHIFT FROM SIALOGLYCOLIPID SYNTHESISTO SIALOGLYCOPROTEINSYNTHESISDURING MALIGNANTTRANSFORMATION? From the foregoing discussions, it appears that on malignant ceIls the sialic acid content of glycoprotein is greater, but in glycolipid the sialic acid content is far less. It is not precisely known whether during malignant transformation there is a shift whereby sialic acid from glycolipid becomes attached to glycoprotein, and this is worth investigating. It is still not known whether this shift may be a safety measure that allows malignant cells to escape the immune surveillance of the body. It is also possible that, during the very early phases of malignant transformation, sufficient sialic acid is not on the surface, and cells are eliminated by the mechanism involving autoantibody described above. During the process of tumor establishment, because

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of the immunosuppressive status of the animal the host cannot destroy the transformed cells immunologically owing to faulty synthesis of the antibody against the exposed p-glycosyl antigen or owing to the possession of very low levels of this naturally occurring antibody-thus, if the transformed cell covers the p-glycosyl antigen it may protect itself from the immune attack of the host. Future research in this direction will bring definitive information.

F. WHY THE NATURALLYOCCURRINGANTIBODYIs ANTICARBOHYDRATE ANTIBODY As we are continuously exposed to various microorganisms, we could be expected to have an appreciable amount of naturally occurring antibody against these carbohydrate antigens. It was demonstrated by Watkins (1972) that the immunodominant group in XIV pneumococcal polysaccharide antigen is Gal p-1,4 Glc NAc, and this is the same as the nonreducing end disaccharide of lacto-N-neotetraose. The antibody against XIV polysaccharide would contain anti-Galp-1,4 Glc NAc antibody. In normal human sera, antibodies against various pneumococcal antigens are available in appreciable amount (David et al., 1968), though this may not be due to subminimal infections with pneumococcal organisms such as we all normally get. These antibodies might function in two ways: (a) as a defense against the human pathogen, (b) to destroy the diseased, defective and/or malignantly transformed cells having the neuraminidase-exposed antigen of p-glycosyl type.

VII. A Probable Mechanism by Which Neuraminidase-TreatedTumor Cells Give Rise to Specific Antitumor Immunity Various types of possible immunological reactions have been discussed above in detail. Now, combining all these possibilities, a working hypothesis regarding the mechanism of induction of antitumor immunity following the inoculation of neuraminidase-treated tumor vaccine may be put forward as follows. Neuraminidase treatment, although it does not unmask H-2 antigens (Ray et al., 1970, 1972; Ray and Simmons, 1971a; Simmons et al., 1970, 1971a,b) on the cell surface, unmasks some specific neoantigens (Ray et al., 1970, 1971, 1972; Ray and Simmons, 1973b; Ray and Sundaram, 1975a,b; Rosenberg and Rosentine, 1972; Rosenberg and Schwarz, 1974; Kassulke et al., 1971; Schlesinger and Gottesfeld, 1971; Hughes et al., 1972). Specificities of these antigens were not found to be present normally on the cell surface (Ray and Simmons, 1973a; Ray and Sundaram, 1975a,b; Rosentine and Plocinik, 1974). As those hidden or crippled antigens are exposed after neuraminidase

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treatment, they may act as potent immunogens triggering the immune reactions against the exposed, and also the previously available, antigens on the cell surface. Originally, Ray and colleagues (1970,1971,1972;Ray and Simmons, 1973b; Ray and Sundaram, 1975a,b) and subsequently Rosenberg and Rosentine (1972) and Rosenberg and Schwarz (1974), showed that some normally cryptic or hidden cellular antigens, when exposed after neuraminidase treatment, may bind with naturally occurring complement-dependent cytotoxic antibodies available even in autologous serum (Ray et al., 1970, 1971, 1972; Rosenberg and Rosentine, 1972; Rosenberg and Schwarz, 1974; Ray and Sundaram, 1975a,b). These exposed antigens may bind with the naturally occurring antibodies and hcilitate the immunological processing of the cellular antigens. It is known that phagocytosis can be enhanced by increased absorption of opsonins or opsoninlike materials onto the surface of cells. In fact, it had been shown earlier that opsonized neuraminidasetreated red blood cells were more easily phagocytosed (Schmidtke et al., 1973; Lee, 1968a,b), and phagocytosis is directly related to antigen handling and/or processing. Ray and colleagues showed earlier that neuraminidasetreated cells are sensitive to immunolysis by autologous (Ray et al., 1970, 1971, 1972; Ray and Sundaram, 1975a,b), allogeneic (Ray et al., 1970, 1971, 1972), and xenogeneic (Ray et al., 1970, 1971, 1972) sera. Thus, phagocytosis or immunolysis of neuraminidase-treated tumor cells may be followed by effective processing of the exposed and the previously available tumor antigens in order to build up strong antitumor immunity, since it is known that by changing the nature of the carrier protein the response against the haptene can be modified. It is not yet certainly established what precisely is the mechanism through which strong antitumor immunity is developed after inoculation of neuraminidase-treated tumor, but the above mechanism may be at least one of the immunological reactions responsible for such antitumor immune reactions. VIII. Summary The enzyme neuraminidase is widely available in a large variety of bacteria and viruses. It is also present in various animal tissues, cells, and body fluids. The substrate of the enzyme is sialic acid-a 9-carbon sugar that is an important constituent of mammalian cell-surface glycoproteins and glycolipids. The occurrence of neuraminidase in bacteria and viruses has been shown to have some relevance to the infectivity and pathogenicity of various microbes. Neuraminidase from various sources differ in substrate specificity. The most enzyme that can reactive one is Vibrio cholerue neuraminidase (VCN)-the hydrolyze 2,3-, 2,6-, and 2,8-a-glycosidic linkages between the sialic acid and

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carbohydrate of the glycoproteins. Although almost all mammalian cells contain sialic acid, it is found in more abundant quantities on malignant cells. Treatment of cells with VCN is associated with the exposure of some hidden or crippled cell-surface antigens, which are otherwise not available on the untreated cell surfaces. These are, however, not histocompatibility antigens, but neoantigens, the specificities of which are normally not present on the cell surface. Various normal sera contain preformed naturally occurring antibody molecules, generally of IgM type, which is specific for the VCNexposed neoantigens. Thus, VCN-treated cells, even of autochthonous type, become susceptible to autologous immune destruction. Chance alteration or modification of cell surface, if associated with the exposure of such hidden or crippled antigens, may lead to immune destruction of those cells. Increased antigenicity of VCN-treated cells is associated with an increase in their immunogenicity. Normal, fetal, and malignant cells all become increasingly immunogenic after VCN treatment both in vitro and in uiuo. Treated cells are processed and/or handled more effectively than the corresponding untreated cells. Tumors previously treated with VCN, when inoculated into animals, show reduced transplantability. Animals that are immune to VCN-treated tumor also become refractory to normal tumor challenge. Animals can be successfully immunized using VCN-treated tumor whose tumorigenicity has been completely abolished either by mitomycin C treatment or X-irradiation. A “tumor vaccine” prepared by VCN treatment followed by X-irradiation can immunize animals against syngeneic tumors. Some successes have been achieved in treating even growing established tumors with such vaccineinduced immunotherapy. Apparently, a very small tumor mass perhaps could be treated by immunotherapeutic procedures. There is a promising prospect of being able to treat secondary tumors or to prevent the recurrence of tumor growth when the primary tumor either has been removed completely or reduced to minimum size by conventional treatment involving chemotherapy, radiotherapy, and surgery. A working hypothesis regarding the mechanism of action of the tumor vaccine is discussed. REFERENCES Ada, G. L., and French, E. L. (1959a).Nature {London) 183, 1740. Ada, G. L., and French, E. L. (1959b).J. Gen. Microbiol. 21, 561. Ada, G. L., and Stone, J. D. (1950). Br. /. Erp. Pathol. 31, 263. Ambrose, E. J. (1966). Prog. Biophys. Mol. Biol. 16, 241. Ambrose, E. J. (1967). Proc. Can. Cancer Res. Con$ 7, 247. Ambrose, E. J., James, A. K . , and Lowick, J. H . (1956). Nature (London) 177, 576

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Pharmacologically Active Compounds from Microbial Origin HEWITTw

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MATTHEWSAND BARBARAFRITCHEWADE

Southern School of Pharmacy, Mercer University, Atlanta, Georgia I. Introduction ........................................... 11. Types of Pharmacological Activities ....................... A. Cardiotonic Effects ...................... B. Hypotensive Effects . . C. Effects on Coagulation .............................. D. Antiinflammatory Ac E. Neuromuscular Blocking Properties . . . . . . . . . . . . . . . . . . . F. Smooth-Muscle Relaxation ........................... G . Effects on Fertility . . . . . . . . . . . . . . H. Effects on Hormone Release I. Diabetogenic Effects ................................ J. Hypocholesterolemic Effects ......................... K. Miscellaneous Activities ............................. 111. Summary ............................................. References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

269 269 269 271 272 275 277 278 279 281 281 283 286 286 287

I. Introduction

Since the historic discovery of penicillin, over 3000 antibiotics have been isolated &om microbial origin. Even today, scientists are successful in their continued search for new antibiotics from untested microorganisms. Although the major interest in the screening of microorganisms has been channeled toward the discovery of metabolities with antibiotic properties, many of these microbial metabolites have been found to possess pharmacological activity. These include: cardiotonic, hypocholesterolemic, antiinflammatory, antihypertensive, diabetogenic, and neuromuscular blockade. In many instances, these compounds showed pharmacological activities comparable to those of drugs in clinical use today. It is the purpose of this review to summarize the more recent studies on the pharmacological activities of microbial metabolites that have been reported in the past 5 years. See Perlman and Peruzzotti (1970)for a summary of earlier literature. II. Types of Pharmacological Activities

A. CARDIOTONIC EFFECTS The positive inotropic and chronotropic actions of a new antibiotic, adriamycin, isolated from cultures of Streptomyces peucetuis caesius, were reported by Kobayashi et al. (1972). This drug is composed of a red269

