Advances in Applied Microbiology, Volume 1

ADVANCES

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Applied Microbiology VOLUME 1

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ADVANCES I N

Applied Microbiology Edited by WAYNE W. UMBREIT Department of Bacteriology Rutgers, The State University New Brunswick, New Jersey

VOLUME 1

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1959

ACADEMIC PRESS, New York and London

Copyright 0,1959, by Academic Press Inc. ALL RIQHTS RESERVED NO PART O F T H I S BOOK MAY B E REPRODUCED IN ANY FORM, BY PHOTOSTAT, MICROFILM, OR ANY OTHER MEANS, WITHOUT WRITTEN PERMISSION FROM THE PUBLISHERS.

ACADEMIC PRESS INC. 111 FIFTHAVENUE NEWYORK3, N. Y.

United Kingdom Edition Published by ACADEMIC PRESS INC. (LONDON) LTD. 40 PALLMALL,LONDONS.W. 1

Library of Congress Catalog Card Number 60-19823

PRINTED I N THE UNITED STATES O F

AMERICA

CONTRIBUTORS M. C. BARTLETT, Department of Bacteriology, The University of Michigan, Ann Arbor, Michigan* W. DEXTERBELLAMY, General Electric Research Laboratory, Schenectady, New Yo& E. 0. BENNETT, Department of Biology, University of Houston, Houston, Texas R. S . DAVIDSON, Battelle Memorial Institute, Columbus, Ohio ARNOLDL. DEMAIN,Merck Sharp and Dohme Research Laboratories, Rahway, New Jersey S . R. DUTKY,Insect Pathology Laboratory, United States Department of Agriculture, Agricultural Research Service, Beltsville, Maryland PHILIPP GERHARDT, Department of Bacteriology, The University of Michigan, Ann Arbor, Michigan MILOB HEROLD,Antibiotics Research Institute, Roztoky near Prague, Czechoslovakia SHUKUQ KINQSHITA, Tokyo Research Laboratory, Kyowa Fermentation I n dustry Company, Ltd., Tokyo, Japan J A N NEEhsEK, Antibiotics Research Institute, Roztoky near Prague, Czechoslovakia D. PERLMAN, Squibb Institute for Medical Research, New Brunswick, New Jersey ARTHURW. PHILLIPS, Biological and Food Research Laboratories, Department of Bacteriology and Botany, Syracuse University, Syracuse, New York DAVIDPRAMER, Department of Agricultural Microbiology, Rutgers, The State University, New Brunswick, New Jersey RADCLIFFE F. ROBINSON, Battelle Memorial Institute, Columbus, Ohio JAMES E. SMITH, Biological and Food Research Laboratories, Department of Bacteriology and Botany, Syracuse University, Syracuse, New York * Present address: Ahhott Laboratories, North Chicago, Illinois.

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PREFACE This volume is the first in a series designed for the publication of critical and definitive reviews of those areas of microbiology which are of interest to the practical microbiologist. They are intended to provide the applied microbiologist with useful and up-to-date information. I n view of the overwhelming amount of original literature that is appearing, the need for additional reviews has been growing steadily. The reviews and essays which are already available have not primarily been written from the viewpoint of the practical microbiologist and therefore it is felt that this present series will serve a useful purpose in presenting such information within the confines of a single publication. The editor expects in future volumes to continue to include reviews by foreign contributors discussing material not readily accessible to thc general reader.

W. W. UMBREIT Rutgers University July, 1959

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CONTENTS Co NTRIB UTORS . . . , . . , , . , . , . , . . . . . . . , , , , , . , . , . , , , . . . . . . . . . . . . . . . . . . . . . . . PREFACE . . . .. . . . . . . . .. . . .. . . . . . . . . , . . , . . . . , . . . .. . , . . . . . . . . . . . . . . . . . . . . . .