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pigmented aglycon, adriamycinone, and a water-soluble aminosugar, daunosamine, connected by a glycoside linkage. The aglycon portion of the molecule is thought to be the active part and contains an anthracycline nucleus. Both positive inotropic and chronotropic actions and the shortening of the action potential duration occurred with administration of this drug. In comparative experiments on daunomycin, an antiumor antibiotic chemically almost identical to adriamycin, similar results were obtained with equal doses, but the positive inotropism of daunomycin was stronger. In addition, daunomycin markedly shortened the action potential duration, especially phase 2 of repolarization. Kobayashi et al. (1972) have suggested that adriamycin and daunomycin might produce their effects at the site of either calcium-stimulated ATPase because of their resemblance to a cardiac glycoside (i.e., aglycon-glycoside linkage-sugar moiety) or the epinephrineresponsive cyclase system because of increased plasma concentrations of epinephrine and norepinephrine. The antibiotic ionophores lasalocid (Hoffmann-La Roche designation Ro22985 and X537A) and A23187 (Lilly) were studied by Schwartz et al. (1974) with respect to their effects on the sarcoplasmic reticulum, the sodiumpotassium ATPase transport systems, and the control of calcium in cardiac muscle cells. Lasalocid, an ionophore with a broad cation-complexing spectrum, was originally isolated in 1951 from a species of Streptomyces, and A23187 was isolated from a strain of Streptomyces chartreusis. Dogs treated with reserpine did not respond to the ionophores; however, lasalocid did produce inotropic effect in both atrial and ventricular muscles in nonreserpinized animals. Lasalocid also inhibited sodium-potassium ATPase activity, which was not inhibited by A23187. It was proposed by Schwartz et al. (1974)that an increased calcium availability, yielding an increased contractile force, could be produced by lasalocid via a calcium-proton exchange as follows: (a) interaction with the sarcoplasmic reticulum to cause calcium release; (b) interaction with calcium binding sites on the nerve terminal, which cause a slow release of a humoral substance; and/or (c) interaction with sodiumpotassium ATPase, which would increase available calcium. They have further suggested from the aforementioned observations that calcium-chelating ionophores might find future use in therapy for congestive heart hilure or cardiogenic shock. DeGuzman and Pressman (1974) confirmed the reports of Schwartz et al. (1974) on the myocardial contractility of lasalocid. In addition, this response was antagonized by propranolol, suggesting that the positive inotropic action of lasalocid is mediated through stimulation of beta receptors. At the same time, the mean aortic pressure increased, indicating a possible stimulation of the alpha receptor in the arteries by lasalocid. However, even with this increased mean aortic pressure, total peripheral resistance decreased. It was

PHARMACOLOGICAL AGENTS FROM MICROORGANISMS

27 1

postulated that the effects of lasalocid may arise from one or more possible transport capabilities: (a) increased availability of calcium to the contractile elements caused by directly or indirectly transporting calcium across membranes; (b) release and/or increased synthesis of neurotransmitter; (c) alteration of membrance potentials by transport of sodium or potassium across membranes; and (d) release of other hormones such as glucagon or histamine. DeGuzman and Pressman (1974) suggested that lasalocid may be of greater value than digitalis in the treatment of myocardial decompensation owing to its earlier and shorter peak of action. In investigating A23187 and cardiac contractility, Holland et al. (1975) reported that the increase in both force of contraction and rate of tension development were dependent on concentration of the ionophore. Pretreatment with reserpine did not change the effects of the drug, and the betablocker propranolol did not change the positive inotropic effect. The presence of A23187 made the atrial muscle more responsive to the effects of external calcium ions, which are necessary for contractile force, but it did not affect the contractile mechanism of the muscle. Holland et al. (1975) also suggested that the ionophore either increases available calcium by aiding in its influx across the myofibril or interacts within the cell to aid in the release of calcium from the sarcoplasmic reticulum. They, too, felt that this drug could be the beginning of a new group of cardiotonic drugs that affect the movement of calcium ions. In subsequent work on the ionophore lasalocid, Hanley et al. (1975) studied the effect of this drug on myocardial function and blood flow in various vascular beds. It was observed that when blood volume was decreased to cause a hypotensive state, lasalocid increased systemic arterial pressure, left and right ventricular force, and the speed with which the myocardial wall shortened. Coronary flow reached a maximum 15 minutes after drug injection, decreased rapidly between 15 and 30 minutes, and then slowly continued to decline. Conversely, renal blood flow increased slowly 30 minutes after injection, reached its maximum between 2.5 and 4 hours, and remained elevated until 5 hours. After producing a chemical sympathectomy with 6-hydroxydopamine, anesthetized dogs responded with increased contractility and increased renal and coronary blood flows to subsequent administration of lasalocid but were unresponsive to tyramine. From these findings the authors concluded that lasalocid does not exert its action on the cardiovascular system by the release of catecholamines.

B. HYPOTENSIVE EFFECTS Wakabayashi and Yamada (1972) investigated the effects of the macrolide antibiotics erythromycin, oleandomycin, spiramycin, and leucomycin on

272

HEWITT W. MATTHEWS A N D BARBARA FRlTCHE WADE

blood pressure depression. All four drugs effectively lowered blood pressure and produced a concomitant increase in the histamine level in blood. It was proposed that the lowered blood pressure was caused not by the antibiotics themselves, but by the fact that they caused the release of histamine. Thoa et al. (1974)studied the effect of the ionophores lasalocid and A23187 on the release of norepinephrine fiom peripheral adrenergic nerve terminals. Both substances caused a depletion of norepinephrine from the nerve terminal, but supposedly by different mechanisms. Ionophore lasalocid, like reserpine, caused the release of deaminated metabolites independent of calcium, and A23187 caused the release of deaminated metabolites by calcium-dependent exocytosis. Johnson and Scarpa (1974) in their work with lasalocid also reported that this substance could directly transport epinephrine and norepinephrine through an organic solvent. Three to six times more norepinephrine was transported than was epinephrine, and this transport was independent of calcium concentration. Similarly, Garcia et al. (1975), using the calcium ionophore A23187 in experiments on perfused cat adrenals, indicated that catecholamine release was directly proportional to the calcium concentration of the extracellular medium; release was suppressed by high concentrations of magnesium and reversed again by high concentrations of calcium. Other reports related hypotensive effects of microbial metabolites to the inhibition of enzymes, such as monoamine oxidase. Shown in Table I are the various metabolites isolated from microorganisms and their means of causing or potentially causing a hypotensive effect.

C. EFFECTSON COAGULATION Polson and Wosilait (1969) attempted to determine which of several antibiotics would stimulate the action of the coumarin anticoagulant (warfarin). Both nogalamycin and actinomycin D produced a prolongation of the prothrombin time after a lag period of several hours. The effective doses were found to be lethal to both rats and hamsters. Puromycin and cycloheximide when administered in reversible, nonlethal doses, produced results similar to warfarin. The optimum medication regimen of puromycin and cycloheximide was six hourly injections of 25 mg/kg. Puromycin was as effective as warfarin in significantly lowering the circulating levels of factor VII. Because both nogalamycin and actinomycin D inhibit most of the DNAdependent RNA synthesis, it is possible that the lag time observed in the prolongation of the prothrombin time might represent the time required for degradation of the messenger RNA present. It is also possible that nogalamycin and actinomycin D have no effect on prothrombin time other

TABLE I INHIBITION OF ENZYMES BY MICROBIAL METABOLITES WITH ANTIHYPERTENSIVE ACTIVITY

Organism

Enzyme inhibited

Oudenone Oosponol Dopastin Methylspinazarin

Oudemansiella radicata Gloeophyllum striatum Pseudomas sp. streptomyces $lipinensis

Tyrosine hydroxylase Dopamine-P-hydroxylase (D-P-H)

Dihydromethylspinazarin” Pimprinine trans-Cinnanic acid amide Phenethylamine Harman 7-0-methylspinochrome Bb 6-(3-Hydroxy-n-butyl) 7-0-methylspinochromeBb Phenopicolinic acid 3‘,5,7-Trihydroxy4’,6-dimethoxyisoflavone 3‘,5,7-Trihydroxy4’,8-dimethoxyisoflavonen

Streptomyces Flipinensis Actinomycetes Actinomycetes Actinomycetes Coriolus marimus Fungi imperjecti

Catechol-0-meth yltransferase (COMT) COMT Monoamine oxidase (MAO) MA0 MA0 MA0 COMT

Fungi imperfecti Paecilomyces sp. AF-2562

Metabolite

Dose (mg/kg)

References

3

j:

100 6.25 20 50

Umezawa et al. (1970) Umezawa et al. (1972) Iinuma et al. (1972)

25 -

-

Chimura et al. (1973a) Takeuchi et al. (1973) Takeuchi et al. (1973) Takeuchi et al. (1973) Takeuchi et al. (1973)

12.5

Chimuraet al. (1973b)

COMT D-P-H

12.5 50

Chimuraet al. (1973b) Nakamuraet al. (1975)

Streptomyces roseolus

COMT

12.5

Chimura et al. (1975)

Streptomyces roseolus

COMT

50

Chimura et al. (1975)

“Could also inhibit dopamine-P-hydroxylase. b e o d d also inhibit tyrosine hydroxylase.

D-p-H

Chimura et al. (1973a)

!$ 8

F r g z 2 a

2 3.

0”

5z

g

274

HEWITT W. MATTHEWS AND BARBARA FRlTCHE WADE

than nonspecific toxic effects, considering that only lethal doses were effective. On the other hand, since puromycin and cycloheximide inhibit the biosynthesis of protein at the ribosomal level, and since they produced results similar to warfarin, Polson and Wosilait (1969)suggested that warfirin also might act at the ribosomal level or at a subsequent step in the synthesis of the clotting factors. Since it was known that the polyene antibiotic filipin inhibits blood coagulation along the extrinsic pathway, Van der Plas et al. (1974) were interested in the effect of this substance on the lipid-dependent intrinsic pathway of blood coagulation. Of special interest was the influence of filipin on the adsorption of factors VIII and IX, into phospholipid in the presence of several calcium concentrations. It was observed that the calculated adsorption of factor VIII was enhanced, especially when recovery from the supernatant fluid was already diminished by the absence of calcium ions. Factor IX,, however, was more readily adsorbed at higher calcium concentrations. Despite the fact that more factor VIII was adsorbed onto the lipid surface, filipin invariably inhibited intrinsic clotting. Thus, the enhanced adsorption probably takes place in an unfavorable way, resulting in complexes that are not suitable for &tor conversion. Van der Plas et al. (1974) also concluded that the inhibition of the intrinsic pathway depends upon the sterol content of the lipids used for the formation of the factor VII1,-IX, lipid complex. Gerrard et al. (1974) studied the effects of the ionophore A23187 on blood platelets with respect to ultrastructure. This was undertaken to determine the nature of the physical changes that develop in the cells as a direct result of an increased level of calcium ions. It was found that A23187 caused concentration-dependent physical alterations in blood platelets that were identical to those stimulated by potent aggregating agents. With moderate amounts of A23187, platelets lost their discoid form, became irregular in shape, and developed internal transformation. This transformation consisted of movements of randomly dispersed organelles to the center of the platelets where they were surrounded by microtubules and microfilaments. At high concentrations of A23187 (100 pdml), the cytoplasmic gel constricted into a dense central mass and irreversible injury to the cell membrane occurred. Gerrard et al. (1974)suggested that A23187 caused the movement of calcium ions from channels of the dense tubular system into the platelet cytoplasm and that these increased levels of calcium triggered the contractile processes, causing secretion and aggregation. In related studies on the effects of A23187 on blood platelets, White et al. (1974) investigated the influence of this ionophore on platelet aggregation and secretion. At concentrations of 20 pg or more of ~23187,rapid irreversible aggregation of platelets occurred. At all concentrations of A23187 where

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275

aggregation occurred, there was a significant release of [14C]serotonin.Both the above responses were concentration dependent. Evaluation of the uptake of isotopic 45Cainto the platelets stimulated by the ionophore showed a rapid increase in cell-associated calcium. When calcium ions were added to divalent-free suspensions of platelets, aggregation with A23187 was accelerated. Magnesium ions could also improve the effects of ~ 2 3 1 8 7but , greater concentrations of magnesium were needed to obtain the same effect obtained with the calcium. A23187 could activate platelets even in the absence of calcium in the suspensions, and aggregation could be stimulated even when the uptake of extracellular cations was blocked by lanthanum chloride. White et al. (1974) proposed that these findings give added support to the idea that platelets are muscle cells and that contractile physiology mediates their response to an aggregation reaction. O’Reilly (1975) studied the interaction of chronic daily warfarin therapy and rifampin. Normal human subjects were given daily doses of warfarin, with or without rifampin. Both hypoprothrombinemia and plasma levels of warfarin were measured. The results indicated that there was a significant increase in the one-stage prothrombin activity toward the normal, and plasma warfarin levels were markedly reduced. O’Reilly (1975) suggested that rifampin enhances the elimination of warfarin from the body rather than inducing its enzyme induction. This hypothesis was based on the fact that although the plasma levels of wa&rin decreased greatly, the increased quantities of metabolic products of warfarin were relatively small.

D . ANTIINFLAMMATORYACTIVITIES Aoyagi et al. (1969) described the biological activity of leupeptins, produced by various species of Actinomycetes. The leupeptins used in these experiments were acetyl- and propionyl-~-leucyl-l-leucyl-~,~~ argininals. These substances were observed to competitively inhibit proteolysis by plasmin, trypsin, and papain. After oral administration of 100 mgkg to rats, these leupeptins produced an anti-inflammatory effect on carragenininduced edema. The effects of a mixture of leupeptins on various enzyme systems compared with those of r-aminocaproic acid, trans-4aminomethylcyclohexanecarboxylic acid, soybean trypsin inhibitor, and trasylol are shown in Table 11. Famaey and Whitehouse (1975) investigated the effects of the membrane permeant antibiotics valinomycin, alamethicin, and gramicidin A on the incorporation of thymidine and uridine into DNA and RNA, respectively. All three substances effectively decreased such protein synthesis in rabbit thymocytes in uitro. Since this mode of action has been attributed to

276

HEWlTT W. MATTHEWS AND BARBARA FRITCHE WADE

TABLE I1 EFFECTSOF LEUPEPTINS,E-ACA, t-AMCHA, SOYBEAN TRYPSIN INHIBITOR AND TRASYLOL ON ENZYME SYSTEMS”

IDm Enzymes

Substrates

Trypsin inhibitor

19.2

Trasylol (KIU)h

500

500

10,000

12,000

10,000 10,Ooo

10,000 10,000

lo00 1000

200 1000

Fibrinogen Fibrin Casein TAME

8 6 16 85

1,000 500 2,250 4,500

170 100 500 1,000

4

5.7 5 80

3 3 10 15

Casein Hemoglobin BAAd TAME BAEE

2 3.6 0.1 65 80

1,000 500 200 10,000 10,Ooo

1,000

0.5 2 1.8 2 2.5

2.5 5 20 30

Thrombin

TAMEb BAEEC

Papain

t-AMCHA’

500

Plasma

Trypsin

E-ACA’

15

Thrombokinase

Plasmin

Leupeptins

(~dml)

Casein Hemoglobin BAA

Kallikrein

BAEE

a-Chymotrypsin

Casein ATEEe

0.5 0.15 0.05 75 500 2,500

500 200 10,Ooo

10,000

5,000 500 200

1,000 500 200

1000 500 200

54 500

10,000

10,000

1000

6

5,000 20,000

2,500 10,000

100 350

5.5 50

84

“From Aoyagi et d.(1969). “Biological activities of leupeptins,” j . Antibiotics 22, 558-568, Table 111 (p. 564). bp-T~l~ene~~lfonyl-~-arginine methyl ester HC1. ‘a-N-Benzoyl-L-arginine ethyl ester HCI. da-N-Benzoyl-~-arginineamide HCI. eN-Acetyl-L-tyrosine ethyl ester. ‘c-Aminocaproic acid. gtrans-4-Aminomethylcyclohexanecarboxylicacid. ’1 KIU = 3 pg.

nonsteroidal and antiinflammatory drugs, Famaey and Whitehouse (1975) suggested that valinomycin, alamethicin, and gramicidin A could potentially have antiinflammatory properties. Various antiinflammatory substances have been isolated from microorganisms and are listed in Table 111.

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PHARMACOLOGICAL AGENTS FROM MICROORGANISMS

TABLE I11 ANTIINFLAMMATORY COMPOUNDS FROM MICROORGANISMS Name of compound

Microbial source

Kinonase BI Retikinonase I A neutral proteinase Three neutral proteinases Alkinonase A, Alkinoase AF Alkaline proteinase A and B A semi-alkaline proteinase Elastatinal

Streptomyces kinoluteus Streptomyces verticillutus Streptomyces griseolus Sheptomyces cacasi Streptomyces violaceo Sheptomyces griseoviridis Sheptomyces cinereoruber Streptomyces griseoruber

Antipain

Planomonospora parontospora

References Nakamura et al. (1969) Nakamura et al. (1970a) Nakamuraet al. (197Oa) Nakamuraet al. (1970b) Nakamura et al. (1972) Nakamuraet al. (1970a) Nakamura et al. (1970b) Umezawa et al. (1973), Okura et al. (1975) Wingenderet al. (1975)

E. NEUROMUSCULAR BLOCKINGPROPERTIES In his review of the literature with respect to neuromuscular blocking properties of antibiotics, Emery (1968) described several cases in which aminoglycoside antibiotics produced respiratory depression following surgery. The most potent blocking agent was neomycin when used in the peritoneal cavity for gastrointestinal surgery. Emery (1968) suggested that the neomycin was rapidly absorbed through the damaged peritoneum, thus producing the observed respiratory depression. Davia et al. (1970) reported of uremia, deafness, and paralysis caused by irrigating solutions of antibiotics. Neomycin and polymyxin B were thought to produce this paralysis by a neuromuscular competitive blockade involving depression of the calcium ion concentration at the myoneural junction. Jenkins et a2. (1970) in their discussion of the treatment of crisis in myasthenia gravis, indicated that many antibiotics can block transmission at the neuromuscular junction and enhance or induce a myasthenia-like state. Since infection is the most common cause of myasthenic crises, the choice of the proper antibiotic is of great importance. Antibiotics with dangerous neuromuscular blocking properties are as follows: neomycin, streptomycin, kanamycin, polymyxin, colistin, bacitracin, viomycin, and gentamicin. These substances should be avoided unless the patient is already receiving artificial respiration. Antibiotics that do not possess dangerous neuromuscular blocking effects are penicillin, ampicillin, cephalosporin, and erythromycin. McQuillen (1970), in a similar discussion, reported on the potentiation of neuromuscular weakness in myasthenic patients treated with the antibiotics listed above.

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HEWITT W . MATTHEWS A N D BARBARA FRITCHE WADE

Dretchen et al. (1972) conducted studies to determine the effects of certain antibiotics on acetylcholine release at the nerve terminal. The antibiotics under investigation were streptomycin, neomycin, kanamycin, polymyxin and gentamicin. They found that within the range of concentrations producing neuromuscular blockade, neomycin, streptomycin, kanamycin, and polymixin B caused no significant change in the amount of acetylcholine released from the presynaptic nerve terminal. Polymyxin was found to be the most potent neuromuscular blocking agent of those substances tested. Gentamicin, however, significantly decreased the amount of acetylcholine released, especially at the concentration of 100 pg/ml. The results obtained with gentamycin suggest that it has a direct effect on the presynaptic nerve terminal. Colistin, one of the polypeptide antibiotics that can produce neuromuscular blockade, was studied by McQuillen and Engbaek (1973)to determine its mechanism of action on neuromuscular transmission. Neomycin was used as a comparator. From their data, McQuillen and Engbaek (1973) concluded that colistin decreases the size of the readily releasable store of acetylcholine, whereas neomycin depresses the probability of acetylcholine release. This proposed mechanism for colistin would explain why it could not block muscle contraction alone. As long as there is enough acetylcholine to cause a muscle response to nerve stimulation, no effect of the antibiotic would be seen.

F. SMOOTH-MUSCLERELAXATION The effect of dicloxacillin on in vitro and in vivo preparations of bile duct and gall bladder were studied by Benzi and Arrigoni (1970). Dicloxacillin induced a relaxation of isolated calf terminal bile duct in both the normal bile duct and bile duct made hypertonic with the spasmogens barium chloride, carbachol, and histamine. At concentrations levels of 20 pg/ml, barium chloride could be inhibited; levels of 80 pg/ml were needed to note any inhibition against carbachol and histamine. In isolated guinea pig and cat gall bladder, dicloxacillin had an opposite effect-that of increasing the tone and motility of the gall bladder. When dicloxacillin in varing concentrations was combined with constant concentrations of rifampin SV or erythromycin, the constrictive action of the latter two antibiotics on isolated calf terminal bile duct was decreased. Data obtained indicated a probable competitive inhibition. Combinations of constant concentrations of aminosidin or ampicillin with various combinations of dicloxacillin increased the relaxing abilities of these two antibiotics. In a subsequent study, Benzi et al. (1972) reported the effect of the an-