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Protected Fermentation b y MI LO^ HEROLDand JAN NEUSEK

. . . . . . . .. . . . . . . . . . . .. . . . . . . . . . . . . . . .. . . . . ting in Fermentation Processes. . . . . . . . . . . . . . . . . . 111. Classification of Fermentation Processes According to the Importance of Contamination. . . , , , , . , , , , , , , . . . . , . , , , , , , . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Sources of Contamination in Aseptic Fermentation Processes V. Application of Antimicrobial Agents for the Protecti Processes. . . . . . . . . . . . . . . . . , . . . . . , . , , . , . , . , . . . . . . . . VI. Biosynthesis of Chlortetracycline without Maintena .. ditions. . . . . . . . . . . . , . , . . . , . , . , . , . . . . . . . . , , , , , , , , . . VII. Perspectives. .................... . . . . . . VIII. Conclusions and Summary.. , . . . , . , . . . . . . , . , . , . . . . . . . . . . . . _ . . . . . . . . . .. . . . . . . . . References. . . . . . . . . . .

1 2

5 7 8 12 13 17 18

The Mechanism of Penicillin Biosynthesis by ARNOLDL. DEMAIN

I. Introduction.. . . . . . . . . . . . . . . , . , . , . , , . , . , , , , , , . . . . . . . . . . , . , . .. .. . . . . . . . . .. 11. Precursors of the Side Chain

23 25

111. Precursors of the 8-Lactam-T ucleus. IV. Condensation of the Precursor Moieties. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Future Experimentation.. . . . . . . , . , . . . , . , . . . . . . . . . . . . . . . . . . . .. . . . . . . .. . . . . . . . . . . . .. . . . . . . . . . . . VI. Summary References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . , . . . . .. . . . . . .

26 38 43 ,44 45

Preservation of Foods and Drugs by Ionizing Radiations by W. DEXTERBELLAMY * ..... I. Introduction and Scope. 11. History.. . . . . . , . . . . . . . . , . . . . . . . . . . . , , , , , , , , . , . . , , , . . . . . . . . . . . . . . . . . . . . _ , . _ . . . . . . . . . . . . .,.. . . . . 111. Nature of High-Energy Radiation IV. Sources of High-Energy Radiation. . . , . . , , . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . V. Dosimetry VI. Physical and Chemical Changes.. . . . . . . . . . . . . . . . . . . . VII. Microbiological Effects. , . . . . . . . . . . . . . . . . . . . . . . . . . . . . VIII. Wholesomeness.. . . . . . . . . . . . . . . . . . . . . . . . . . , . . . . . . . .. . . .. . . . . . . . . . IX. Applications. . . ..................... . . . . . . . . . . . . . . . . . . . . .................... . . . . . . I

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51 54 54 60 63 65

67 68 . . . . . . . . . . . . . . . . . . . . . . . . . . 69 XII. Summary and Conclusions . . . . . . . . . . . . . 70 References. .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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CONTENTS

The Status of Antibiotics in Plant Disease Control by DAvm PRAMER I. Introduction.. . . ........................................... . 11. Selective Toxicit ..... . . . . . . . .... .......................... .. 111. Absorption and Translocation.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . , . , . , . . . . . . . . . . . . . . .. .. ... IV. Mode of Action . . _ _ . . _ . . . . . . . ..................... . . . . . .. . . . . . . . . . . . . . . . . . . V. Summary.. . . . . . .

75 76 77 81 82 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83

Microbial Synthesis of Cobamides by n. PERLMAN I. Introduction.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Analytical Methods for the Determination of Cobamides. . . . . . . . . . , . . . . . 111. Microbial Processes for Synthesis of Naturally Occurring Cobamides. . . ... IV. Microbial Synthsis of “Unnatural Cobamides”. . . . . . . . . . . . . . . . . . . . . . Refererices . . . . . . . , , , , , , , , . . . . . , . , . , . . , . , , . . , , . . . . . . . . . . . . . . . . . , . . . . .