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279

tibiotics gentamicin, aminosidin, and ampicillin on uterine tone and motility. Gentamicin caused increased uterine tone and frequency of uterine contractions and decreased the height and duration of these contractions in both pregnant and nonpregnant guinea pigs in uitro. Aminosidin and ampicillin, on the other hand, inhibited both tone and frequency of uterine contractions but increased height and duration of these contractions. Pretreatment with atropine, chlorpheniramine, methylsergide, cyproheptadine, dibenamine, INPEA, and hexamethonium had no effect on uterine response to the antibiotic, indicating that the antibiotics work directly on the uterine muscle. It was observed that doses per kilogram of the antibiotics tested on uterine activity were within the range of doses used for humans during obstetrical and gynecological infections. In 1973, Benzi et al. studied the effects of certain antibiotics on ureter motor activity. Studies, both in vitro and in situ, on guinea pigs and dogs demonstrated that chloramphenicol, gentamicin, aminosidin, ampicillin, isoxazolyl penicillins, and spiramycin induced both antagonism of spasms produced by spasmogens (barium chloride, histamine, serotonin) and an increase of flow through the ureter, either under normal or spastic conditions. The order of potency of the myolytic activity was chloramphenicol > isoxazolyl penicillins > gentamicin > aminosidin > spiramycin 2 ampicillin. The action of the tetracyclines varied with the specific antibiotic used and with the percentage of the 4-epiderivatives in the tetracycline used. That is, mepicycline inhibited the spasm-producing effect of barium chloride, whereas tetracycline and rolitetracycline increased this effect. The greater the 4-epiderivative concentration, the greater the stimulating action of barium chloride. There were no significant differences in the activities of tetracycline, oxytetracycline, and chlortetracycline. Triggle et aZ. (1975) investigated the effects of the ionophone A23187 on intestinal smooth muscle contraction. This ionophore produced a contraction of guinea pig ileal longitudinal muscle that was dose dependent. The initial contraction was very rapid, followed by a slow return to a normal relaxed state. Moreover, the tissue remained in an enhanced state even after the drug was washed out. Since the presence of calcium was necessary for the activity of ~ ~ 3 1 8it7 is , possible that this ionophore induces a contraction by transporting calcium from the outside to the inside of the smooth muscle or by affecting the release of calcium from sarcoplasmic reticulum. G. EFFECTSON FERTILITY The inhibitory effect of twenty-one antibiotics on the motility of spermatozoa of various animals and man was studied by Fuska et al. (1973). Only frequentin (isolated from Penicillium fj-equentans) and cyclopaldic acid to-

280

HEWITT W. MATTHEWS AND BARBARA FRITCHE WADE

tally stopped the movement of spermatozoa within 5 minutes. Frequentin, in concentrations of 20-80 pg/ml, had the greatest effect on bull's semen but the least effect on semen from rabbits and bucks. An evaluation of the biochemical effect of frequentin on sperm indicated a decreased vitality of the sperm by this substance. Cyclopaldic acid inhibited the movement of sperm only at concentrations from 400 to 500 pglml. The results of the various antibiotics tested are shown in Table IV. Timmermans (1974) reported on the effects of ten antibiotics on the spermatogenesis in animals. From the results obtained, it was possible to classify these substances into three groups based on their effects on sperTABLE IV

EFFECTOF ANTIBIOTICSON

THE

MOTILITYOF SPERMATOZOA" Influence on motility

Antibioticb Bacitracin Chloramphenicol Cycloheximide Erythromycin lactobionate Helvolic acid Kojic acid Neomycin sulfate Penicillin G (potassium salt) Streptomycin sulfate Tetracycline HCI Viomycin sulfate Amphotericin B Chlorotetracycline HCI Citrinin Cyanein Griseofulvin Palitantin Penicillic acid Trichothecin Cyclopaldic acid Frequentin

Mechanism of actionC

P, P

cw

NA P

Toxicity

of

(LDS0,pg/ml)

spermatozoae

342 1320 300 1800

400 250

NA, P

cw NA, P, CW P

P

cw P

cw NA, P NA

4450 None 200 330 240 26 192 200 200 280 250

cw

N N N N N N N N N N N

I I

I

I I I I

I

s S

nFrom Fuska et al. (1973). q h e concenbation of antibiotics in all experiments was 500 pg/ml of semen. 'P = synthesis of proteins; CW = cell wall synthesis; NA = synthesis of nucleic acids; N = motility of spermatozoa not inhibited; I = motility of spermatozoa inhibited within 3 hours by the added substances; S = substances totally stopped the movement of spermatozoa within 5 minutes.

PHARMACOLOGlCAL AGENTS FROM MICROORGANISMS

28 1

The first group, including gentamicin sulfate, N(5-nitrohrfuryllidene)-l-aminohydantoin, and oxytetracycline, stopped spennatogonia division and inhibited spermatocytes I meiosis. Oxytetracycline had the smallest effect in this group. The second group, containing colymyspiramycin, sodium-7-(thiophene-2-acetamido)-cephalo-sporanate, cin methane sulfonate, and framycetin, showed an alteration of mitosis in spermatogonia. In the third group, potassium penicillin G , chloromycetin succinate, and trimethoprim showed partial or complete inhibition of the spermatogonial division and aggravated meiosis in most spermatocytes. Timmermans (1974)observed that the action of the antibiotics seemed to be specifically on germ cells rather than on interstitial tissue. matogenesis.

RELEASE H. EFFECTSON HORMONE Hertelendy et al. (1971)found that valinomycin inhibited the in uitro responses of prostaglandin El dibutyryl cyclic AMP, theophylline, and K+ on the stimulation of growth hormone release. Based on experimental results, it was postulated that the site of action was not at the adenyl cyclase level, but rather distal to this site. It is possible that this antibiotic interferes with the energy metabolism of the pituitary cells. Russell et a2. (1974)observed the effect in uitro of the ionophore A23187 on release of vasopressin from the neurohypophysis. The neurohypophyses of rats were incubated in a calcium-&eemedium with A23187.When calcium was added to the medium, there was a significant increase in vasopressor release. When labeled 45Ca was used, addition of the ionophore to the medium caused an increased efflux of the ion. From these experiments, it seemed that A23187 increased the transport of calcium &om outside the cell to the inside, a step that is necessary for vasopressin release. Grenier et al. (1974)studied the effect of A23187 on thyroid secretion. They found that those thyroid functions needing extracellular calcium for their actions were positively affected by A23187. These functions were the activation of glucose carbon-1 oxidation and the binding of iodide to proteins.

I. DIABETOGENIC EFFECTS Schein and Bates (1968)compared plasma glucose levels in mice treated with streptozotocin with those pretreated with nicotinamide. Streptozotocin, an antitumor agent produced by Streptomyces achromogenes, had been observed to destroy the pancreatic beta cells of test animals. In this experiment both control and nicotinamide-protected mice showed an initial hyperglycemic phase after administration of 175 mg of streptozotocin per kilogram. This early elevation of blood sugar was not dependent on the

282

HEWITT W. MATTHEWS AND BARBARA FRITCHE WADE

presence of an adrenal gland but was drug related. After 7 hours, the animals treated with only streptozotocin showed a hypoglycemic phase, associated with increased plasma levels of immunoreactive insulin. After 24 hours, these mice were permanently diabetic. The group receiving 500 mg of nicotinamide per kilogram remained protected and showed no evidence of diabetes. Beloff-Chain and Rookledge (1968) studied the metabolism of glucose in diaphragm muscle obtained from normal rats, streptozotocin-treated diabetic rats, and rats treated with anti-insulin serum. It was found that the formation of glycogen and oligosaccharides from labeled [ 14C]glucosewas decreased in both the diabetic and anti-insulin-treated rats. Neither state influenced the oxidation of glucose or the formation of lactate and hexose phosphate esters from glucose. In the presence of insulin, the diabetic muscle resumed its ability to incorporate glucose into glycogen. Although its ability to form oligosaccharides increased somewhat, these levels never returned to normal values. Kushner et al. (1969) found that rabbits and guinea pigs receiving a maximum dose of 130 mg of streptozotocin per kilogram were resistant to the diabetogenic effects of streptozotocin, whereas rats became diabetic. This effect in rabbits and guinea pigs was apparently not due to rapid metabolism or lack of absorption of streptozotocin, because the blood levels of the drug remained high. Thus, it is possible that the drug may exert its effect on a specific metabolic pathway that is species dependent. Since nicotinamide administration prevents the formation of diabetes in rats, it is possible that streptozotocin blocks the synthesis of N A D from nicotinamide in rats but has little or no effect on N A D synthesis from nicotinic acid, the pathway that may be preferentially employed by rabbits and guinea pigs. Veleminsky et al. (1970) compared the early metabolic events resulting from administration of the two diabetogenic agents alloxan and streptozotocin. Either 65 mg of streptozotocin or 60 mg of alloxan per kilogram produced similar degrees of pancreatic insulin depletion 48 hours after administration, resulting in metabolic changes indicative of acute diabetes. These metabolic changes were ultimately the result of beta-cell destruction. In a similar study, Forster and Rudas (1969) showed that rats made diabetic with streptozotocin developed a transient ketosis which disappeared after 7 days. Woods et al. (1970) used strepotozotocin to produce a diabetic state for their studies on conditioned insulin secretion in the albino rat. Pitkin and Reynolds (1970)observed that doeses of 60 mg of streptozotocin per kilogram could produce diabetes in rhesus monkeys. However, the diabetogenic dose of streptozotocin in these animals was very close to the toxic dose. Histological studies revealed decreased numbers of pancreatic islets, and during the first few days after streptozotocin administration the islets contained

PHARMACOLOGICAL AGENTS FROM MICROORGANISMS

283

virtually no beta cells. Other accounts of the diabetogenic activity of streptozotocin include those of Golob et al. (1970a,b), Wyse and Dulin (1971), Beloff-Chain et al. (1971), and Beloff-Chain and Rookledge (1972).