87 92 97 106 112

Factors Affecting the Antimicrobial Activity of Phenols by E. 0. BENNETT I. Introduction.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , . . . . . . . . . . . . . . . . . 11. The Effect of Temperature.. . . . . . . . . . . . , . .................... .. 111. The Effect of Oxygen Tension. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , , , , , , , . . .. . ............................................. 1V. The Effect of .. . V. The Effect of eous Materials.. . . . . .................. VI. The Effect of the Size of the Bacterial P on. . . . . . . . . . . . . . . . . . . . . . VII. The Revival of Bacteria Aft,er Treatment with Phenol.. , . . . . . , . . . . . . VIII. The Development of Resistance t o Phenols.. . . . . . . . , . , , , , . , , , , , , , , . . . . . .. . I X . The Oxidation of Phenols by Bacteria. ............... ... X. The Use of Phenols with Other Inhibit ... .. . . .. . . . . . . . . . . . , .. . . .. . . . . , . References. . . . . . . . .

123 124 125 126 126 131 132 133 134 136 137

Germfree Animal Techniques and Their Applications by ARTHURW. PHILLIPS and JAMES E. SMITH .. . . . . . . . . . . . . .. .. . . . . . . . . ., . . . . I. Introduction.. . . . ..................... . . . ... 11. Methods and Equipmen ... 111. Characteristics of IV. Applications of Germfree Animals . . . . . . . . . . . . . . .. . . V. Additional Research Needs.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , . . . . . . . . . .. . . . .. .. . . . . VI. Summary. . . . . . . . References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

141 142 148 153 167 171 171

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Insect Microbiology by S. R . DUTKY

I. Introduction . . . . . . .. .. . . .. .. . . . . . . . . . . . . , _ .. _ .. _. . . . . , , . . , _ ._ .. . , . , . . . . 11. Bacterial Diseases.. . . . . . . . . . . . . . . . . . . . . . . , , , , . . . . . . . . . . . . . . , , . , . . . . . . . . 111. Fungous Diseases. .. .. . , . . . . . . . . . . . , . , . , . , , , , . . . , . . . . , . , . , . , . . . . . . . , , , , IV. Protozoan Diseases.. . . . . . . . . . . . . . . . . . . . . . . . , , . . . . . . . . . . . . . . . . , . . . . . . . . .

175 176 179 181

CONTENTS

V. VI. VII. VIII.

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Rickettsia1 Diseases. ............. . . . . . . . . . . . . . 185 Virus Diseases. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 188 Nematode Diseases. . . . . . . . . . . . . . . . Summary and Conclusions.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 198 References. . . . . . . . . . . .

The Production of Amino Acids by Fermentation Processes by SHUKUO KINOSHITA I. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Possibility of Amino Acid Production by Various Microorganisms. . . . . . . . . 111. Glutamic Acid Fermentation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Lysine Fermentation.. . . . . . . . . . . . . ............................... V, Ornithine Fermentation.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI. Miscellaneous Amino Acid Fermentations. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

201 202 203 208 210 211 212

Continuous Industrial Fermentations by PHILIPGERHARDT and M. C. BARTLETT I. Introduction.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 215 11. Classification of Continuous Operations and Processes. . . . . . . . . . . . . . . . . . . . 217 111. Summary, Predictions, and Experimental Tests of Continuous Fermentation Theory.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 237 IV. Continuous Fermentation in Practice. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .......................

The Large-Scale Growth of Higher Fungi I. 11. 111. IV. V. VI. VII.

by RADCLIFFE F. ROBINSON and R. S. DAVIDSON Introduction.. ....................... . . . . . . . . . . . 261 Culture. . . . . . . . . . . . . . . History of Mu Research in the Mushroom Industry.. . . . . . . . Development of Deep-Vat Fermentation Met 269 Food Yeasts and Molds in Submerged Culture.. . . . . . . . . . . . . . . . . . . . . . . . . Mushrooms in Submerged Culture.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 270 Problems to be Met in the Large-Scale Production of Higher Fungi in Sub....................... 275 merged Culture. . . . . . . . . . . . . . . . . . . . . . . . . . . References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ....................... 277

AUTHORINDEX ............................................................... SUBJECT INDEX ...............................................................

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Protected Fermentation MILO&HEROLD AND JAN NEEASEK Antibiotics Research Institute, Roztoky near Prague, Czechoslovakia

I. Introduction.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ...................... cesses . . . . . . . . . . 11. Problems of Sterility Testing in Fermentati 111. Classification of Fermentation Processes According to the Importa ..... ................................... IV. Sources of Contamination in Aseptic Fermentation Processes . . . . . . . . . . . V. Application of Antimicrobial Agents for the Protection of Fermentation Processes . . . . . . . . . . . . . . . . . . . . . . . . . . . . ............................ VI. Biosynthesis of Chlortetracycline with aintenance of Aseptic Conditions . . . . . . . . ................................................ VII. Perspectives.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ................... VIII. Conclusions and Summary, . . . . . . . . . . . . . . . . ................. References, . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ...................

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I. Introduction This article deals with methods used for protecting industrial fermentation processes against unfavorable effects caused by contaminating microflora, by means of antimicrobial agents. The article is to be considered more of an essay rather than a review, due to the fact that the topic concerned has so far not been studied in great detail. Especially in the field of aseptic fermentation processes, the state of our experience in this respect in still only fragmentary. The influence of microbial contamination in fermentation processes and the problems of its elimination have to be considered from several different viewpoints. The most important of these are the following: 1. Type of metabolite being synthesized. 2. Specific properties of the production culture. 3. Construction of the fermentation equipment. 4. Type of nutrient medium and raw materials used for its preparation. 5. The technology of conducting the fermentation process. The over-all degree of the interference of contamination in a given fermentation process is a result of simultaneous action of all the above listed factors coming into play. Some examples may be cited: 1. Because of the inactivation of penicillin by penicillinase (Abraham and Chain, 1940), the effect of contamination is generally much more pro1

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found in penicillin fermentation than in fermentations of other products resistant to similar microbial enzymes. 2. I n the fermentation of streptomycin, difficulties will be more pronounced with an actinophage-sensitive strain than with a resistant one (Abe et al., 1952). 3. Fermentations performed in unsuitable equipment, disregarding technological requirements, can cause considerable trouble; it is usually very difficult to localize ex post the source of contamination caused by the unsuitability of the equipment. Even with suitable detection methods this task is usually very time consuming. 4. Fermentation media containing as nutrients insoluble, difficult sterilizable materials, or materials with a high microbial count cause much more trouble due to contamination than soluble media and raw materials with a relatively low microbial count. 5 . From the technological viewpoint, susceptibility to contamination and the degree of their unfavorable effect is determined by a number of cooperating factors, e.g., the p H of fermentation, fermentation time, intensity of aeration, etc. The contamintitirig cultures may unfavorably affect the fermentation process in different ways. They can (a) directly destroy the cells of the production culture; ( b ) inactivate the metabolite being produccd; (c) produce substances unfavorably affecting the metabolism of the production culture and thus the formation of the desired metabolite; ( d ) consume components of the medium which are qualitatively or quantitatively indispensable for the maximum efficiency of the production culture. The presence of the contaminating culture need not necessarily be manifested by a decrease of production of the required metabolite. It may in such cases be difficult to detect contamination in the production culture. This occurs, for example, in the production of some broad-spectrum antibiotics where the antibiotic being produced more or less suppresses the development of microbial contaminants and thus protects the fermentation from the effects caused by the presence of alien microflora. Under the term “protected fermentations,” we therefore understand such fermentation processes in which foreign contamination is suppressed by the presence of a substance with antimicrobial activity; the antimicrobial substance may, under some circumstances, be produced by the same culture which is being protected.