J. HYPOCHOLESTEROLEMIC EFFECTS Van den Bosch and Claes (1967) attempted to correlate the bile saltprecipitating capacity of derivatives of basic antibiotics in vitro and their plasma cholesterol-loweringeffectsin oiuo. Derivatives of streptomycin and neomycin were synthesized to observe whether increasing the number of basic groups on the molecule would form a compound more active than the parent compound. Their effects on bile salt solutions, on resorption of lithocholic acid, and on plasma cholesterol levels in chickens were investigated. The effects of only one derivative (N-methylatedneomycin) on plasma levels and on fecal bile salt excretion in humans was also observed. The following derivatives of neomycin were prepared: N-methylated neomycin, N-methylated neomycin methochloride, and N-hexaacetylneomycin. The two derivatives of streptomycin were synthesized by condensation of streptomycin trihydrochoride with di- and triaminoguanidine hydrochloride to obtain distreptomycylidene-diaminoguanidine heptahydrochloride and tristreptomycylidene-diaminoguanidine decahydrochloride. When dilutions of these substances were added to solutions of sodium glycodeoxycholate, precipitates of the bile salt were observed for all substances except streptomycin and N-hexaacetylneomycin. These results were consistent with the basicity of the molecules, since streptomycin contains only three basic groups and N-hexaacetylneomycin is not basic. In the streptomycin series there was a sharp increase in the precipitating capacity with an increased number of basic groups per molecule. In tests on newly hatched chicks fed a semisynthetic casein sucrose diet supplemented with 0.25% cholesterol, again those substances which initially precipitated bile salts also decreased plasma cholesterol levels. A diet with 2% tristreptomycincylidenediaminoguanidine decahydrochloride lowered plasma levels nearly to normal values. Human volunteers given doses of either 3 or 6 gm/day of N-methylated neomycin showed a sharp decrease in plasma cholesterol levels after 20 days. It was also observed that feces of subjects given N-methylated neomycin contained increased amounts of bile salts and sterols. No objective or subjective side effects of N-methylated neomycin were observed. In a similar study, Eyssen et aZ. (1971) prepared N-methylated, N-acetylated, and dimethylaminopropyl derivatives of neomycin to observe the effect of these polybasic antibiotics on absorption and excretion of cholesterol and bile salts. These derivatives were chosen for this experiment in an

284

HEWITT W . MATTHEWS A N D BARBARA FRITCHE WADE

TABLE V EFFECTOF NEOMYCIN, STREPTOMYCIN, AND THEIR DERIVATIVES ON SERUM AND LIVERCHOLESTEROL LEVELSOF NEWBORN CHICKS

Treatment' N-Methylated neomycin N-Acetylated neomycin DimethylaminoProPYl neomycin Streptomycin Distreptomycinb TristreptomycinC

Serum cholesterol

Liver cholesterol

-22%

-44%

+4%

-59%

--10% -78%

+8%

-5%

-20% -45%

-26%

-75%

aTwenty chicks per group after 2 weeks. Diets contained 0.2%of the test substance. *Distreptomycylidene-diaminoguanidineheptahydrochloride. Tristreptomycylidene-triaminoguanidine decahydrochloride.

attempt to show that their hypocholesterolemic effect was a result of their basicity, not of their antimicrobial properties. Thus, the N-methylated compound contained six basic groups, the N-acetyl substance was neutral, and the dimethylaminopropyl derivative possessed more basicity than the N-methylated compound. Similarly, two basic derivatives of streptomycin were prepared: (a) distreptomycin containing seven basic groups, and (b) tristreptomycin containing ten basic groups. Streptomycin, with only three basic groups, was not expected to have any effect on cholesterol concentrations. The data are summarized in Table V. It can be seen that, as expected, streptomycin and N-acetylated neomycin had very little effect on serum and liver cholesterol levels, whereas the most basic substances, tristreptomycinand the dimethylaminopropylneomycin had the greatest effect. In addition, fecal concentrations of bile salts increased with those substances that showed a hypocholesterolemic effect. Experiments with neomycin and germfree chicks also showed an increased fecal excretion of bile salts and a lowering of the serum and liver cholesterol concentrations. This would indicate that the antibiotic activity, yielding alterations of intestinal microflora, was not responsible for the observed results. These polybasic substances must have a direct effect on cholesterol and bile salts.

285

PHARMACOLOGICAL AGENTS FROM MICROORGANISMS

Samuel et a2. (1973)observed the effect of neomycin on serum cholesterol levels and the 7-a-dehydroxylationof bile acids by fecal bacteria in humans. Oral administration of 2 gm of neomycin lowered serum cholesterol levels from 316 mg/100 ml of plasma to 237 mg/100 ml of plasma. In addition, 7-a-dehydroxylation was decreased from 89% to 9%. Similar results were obtained with kanamycin, chloramphenicol, and chlortetracycline. In several patients where these various drugs did not lower serum cholesterol, there was also observed to be no bile acid degradation. Sasaki et al. (1973), in screening for hypolipidemic agents, isolated ascofuranone and ascofuranol from an ascochlorin-producing fungus, Ascochyta viciae. Ascofuranone was found to lower lipid levels in mice and rats. In a subsequent paper, Sawada et al. (1973)reported on the pharmacological properties of this new hypolipidemic agent, ascofuranone. Its LD5,, was found to be high for both mice and rats; diarrhea was the only side effect observed with large doses. After a single oral dose of ascofuranone, serum cholesterol levels were observed to be as low as or lower than those of the positive control agent clofibrate. Doses for each drug were approximately 108 mgkg. Serum triglyceride, phospholipid, and free fatty acid levels were also determined for both ascofuranone and clofibrate. It was observed that ascofuranone lowered serum triglyceride and free fatty acid levels more than the control clofibrate. Serum phospholipid levels after ascofuranone administration were only 2%higher than the clofibrate control. To test the effects of ascofuranone and clofibrate on long-term treatment, rats were given doses of 20 and 30 mg/kg, respectively, for 10 days. Clofibrate lowered serum lipid levels more than did ascofuranone. The results of this treatment are shown in Table VI. When the organ weights of these rats were measured, it was found that hepatomegaly, accompanied by atrophy of the spleen and heart, occurred with clofibrate. Although ascofuranone treatment slightly reduced the 1iver:body weight ratio, liver function was normal. When heart cholesterol content was deterTABLE VI BY ASCOFURANONE EFFECTOF CONSECUTIVE 10-DAYTREATMENT ON SERUM LIPIDLEVELS

Agents

Serum cholesterol (% change)

Serum triglyceride (% change)

phospholipid (% change)

Serum free fatty acid (% change)

Ascofuranone Clofibrate

-16.5 -23.9

-44.3 -48.4

-33.8 -40.8

-20.2 -24.3

Serum

286

HEWITT W. MATTHEWS A N D BARBARA FRlTCHE WADE

mined, it was found that ascofuranone lowered total heart cholesterol about 14% whereas clofibrate had no effect. Micklewright and Trigger (1974) observed that cholesterol absorption was reduced in small laboratory animals fed the antihngal agent candicidin. With a candicidin dose of 100 mg/kg for rats, cholesterol absorption was reduced to an average value of 23.7%. Studies with mice indicated that absorption of cholesterol was reduced to 8.8%. Similar results were obtained with hamsters, guinea pigs, and rabbits. Oral and intraperitoneal administration of the antibiotic hamycin caused a decrease in plasma cholesterol levels to about 40% of the initial levels after 120 hours (Dave and Parekh 1975). Hamycin was not thought to prevent absorption of cholesterol levels in rats. Doses of 1 m&g per day resulted in a maximum decrease in serum cholesterol of 25% after 4 days. Thereafter, serum cholesterol levels returned to normal, and no dgerence was observed with continued treatment. On autopsy no gross morphological or histological changes in the organs were observed, indicating that amphotericin B’s mode of action is direct rather than through organ damage.

K. MISCELLANEOUSACTIVITIES Phansalkar and Balwani (1970) observed the effects of various antibiotics on hexobarbitone sleeping time in rats. Streptomycin, erthromycin, and spiramycin were observed to significantly shorten the mean sleeping time of these animals. Ogata et al. (1974) reported of a cholinesterase inhibitor produced by Asper-gillus terreus. This inhibitor, labeled 1-6123, was tested on esterases from Pseudomnas aer-uginosa, Electrophorus electricus, horse serum, and pig liver, under conditions where substrate inhibition does not occur. 1-6123 strongly inhibited the nonspecific pig-liver esterase and only weakly inhibited the others. 111. Summary

Since microorganisms provide such a wide variety of metabolic metabolites (i.e., amino sugars, macrolides, pyridines, etc.), it would seem reasonable to assume that microbial metabolites would be excellent sources of pharmacological compounds. Some of the aforementioned compounds reviewed were found to have pharmacological properties secondary to their antibiotic properties. Perhaps if greater emphasis were placed on seeking microbial products with pharmacological activities rather than antibiotic activities, a greater variety of compounds would be available for clinical use.

PHARMACOLOGICAL AGENTS FROM MICROORGANISMS

287

REFERENCES Aoyagi, T., Miyata, S., Nanbo, M., Kojima, F., Matsuzaki, M., Ishizuka, M., Takeuchi, T., and Umezawa, H. (1969).J . Antibiot. 22, 558-568. Beloff-Chain, A., and Rookledge, K. A. (1968) Biochem. J . 110, 5 2 W 2 . Beloff-Chain, A., and Rookledge, K. A. (1972). Zsr. J . Med. Sci. 8, 808409. Beloff-Chain, A., Chain, E. B., and Rookledge, K. A. (1971). Biochem. J. 125, 97-103. Benzi, G., and Arrigoni, E. (1970).Arch. Znt. Pharmucodyn. Ther. la,213-226. Benzi, G., Arrigoni, E., Panceri, P., and Berte, F. (1972).Jpn. J. Pharmucol. 22, 571576. Benzi, G.. Amgoni, E., Panceri, P., Panzarasa, R., Berte, F., and Crema, A. (1973). I p n . 1. Phannacol. 23, 559-607. Chimura, H., Sawa, T., Takita, T., Matsuzaki, M., Takeuchi, T., Nagatsu, T., and Umezawa, H . (1973a).J . Antibiot. 26, 112-115. Chimura, H., Sawa, T., Kumade, Y., Nakamura, F., Matsuzaki, M., Takita, T., Takeuchi, T., and Umezawa, H. (197313).J . Antibiot. 26, 618-620. Chimura, H., Sawa, T., Kumada, Y.,Naganawa, H., Matsuzaki, M., Takita, T., Hamada, M., Takeuchi, T., and Umezawa, H. (1975).J . Antibiot. 28, 619-626. Dave, C. V., and Parekh, A. C. (1975). Proc. SOC. E x p . B i d . Med. 149, 299-301. Davia, J. E., Siemsen, A. W., and Anderson, R. W. (1970).Arch. Intern. Med. 125, 135-139. DeGuzman, N. T., and Pressman, B. C. (1974). Circulation 49, 1072-1077. Dretchen, K. L., Gergis, S. D., Sokoll, M. D., and Long, J. P. (1972).Eur. J. Phurmacol. 18, 20-203. Emery, E. R. (1968).Znt. Anesth. Clin. 6, 665473. Eyssen, H . , Vanderhaeghe, H., and D e Somer, P. (1971).Fed. Proc., Fed. Am. SOC. E x p . Biol. 30, 1803-1807. Famaey, J. P., and Whitehouse, M. W. (1975).Agents Actions 5, 133-136. Forster, O . , and Rudas, B . (1969). Lancet 1, 1321-1322. Fuska, J., Podany, J . , and Muzikant, J. (1973). Biologia (Brutislaoa) 28, 463-467. Garcia, A. G., Kirpekar, S. M., and Prat, J. C. (1975).I . Physiol. (London) 244, 253-262. Gerrard, J. M., White, J. G., and Rao, G. H. (1974).Am. J . Pathol. 77, 151-166. Golob, E. K., Rishi, S., Becker, K. L., Moore, C., and Shah, N. (197Oa).Diabetes 16,610-613. Golob, E. K., Rishi, S., Becker, K. L., and Moore, C. (1970b). Metab., Clin E r p . 19, 10141019. Grenier, G., Van Sande, J., Glick, D., and Dumont, J. E. (1974). FEBS Lett. 49, 96-99. Hanley, G. G., Lewis, R. M., Hartley, C. J., Franklin, B., and Schwartz, A. (1975).Cir. Res 37, 215-225. Hertelendy, F., Peake, G., and Todd, H. (1971). Biochem. Biophys. Res. Commun. 44, 253260. Holland, D. R., Steinberg, M. I., and Armstrong, W. M. (1975). Proc. Sac. E x p . B i d . Med. 148, 1141-1145. Iinuma, H., Takeuchi, T., Kondo, S., Matsuzaki, M., and Umezawa, H. (1972).J . Antibiot. 25, 497-500. Jenkins, R. B., Witorsch, P., and Smyth, N. P. (1970). South. Med. J . 63, 1127-1130. Johnson, R. G., and Scarpa, A. (1974). FEBS Lett. 47, 117-121. Kobayashi, T., Nakayama, R., Takatani, O., and Kimura, K. (1972).J p n . Circ. J . 36, 259-265. Kushner, B., Lazar, M., Furman, M., Lieberman, T. W., and Leopold, I. H. (1969).Diabetes 18, 542-544. McQuillen, M. P. (1970).Ann. Intern. Med. 73, 487-488.

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W.

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McQuillen, M. P., and Engback, I. (1973). Trans. Am. Neurol. Assoc. 98, 86-89. Micklewright, P. F., and Trigger, D. J. (1974). J . Pharm. Pharmacol. 26, 10&109. Nakamua, S., Marumoto, Y., Miyata, H., Tsukada, I., Tanaka, N., Ishizuka, M., and Umezama, M. (1969). Chem. P h a m . Bull. 17, 2044-2048. Nakamura, S., Hamada, M., Ishizuka, M., and Umezawa, H. (1970a). Chem. Phunn. Bull. 18, 2112-2118. Nakamura, S., Hamada, M., and Umezawa, H. (1970b). Chem. Fhann. Bull. 18, 2577-2581. Nakamura, S., Fukuda, H., Yamamoto, T., Ogura, M., Hamada, M., Matsuzaki, M., and Umezawa, H. (1972). Chem. P h a m . Bull. 20, 385-390. Nakamura, T., Yasuda, H., Obayashi, A,, and Tanabe, 0. (1975). J . Antibiot. 28, 477-478. Ogata, K., Ueda, K., Nagasawa, T., and Tani, Y. (1974). J. Antibiot. 27, 343-345. Okura, A,, Morishima, H., Takita, T., Aoyagi, T., Takeuchi, T., and Umezawa, H. (1975). J. Antibiot. 28, 337439. O'Reilly, R. A. (1975). Ann. Intern. Med. 83, 506-508. Perlman, D., and Peruzzotti, G. P. (1970). Adu. Appl. Microbial. 12, 277-294. Phansalkar, A. G., and Balwani, J. H. (1970). Hind. Antibiot. BuU. 12, 179-181. Pitkin, R. M., and Reynolds, W. A. (1970). Diabetes 19, 8590. Polson, J. B., and Wosilait, W. D. (1969). Proc. SOC. E x p . Biol. Med. 132, 963-967. Russell, J. T., Hansen, E. L., and Thorn, N. A. (1974). Acta Endom'nol. (Copenhagen) 77, 44-50, Samuel, P., Holtzman, C. M., and Meilman, E. (1973). Circ. Res. 33, 393-402. Sasaki, H., Hosokawa, T., Sawada, M., and Ando, K. (1973). J. Antibiot. 26, 67-80, Sawada, M., Hosokawa, T., Okutomi, T., and Ando, K. (1973). J. Antibiot. 26, 681486. Schein, P. S., and Bates, R. W. (1968). Diabetes 17, 760-765. Schwartz, A., Lewis, R., Hanley, H . G., Munson, R. G., Dial, F. D., and Ray, M. V. (1974). Circ. Res. 34, 102-111. Takeuchi, T., Ogawa, K., Iinuma, H., Suda, H., Ukita, K., Nagatsu, T., Kato, M., and Umezawa, H. (1973). J. Antibiot. 26, 162-167. Thoa, N. B., Costa, J. L., Moss, J., and Kopin, I. J. (1974). Lije Sci. 14, 1705-1719. Timmermans, L. (1974). J . Urol. 112, 348-349. Triggle, C. R., Grant, W. F., and Triggle, D. J. (1975). J . P ~ Q T - T M Z C194, O ~ .182-190. Umezawa, H., Takeuchi, T., Iinuma, H., Suzuki, K . , Ito, M., and Matsuzaki, M. (1970). J. Antibiot. 23, 514-518. Umezawa, H., Iinuma, H., Ito, M., Matsuzaki, M., and Takeuchi, T. (1972). J . Antibiot. 25, 239-242.

Umezawa, H., Aoyagi, T., Okura, A., Morishima, H., Takeuchi, T., and Okami, (1973). /. Antibiot. 26, 787-789. Van den Bosch, J. F., and Claes, P. J. (1967). Frog. Biochem. Phannacol. 2, 97-104. Van der Plas, P. M., Kraan, L., van Es, G., Stibbe, J., and Hemker, H. C. (1974). Huemostusis 3, 1-7. Velerninsky, J., Burr, I. M., and Stauffacher, W. (1970). Eur. J. Clin. Inuest. 1, 1W108. Wakabayashi, K., and Yamada, S. (1972). Jpn. J . Phurmacol. 23, 7-07, White, J. G., Rao, G. H., and Gerrard, J. N. (1974). Am. J. Patho[. 77, 135-149. Wingender, W., von Hugo, H., and Frommer, W. (1975). I. Antibiot. 28, 611. Woods, S. C., Hutton, R. A,, and Makous, W. (1970). Proc. SOC. E r p . B i d . Med. 133, -968. Wyse, B. M., and Dulin, W. E. (1971). Proc. SOC. Exp. B i d . Med. 136, 70-72.

SUBJECT INDEX

Antihypertensive substances from microorganisms, 269 Antiinflammatory substances from microorganisms, 269 Antimycoin A, 5, 33 Antipain, 277 Antipiriculin, 61 Ascofuranol, 285 Ascofuranone, 285 Ascosin, 2, 9, 12, 25 Aurenin, 6 Aureofacin, 9 Aureofungin, 9, 12, 25 Aureothricin, 61 Avenomycin, 13 Axenomycins, 3 Azacolutin (F-17-C), 9, 12, 25

A

Aabomycin A, 82 Actinomycin D, 272 Actinophenol, 45 Adriamycin, 270 Aeromycin B, 4 Akitamycin (toyamycin), 5 Alamethicin, 275, 276 Albotetraene, 5 Aliomycin, 7 Alkinonase A, 277 Alkinonase AF, 277 Alkyl arsines, 191, 195 Amethylfungimycin, 26 Aminomycin, 10 Aminosidin, 279 Amphotericin A, 5 Amphotericin B, 13, 22, 27, 28, 33, 38, 43, 44 Ampicillin, 279 Anisomycin, 80 Antibiotics 0371, 7 17-41 B, 7 A-3, 11 A-228, 7 A-1404, 9 A-5283, 6 A23187, 270, 271, 272, 274, 279, 281 AC435, 6 HA-106, 6 HA-135, 7 HA-145, 7 HA-176, 7 J4-B. 6 LIA 0777, 6 MM-8, 4 PA-153, 7 PA-166, 4 RP-7071, 6 RP-9971, 5 x-63, 11 Antiblastin, 61 Antifungin 4915, 9

B

Bacitmcin, 277 Bihoromycin, 82 Biological methylation, 189 Blasticidin A, 61 Blasticidln S, 54, 56, 62, 63,64, 65, 66, 81 Blastmycin, 61 C

Cabicidin, 6 Candicidm, 286 Candicidin (G-252, PA-150), 2, 9, 12, 15, 16, 17, 19, 22, 24, 25, 26, 27, 28, 29, 30, 32, a34 Candidin, 10, 13, 15, 28, 34, 35, 38, 40 Candidinin, 10 Candihexin, 15, 35, 38, 40, 44, 45 Candihexin A,B, 8 Candihexin E , F , 8 Candimycin, 10, 12, 25 Capacidm, 7 Cardiotonic substances from fermentations, 269 Cellocidin

289

290

SUBJECT INDEX

Cephalosporin acetylesterase, 115 Cephalosporin acylases, 114 Cephalosporins, 90, 92, 96 Cephamycins, 92 Cerulenin, 26 Chainin, 6, 13 Chloramphenicol, 55, 57, 59, 279 Cholesterol-amphotericin B, 40 Cholesterol-levorin. 40 Chromin, 5 tram-Cinnamic acid amide, 273 Citrinin, 82 Colistin, 277 Cryptocidin, 8 Cycloheximide, 42, 45, 55, 56, 59, 272 D

Daunoniycin, 270 Dermostatin (viridohlvin), 9, 13 Detoxin, 65 Diabetogenic agents from microorganisms, 269 Dicloxicillin, 278 Dienes, 3 4,6-Dimethoxyisoflavone, 273 4,8-Dimethoxyisoflavone, 273 Dimethylarsenic acid, 215 Dimethylselenide, 191 Dimethyltelluride, 191 Distamycin C, 7 Distreptomycin, 284 Dopastin, 273 E

Elastatinal, 277 Endomycin A (helixin A), 5 Endomycin B (helixin B), 8 Erythromycin, 271, 278 Etruscomycin, 4, 13 Eurocidin A, 71, 13, 61 Eurocidin B, 7, 13, 61 Ezomycin, 57, 79

Flavoviridomycin, 5 Fradicin, 8 Fumagillin, 39 Furnanornycin, 7 Fungichromatin, 67 Fungichromin, 2, 6, 13, 22 Fungicidin, 5, 13, 38, 41, 42 Fungimycin, 10, 12, 17 0

Gangtokumycin, 7 Genimycin, 8 Gentamicin, 277, 279 Gerobrecin, 9 Gramicidin A, 275, 276 Griseofulvin, 55, 56, 60 Grubilin. 11

ti Hamycin, 2, 9, 12, 25, 286 Harman, 273 Hepcin, 11 Heptaenes, 3, 9, 11 DJ-400 B,, 10, 12, 13, 25 DJ-400 Bt, 10, 12, 13, 25 Eurotin A, 9 Sch 16656, 10, 12, 14, 25 Heptafungin A, 9 Heptamycin, 11, 12, 25 Hexaenes, 3, 8 Hexin, 8 Hygroscopin, 61 Hypocholestolemic agents from microorganisms, 269 I