II. Problems of Sterility Testing in Fermentation Processes Microscopic arid cultivation methods are currently used for the identification of microbial contaminations in fermentation processes. I n massive contamination of the production culture by foreign microflora, microscopic ulldysis is of great value because of the practically instantaneous result.

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With less intense contamination, foreign microbes may be determined microscopically only in certain special cases, e.g., when contaminating microbes are outstanding by their form or mobility. I n most cases, however, it is not possible to differentiate safely between living bacterial cells and the very varied microflora of the nutrient medium which had been killed by sterilization, originating in the complex organic materials used as sources of nutrients. Viability tests are of some help in these cases, although the results obtained are never quite reliable (Herold et al., 1957). The main disadvantage of identification of contaminating microflora by cultivation methods is the fact that it takes a t least 24 hours before the results may be obtained. In many cases it would be necessary to obtain the result sooner, if not immediately, e.g., in decisions about the quality of unstorable inocula. Although relatively very reliable results may be obtained by cultivation tests, it has to be kept in mind that contamination present in the medium need not necessarily be proved even by such a test. The reasons are the following (Herold et al., 1957): 1. Contamination need not be picked up in sampling, if the number of foreign microorganisms per unit volume of the medium is low. 2. Contamination need not be picked up during inoculation of the Samples on the sterility testing media, as in this operation the material is further diluted. 3. The contaminating microorganism need not, in the given incubation period and on the given medium, multiply to such an extent as to be microscopically or macroscopically evident. 4, The growth of the contaminating microbes in the fermented medium may be inhibited by some metabolite of the production culture, e.g., an antibiotic, the presence of which may render the proof of contamination impossible even after inoculation into test media. Let us therefore define a fermentation process as sterile, if presence of foreign microorganisms cannot be proved under given circumstances by suitable testing methods. In this connection it is necessary to call attention to the fact that sterilization of the nutrient medium on an industrial scale is usually performed in such a way that the medium after sterilization is not strictly sterile, but “production sterile” (Perlman et al., 1953). This is caused by the requirement of minimum destruction of proteins and minimum formation of toxic substances by reactions of carbohydrates with amino acids. The contaminating microorganisms are either bacteria, fungi, or bacteriophages. Of these, bacteria are the most important, being able to attack a majority of fermentation processes, due to their small dimensions, their omnipresence, and their ability to utilize the most varied substrates as sources of nutrients. In other words, they are widely found as contami-

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nan ts because of their general morphological and ecological properties and their metabolic abilities. I n the majority of aseptic fermentation processes, contamination by gram-positive bacteria usually originates from the insufficient tightness of the fermentation equipment and from the insufficient sterilization of air used in aeration; gram-negative bacteria usually come from nontight cooling coils or duplicators (Rlechta, 1955). Fungi are of much more limited importance, with the exception of yeast-like organisms, originating from insufficiently sterilized medium containing, e.g., corn-steep (Slechta, 1955). Of the filamentous fungi, Cephalosporium acremonium was detected in the fermentation of penicillin in one case (Taira and Yamatodani, 1953). It can be assumed that contamination by filamentous fungi in penicillin fermentations can only rarely be observed due to the difficult microscopical differentiation of the mycelia; besides that, sterility tests are usually aimed exclusively a t the identification of bacteria. I n surface fermentation of citric acid by strains of Aspergillus niger, infection by parasitic penicillia of the species Penicillium rugulosum and P. purpurogenum is sometimes observed (Leopold, 1953; Smith, 1954). In the production of feed-yeast, infections by members of the genera Monilia and Oidium and by so-called mycoderms have been dcscribed (Underkofler and Hickey, 1954). In the production of itaconic acid, infection by strains of Aspergillus niger was described in two of eighty fermentations (Pfeifer et al., 1952). Monilia sitophila can cause serious difficulties in the production of technical chlortetracycline during cultivation of the production strain on shredded grain (Herold and NeEQsek, 1952; Herold et al., 1958). Phage contamination is relatively frequent in some classic fermentation processes, especially in the production of butanol-acetone by clostridia (Legg, 1928; Hanson, 1937; McCoy, 1946; Beesch, 1952), in the production of 2-ketoglutaric acid by strains of Pseudomonas aeruginosa (Underkofler and Hickey, 1954) and in the production of 2,3-butanediol by strains of Bacillus polymyxa (Katznelson and Lochhead, 1944). Also in the biosynthesis of vitamin B12 by some bacteria, e.g., Bacillus megatherium, phage Contamination may cause difficulties (Garibaldi et al., 1953). Phage contamination aroused further attention in connection with the production of streptomycin by strains of Streptomyces griseus where phage contamination was first observed in 1947 (Reilly et al., 1947; Saudek and Collingsworth, 1947; Woodruff et al., 1947). Although difficulties caused by actinophage contamination were expected to be great, and although actinophages are able to attack many Actinomycetes producing antibiotics and vitamin Bl2 , they seem a t the moment to be not as dangerous as originally expected (Carvajal, 1953; Rautengtejn, 1955; Welsch, 1957).