Isoxazidyl penicillins, 279 K

Kanamycin, 277 Kasugamycin, 54, 56, 62, 66, 67, 68, 69 Kinonase. 277

F

L

Filipin, 22, 39, 274 Filipin complex (durhamycin), 6, 13 Flavacid, 8 Flavofungin (mycoticin A), 2, 8, 13 Flavomycoin, 2, 8

Lagosin, 2, 6, 13 Lasalocid, 270, 272 Laurusin, 82 Leucensomycin, 4, 13

SUBJECT INDEX

Leucomycin, 271 Leupeptins. 275 Levorin &, A,, A2, As, B(26/1), 2, 9, 12, 25, 28, 36, 37, 40, 42, 43, 44, 45, 46 Levoristatin, 43, 44 M

Mediocidm, 8 Mercury methylation, 197 Methylmercury, 190, 192, 196, 214, 216 Methylspinazarin, 273 7-0-Methylspinochrome B, 273 Miharamycin, 82 Milbemycins, 80 Moldicidin A, 7 Moldicidin B, 6, 13 Monicamycin, 11 Mycelin, 8 Mycelin IMO, 9 Mycoheptin (2814 H), 10, 13, 28, 36, 42 Mycoticin B, 2, 8, 13 Mycotrienin, 4 N

Neoheptane, 11 Neomycin, 277, 285 N-acetylated, 284 dimethylaminopropylneomycin, 284 N-hexaacetylneomycin, 283 N-methylated neomycin, 283, 284 Neopentaene. 6 Neuramidase, 228, 229, 231, 232, 233, 239, 243, 244, 250, 251 Neuromuscular blockade, 269 Nogalamycin, 272 Novobiocin, 57, 60 Nursimycin, 11 Nystatin A,, Az, A3, 2, 5, 13, 27, 28, 29, 35, 38, 39, 42, 43, 44, 45 0

Oleandomycin, 271 Onomycin I, 7 Oosponol, 273 Ornamycin (17731), 5 Oudenone, 273 Oxytetracycline, 55

29 1 P

Penicillin acylase, 97, 99, 101, 105, 110, 111 G acylase, 108 V acylase, 108 acyltransferases, 108, 111 transacylase, 111 Pentaenes, 3, 6 G-8, 7 Glaxo-A246, 6 Pentafungin, 7 Pentamycin, 6, 13, 61 Pentaneucin, 6 Perimycin, 12 Perimycin (NC-968), 10, 12, 25 Phenethylamiue, 273 Phenopicolinic acid, 273 Photosynthetic bacteria, 163, 166 Photosynthetic SCP process, 172 Pimaricin (tennecetin), 2, 4, 13, 39 Pimprimine, 273 Pioticin, 14 Plumbomycin A, 5 Plumbomycin B, 5 Polifungin, 5, 13, 19, 38, 45 Polifungin A, 2 Polymyxin B, 277 Polyoxins, 54, 55, 56, 69, 70, 71, 72 Proteinases as pharmacological agents alkaline A and B, 277 neutral, 277 semi-alkaline, 277 Protocidin, 5 Puromycin, 272 Q

Quinquamycin, 7 R

Resistaphyllin, 4 Retikinonase I, 277 Rhizopchin, 14 Rifampin, 275, 278 Rimocidin (PA-%), 5, 13, 39 Rubrochlorine. 6 S

Shikimic acid, 26 Sialic acid, 231, 232, 235

292

SUBJECT INDEX

Sialoglycoprotein, 235, 237 Sialolipids, 236 Sistomycosin, 5 Spiramycin, 271, 279 Streptomycin, 55, 57, 58, 59, 277, 284, 286 Streptozotocin, 281, 282 Surgomycin, 8

Thiorhodaceae, 164 Toyokamycin, 80 Trichomycin A,B, 2, 3, 10, 12, 25, 43, 44, 61 Triene, 3, 4 Trienine, 4 Tristreptomycin, 284 U

T

Unamycin A, 6 Takamycin, 11 Tblimycin, 11 Tetraenes, 3, 4 Tetraenin A,B, 5 Tetrahexin, 8 Tetramedyn, 5 Tetramethyllead, 191 Tetramycin, 4 Tetramycoin A,B, 5 Tetranactin, 57, 62, 76, 77, 78 Tetrin A,B, 4, 13

V

Validamycin, 54, 57, 62, 73, 74, 75, 76 Valinomycin, 275, 276, 281 Viomycin, 277 Vitamin B,*, 167 X

Xantholycin B, 6

CONTENTS OF PREVIOUS VOLUMES Volume 1

Aerosol Samplers Harold W . Batchelor

Protected Fermentation

A Commentary on Microbiological Assaying

Miloi HeroEd and Jan NeEasek

F . Kauanagh

The Mechanism of Penicillin Biosynthesis

Application of Membrane Filters

Arnold L. Demain

Richard Ehrlich

Preservation of Foods and Drugs by Ionizing Radiations W . Dexter Bellamy

Microbial Control Methods in the Brewery

Gerhard

1. Hass

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

The State of Antibiotics in Plant Disease Control

David Pramer

brandt

Microbial Synthesis of Cohamides

The Microbiological Transformation of Steroids

D. Perlman

T.H . Stoudt

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

Biological Transformation of Solar Energy William J . Oswald and Clarence 6 .

Golueke

Germfree Animal Techniques and Their Applications

SYMPOSIUM ON ENGINEERING hVANCES FERMENTATION PRACTICE

Arthur W . Phillips and lames E . Smith Insect Microbiology

Rheological Broths

S . R. Dutky The Production of Amino Acids by Fermentation Processes

Shukuo Kinoshita

Fred .'

Properties

of

Fermentation

Deindoerfer and John M . West

Fluid Mixing in Fermentation Processes

1. Y. Oldshue

Continuous Industrial Fermentations Philip Gerhardt and M . C . Bartlett

Scale-up of Submerged Fermentations W . H . Bartholemew

The Large-Scale Growth of Higher Fungi Radclqfe F . Robinson and R. S . Daoidson

Air

AUTHOR INDEX-SUBJECT

IN

INDEX

Volume 2

Newer Aspects of Waste Treatment Nandor Parges

Arthur E , Humphrey Sterilization of Media for Biochemical Processes

Lloyd L. Kempe Fermentation Kinetics and Model Processes

Fred H . Deindoerfer 293

294

CONTENTS OF PREVIOUS VOLUMES

volume 4

Continuous Fermentation W. D . Maxon Control Applications in Fermentation George 1. Fuld AUTHOR INDEX-SUBJECT

INDEX

volume 3

Preservation of Bacteria by Lyophilization Robert]. Heckly

Induced Mutagenesis in the Selection of Microorganisms S . I. Alikhanian The Importance of Bacterial Viruses in Industrial Processes, Especially in the Dairy Industry F . 1. Babel Applied Microbiology in Animal Nutrition Harlow H . Hall

Sphaerotilus, Its Nature and Economic Significance Norman C. Dondero

Biological Aspects of Continuous Cultivation of Microorganisms T . Holme

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

Maintenance and Loss in Tissue Culture of Specific Cell Characteristics Charles C. Morris

Protection Against Infection in the Microbiological Laboratory: Devices and Procedures Mark A . Chatigny Oxidation of Aromatic Compounds by Bacteria Martin H . Rogoff Screening for and Biological Characterizations of Antitumor Agents Using Microorganisms Frank M . Schabel, Jr., and Robert F . Fittillo The Classification of Actinomycetes in Relation to Their Antibiotic Activity Elio Baldacci 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 . Hulme AUTHOR INDEX-SUBJECT

INDEX

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

Volume 5

Correlations between Microbiological Morphology and the Chemistry of Biocides Adrian Albert Generation of Electricity by Microbial Action J . B . Davis Microorganisms and the Molecular Biology of Cancer G. F. Gause Rapid Microbiological Determinations with Radioisotopes Gilbert V. Levin The Present Status of the 2,3-Butylene Glycol Fermentation Sterling K . Long and Roger Patrick Aeration in the Laboratory W. R. Lockhart and R. W. Squires

CONTENTS OF PREVIOUS VOLUMES

295

Stability and Degeneration of Microbial Cultures on Repeated Transfer Fritz Reusser

Biodegradation: Problems of Molecular Recalcitrance and Microbial Fallibility M . Alexander

Microbiology of Paint Films Richard T. Ross

Cold Sterilization Techniques John B . Opfell and Curtis E . Miller

The Actinomycetes and Their Antibiotics Selman A. Waksman

Microbial Production of Metal-rganic pounds and Complexes D.Perlman

Fuse1 Oil A. Dinsmoor Webb and John L. Zngraham AUTHOR INDEX-SUBJECT INDEX

Com-

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

Volume 6

Effects of Microbes on Germfree Animals Thomas D . Luckey

Global Impacts of Applied Microbiology: An Appraisal Carl-G&an Hede'n and Mortimer P. Starr

Uses and Products of Yeasts and Yeast-like Fungi Walter J. Nickerson and Robert 6 . Brown

Microbial Processes for Preparation of Radioactive Compounds D. Perlman, A d s P. Bayan, and Nancy A. Giufie Secondary Factors in Fermentation Processes P. Margalith Nonmedical Uses of Antibiotics Herbert S . Goldberg

Microbial Amylases Walter W . Windish and Nagesh S . Mhatre The Microbiology of Freeze-Dried Foods Gerald J . Silverman and Samuel A. Goldblith Low-Temperature Microbiology Judith Farrell and A. H . Rose AUTHOR INDEX-SUBJECT INDEX

Microbial Aspects of Water Polhpon Control K . Wuhnnann

Volume 8

Microbial Formation and Degradation of Minerals Melvin P. Siloennan and Henry L. Ehrlich

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

Enzymes and Their Applications Zrwin W. Sizer

Genetics in Applied Microbiology S . G. Bradley

A Discussion of the Training of Applied Mi-

Microbial Ecology and Applied Microbiology Thomas D. Brock

crobiologists B. W .Koft and Wayne W . Umbreit AUTHOR INDEX-SUBJECT INDEX

Volume 7

Microbial Carotenogenesis Alex Ciegler

The Ecological Approach to the Study of Activated Sludge Wesley 0. Pipes Control of Bacteria in Nondomestic Water Supplies Cecil W . Chambers and Norman A. Clarke

296

CONTENTS OF PREVIOUS VOLUMES

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

Malo-lactic Fermentation Ralph E. Kunkee

Oral Microbiology Heiner Hoffman

Volume 10

Media and Methods for Isolation and Enumeration of the Enterococci Paul A. Hartman, George W .Reinbold, and Devi S. Saraswat Crystal-Forming Bacteria as Insect Pathogens Martin H . Rogoff Mycotoxins in Feeds and Foods Emanuel Borker, Nino F . Insalata, Colette P. k o i , a n d l o h n S . Witzeman AUTHOR INDEX-SUBJECT

INDEX

AUTHOR INDEX-SUBJECT

INDEX

Detection of Life in Soil on Earth and Other Planets. Introductory Remarks Robert L. Starkey For What Shall We Search? Allan H . Brown Relevance of Soil Microbiology to Search for Life on Other Planets 6 . Stotzky Experiments and Instrumentation for Extraterrestrial Life Detection Gilbert V . L o i n Halophilic Bacteria D . 1. Kushner

Volume 9

The Inclusion of Antimicrobial Agents in Pharmaceutical Products A. D. Russell, June Ienkins, and 1. H . Harrison Antiserum Production in Experimental Animals Richard M . Hyde Microbial Models of Tumor Metabolism G . F . Gause Cellulose and Cellulolysis Brigitta Norkrans Microbiological Aspects of the Formation and Degradation of Cellulose Fibers L. Iuraiek, I. Ross Coluin, and D. R. Whitaker

Applied Significance of Polyvalent Bacteriophages S. G . Bradley Proteins and Enzymes as Taxonomic Tools Edward D. Garber and John W . Rippon Mycotoxins Alex Ciegler and Eioind B . Lillehoj Transformation of Organic Compounds by Fungal Spores Claude Vbzina, 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

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

AUTHOR INDEX-SUBJECT INDEX

Bulking of Activated Sludge Wesley 0. Pipes

CUMULATIVE AUTHOR INDEX~UMULATIVE TITLE INDEX

Antibiotics in the Control of Plant Pathogens M . J . Thirurnalachar

297

CONTENTS OF PREVIOUS VOLUMES

Volume 11

Collection of Microbial Cells Daniel I . C . Wang and Anthony]. Sinskey

Successes and Failures in the Search for Antibiotics Selman A . Waksman

Fermentor Design R. Steel and T . L. Miller

Structure-Activity Relationships of Semisyn. thetic Penicillins K . E. Price

The Occurrence, Chemistry, and Toxicology of the Microbial Peptide-Lactones A . Taylor

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

Microbial Metabolites as Potentially Useful Pharmacologically Active Agents D. Perlman and C. P . Peruzzotti

Micromonospora Taxonomy George Luedemunn

AUTHOR INDEX-SUBJECT INDEX

Dental Caries and Periodontal Disease Considered as Infectious Diseases William Gold The Recovery and Purlfication of Biochemicals Victor H . Edwards

Volume 13

Chemotaxonomic Relationships Among the Basidiomycetes Robert G. Benedict

Ergot Alkaloid Fermentations William J. Kelleher

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

The Microbiology of the Hen’s Egg R. G . Board

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

Training for the Biochemical Industries I . L. Nepner

Large-Scale Bacteriophage Production K . Sargent

AUTHOR INDEX-SUBJECT INDEX

Microorganisms as Potential Sources of Food Jnanendra K . Bhattacharjee

Volume 12

History of the Development of a School of Biochemistry in the Faculty of Technology, University of Manchester Thomas Kennedy Walker Fermentation Processes Employed in Vitamin C Synthesis Milod Kulhanek Flavor and Microorganisms P. Margalith and Y . Schwartz Mechanisms of Thermal Injury in Nonspomlating Bacteria M . C. Allwood and A. D. Russell

Structure-Activity Relationships Semisynthetic Cephalosporins M. L. Sassiljer and Arthur Lewis

Among

Structure-Activity Relationships in the Tetracycline Series Robert K . Blackwood and Arthur R. English Microbial Production of Phenazines J. M . lngram and A. C . Blackwood The Gibberellin Fermentation E . G . ]ef&eys Metabolism of Acylanilide Herbicides Richard Bartha and David Pramer

298

CONTENTS OF PREVIOUS VOLUMES

Therapeutic Dentrifrices J . K . Peterson

Fermentation Equipment G . L. Solomons

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

The Extracellular Accumulation of Metabolic Products by Hydrocarbon-Degrading Microorganisms Bernard J . Abbott and William E . Gledhill

Microbiological Patents in International Litigation John V. Whittenburg

AUTHOR INDEX-SUBJECT INDEX

Industrial Applications of Continuous Culture: Pharmaceutical Products and Other Products and Processes R. C . Righelato and R. Elsuxnth

Medical Applications of Microbial Enzymes I N i n W . Sizer

Mathematical Models for Fermentation Processes A. 6 . Frederickson, R. D. Megee, Ill, and H . M . Tsuchija AUTHOR INDEX-SUBJECT INDEX

Volume 14

Development of the Fermentation Industries in Great Britain John J . H. Hastings Chemical Composition as a Criterion in the Classification of Actinomycetes H. A . Lechevalier, M a y P. Lechevalier, and Nancy N. Gerber Prevalence and Distribution of AntibioticProducing Actinomycetes John N . Porter Biochemical Activities of Nocardia R. L. Raymond and V. W.Jamison Microbial Transformations of Antibiotics Oldrich K . Sebek and D. Perlmn

Volume 15

Immobilized Enzymes K . L. Smiley and G . W . Strandberg Microbial Rennets Joseph L. Sardinas Volatile Aroma Components of Wines and Other Fermented Beverages A. Dinsmoor W e b b and Carlos J . Muller Correlative Microbiological Assays Ladislav 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 . Godfiey and Kenneth E . Price Chloramphenicol Vedpal S . Malik Microbial Utilization of Methanol Charles L. Cooney and David W . Levine

In Vivo Evaluation of Antibacterial Chemotherapeutic Substances A. Kathrine Miller

Modeling of Growth Processes with Two Liquid Phases: A Review of Drop Phenomena, Mixing, and Growth P. S . Shah, L. T . Fan, 1. C . Kao, and L. E. Erickson

Modification of Lincomycin Barney J . Magerhn

Microbiology and Fermentations in the Prairie Regional Laboratory of the National

CONTENTS OF PREVIOUS VOLUMES

Research Council of Canada 19461971 R. H . Haskins AUTHOR INDEX-SUBJECT

299

Fungal Sterols and the Mode of Action of the Polyene Antibiotics J . M. T. Hamilton-Miller

INDEX

Volume 16

Public Health Significance of Feeding Low Levels of Antibiotics to Animals Thomas H . Jukes Intestinal Microbial Flora of the Pig R. Kenworthy Antimyciu A, a Piscicidal Antibiotic Robert E . Lennon and Claude Vbzina Ochratoxins Kenneth L. Applegate and John R. Chipley Cultivation of Animal Cells in Chemically Defined Media, A Review Kiyoshi Higuchi Genetic and Phenetic Classification of Bacteria R. R. Colwell Mutation and the Production of Secondary Metabolites Arnold L. Demain Structure-Activity Relationships in the Actinomycins Johannes Meienhofer and Eric Atherton Development of Applied Microbiology at the University of Wisconsin William B. Sarks

Methods of Numerical Taxonomy for Various Genera of Yeasts 1. Campbell Microbiology and Biochemistry of Soy Sauce Fermentation F . M . Young and B. J . B . Wood Contemporary Thoughts on Aspects of Applied Microbiology P. S . S . Dawson and K . L. Phillips Some Thoughts on the Microbiological Aspects of Brewing and Other Industries Utilizing Yeast G . 6. Stewart Linear Alkylbenzene Sulfonate: Biodegradation and Aquatic Interactions WilliamE . GledhiU The Story of the American Type Culture Collection-Its History and Development (1899-1973) WillinmA. Clark and Dorothy H . Geary Microbial Penicillin Acylases E . J . Vandamme a n d ] . P. Voets SUBJECT INDEX

Volume 18

Microbial Formation of Environmental Pollutants Martin Alexander

AUTHOR INDEX-SUBJECT INDEX

Volume 17

Microbial Transformation of Pesticides Jean-Marc Bollag

Education and Training in Applied Microbiology Wayne W. Umbreit

Taxonomic Criteria for Mycobacteria and Nocardiae S . G . Bradley and J . S . Bond

Antimetabolites from Microorganisms Daoid L. Pruess and James P. Scannell

Effect of Structural Modfications on the Biological Properties of Aminoglycoside Antibiotics Containing 2-Deoxystreptamine Kenneth E . Price, John C . Godfi-ey, and Hiroshi Kawaguchi

Lipid Composition as a Guide to the Classification of Bacteria Nwman Shaw

300

CONTENTS OF PREVIOUS VOLUMES

Recent Developments of Antibiotic Research and Classification of Antibiotics According- to Chemical Structure @nos B b d y

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

SUBJECT INDEX

Volume 19

Culture Collections and Patent Depositions T . G. Pridham and C . W. Hesseltine Production of the Same Antibiotics by Members of Different Genera of Microorganisms Hubert A . Lechevalier Antibiotic-Producing Fungi: Current Status of Nomenclature C . W. Hesseltine and 1.J. Ellis Significance of Nucleic Acid Hybridization to Systematics of Actinomycetes S . G. Bradley Current Status of Nomenclature of AntibioticProducing Bacteria Erwin F . Lessel Microorganisms in Patent Disclosures lming Marcus Microbiological Control of Plant Pathogens Y. Henis and I . C h t Microbiology of Municipal Solid Waste Composting Melvin S. Finstein and Merry L. Morris

Nitrification and Deuitdcation Processes Related to Waste Water Treatment D . D. Focht and A. C . Chang The Fermentation Pilot Plant and Its Aims D. I. D. HockenhuU The Microbial Production of Nucleic Acid-Related Compounds Koichi Ogata Synthesis of L-Tyrosine-Related Amino Acids by @-Tyrosinase Hideaki Y a m & and Hidehiko Kumagai

Volume 20

The Current Status of Pertussis Vaccine: An Overview Charles R. Manclark Biologically Active Components and Properties of Bordetella pertussis Stephen I. Morse Role of the Genetics and Physiology of Bwdetella 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 Problems Associated with the Control Testing of Pertussis Vaccine Jack Cameron Vinegar: Its History and Development Hubert A . Conner and Rudolph J . AUgeier Microbial Rennets M . Sternberg Biosynthesis of Cephalosporins Toshihiko Kanzaki and Yukio Fujisawa Preparation of Pharmaceutical Compounds by Immobilized Enzymes and Cells Bernard J. Abbott Cytotoxic and Antitumor Antibiotics Produced by Microorganisms

I . Fuska and B . Proksa SUBJECT INDEX

A

7

c D E F G H

9 0 l 2 3 4

-R R-

1 5 J 6