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TABLE I FERMENTATION PROCESSES RELATIVELY INSENSITIVE (GROUP1) A N D VERY SENSITIVE(GROUP2) TO CONTAMINATING MICROFLORA Group 1 (products) Organic acids Organic solvents Baker’s and fodder yeast Steroids

Group 2 (products) Antimicrobial antibiotics Cytostatic and antivirus antibiotics Vitamin 31% Vitamin Bs

111. Classification of Fermentation Processes According to the Importance of Contamination Fermentation processes for the production of cell substance or for the production of biological preparations may be classified as follows: 1. Processes in which penetration of foreign microflora into the fermentation medium is not dangerous. 2. Processes requiring maintenance of strictly aseptic conditions. These processes will hereafter be called aseptic. Examples of fermentations belonging in the first or second group are given in Table I. The first group is formed first of all by fermentation processes proceeding in relatively strongly acid medium, e.g., production of citric and other organic acids, production of some organic solvents, e.g., ethanol and acetone-hutanol. From this standpoint fermentation processes cannot, however, be classified unambiguously, because for the attainment of maximum yields it is sometimes necessary to conduct the fermentation a t a pH higher than the pK, of the respective acid or a t an extraordinarily strong aeration. This is the case in the production of lactic acid (Sakaguchi et al., 1942), or in the production of gluconic acid by members of the genus Pseudomonas (Katagiri and Itagaki, 1950). Processes belonging in the first group are further characterized by rapid multiplication and high concentration of cells of the production culture per unit volume of the fermentation medium; consequently the contaminating microflora is usually not able to multiply in the production culture to such an extent as to influence considerably the fermentation process. This is the case, e.g., in the production of cell substance by means of bacteria of the genus Bacillus where, in the final stage of propagation, it is not necessary to maintain aseptic conditions (Lewis e2 al., 1953). Finally we may place in this group those biosynthetic processes which proceed very rapidly, e.g., some transformation steroids (Peterson et al., 1952), or the ribosidation of 6-azauracil (Skoda et al., 1957),

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where the biosynthetic process itself is so short that the contaminating microflora is not able to develop beyond its lag-phase. Because of these reasons the processes of the first group do not require any special equipment or measures for the inhibition of penetration and further multiplication of foreign microflora. The second group is formed especially by the production of antibiotics arid some vitamins and organic solvents requiring strong aeration, proceeding a t an approximately neutral p H and with a long fermentation time. Even in this group there exist differences in the necessity of maintenance of strict asepsis. The differences in requirements are dependent first of all on the propertics of the production microorganisms and on the character of the substances produced. Submerged fermentation processes of this group usually require, for the maintenance of aseptic Conditions, special closed fermentation equipment which has evolved to its present state of considerable perfection during the development of the production of penicillin by submerged ferment at’ion. Biosynthetic processes belonging in the second group may further be divided into biosynthesis of antibiotics with antimicrobial activity, and the biosynthesis of other products without antimicrobid activity. Especially the broad spectrum antibiotics can, after attainment of a certain concentration in the medium, i.e., from n certain stage of culture development, protect its own production microorganism against multiplication of contaminating microflora. Periodic reviews of fermentation technology (Silcox and Lee, 1948; Lee, 1949, 1950, 1951; Perlman E t al., 1952, 1953; Perlman and Kroll, 1954; Beesch and Shull, 1955, 1956, 1957) contain relatively few data concerning the problems of protecting the fermentation medium and the production culture against contamination by means of antimicrobial substances. Also the overwhelming majority of scientific, technical, and patent literature on the biosynthesis of antibiotics, vitamins, and similar products stresses the necessity of performing biosyrithesis under aseptic conditions achieved exclusively by suitable machinery or equipment. The course of biosynthesis of the above products is undoubtedly optimum with maintenance of all these conditions using techniques currently available in the laboratory and with machinery; in lnrge-scale production, however, complete prevention of contamination is sometimes very difficult, and a certain number of batches are discarded because contamination has to be taken for granted. This is true especially with long-lasting fermentations, and with continuous cultivation, the importance of which is increasing (Mitlek, 1958). Some authors, therefore, tried to diminish the danger of contamination of the production culture, even with current machinery or equipment, by

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the addition of certain antimicrobial substances to the fermentation medium. Addition of substances protecting the production culture against contamination under normal technological conditions may lead to a lowering of the number of contaminated batches; in some cases protection of the fermentation medium by chemicals may lead to a considerable simplification of the fermentation equipment required and Q lowering of production costs (BBlik et al., 1958).

IV. Sources of Contamination in Aseptic Fermentation Processes I n order to stress the importance of the protection of fermentation processes against contamination by means of antimicrobial substances, it is necessary to describe the technological measures usually taken in normal aseptic fermentations. Contamination may get into the production culture from the stock culture of the production microorganism, from incorrect manipulation during multiplication of the culture in the laboratory or in seed tanks, from insufficiently sterilized fermentation medium, from air used for aeration, from cooling water in insufficiently tight coils and tank duplicators, from insufficient defoaming during fermentation, and finally by omission of some of the basic rules of preventing outside contamination from penetrating into the tank. The following points especially are of importance (Perlman, 1950; Herold et al., 1957): 1. There must be no direct connection between the sterile and nonsterile parts of the piping and armatures. 2. Where connections and flanges are indispensable, these must be made of first-grade rubber without a textile layer. 3. Use of welded construction wherever possible. 4. The types of valves and armatures must be so selected as to be easily sterilizable and able to maintain sterility throughout the whole process. 5. After sterilization all parts of the equipment and piping junctions which are to remain sterile, have to be maintained under constant overpressure of sterile air. 6. Every part of the equipment must be capable of independent sterilization which must not interfere with the function of the rest of the equipment. 7. The entrance and outlet valves and sections of tubing which are not otherwise protected must be connected to sharp vapor tubing. 8. Before every new batch it is necessary to test the tightness of tank closures, tubing connections, valves, and similar parts of the equipment. I n spite of all these pretentious requirements the technology of aseptic fermentation has been brought, especially in the pharmaceutical industry, to such a state that the number of contaminated batches does not exceed 5 % of the total number of fermentations (Saudek, 1956).

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Protection of the production culture by technological measures, especially by specialized machinery or equipment, has been described in a number of books (Florey et al., 1949; Foster, 1949; Prescott and Dunn, 1949; Underkoffler and Hickey, 1954; Herold et al., 1957) and in a number of reviews (Silcox and Lee, 1948; Lee, 1949, 1950, 1951; Perlman et aZ., 1952, 1953; Campbell, 1953; Chain et al., 1954a, b; Paladin0 et al., 1954; Perlman and Kroll, 1954; Beesch and Shull, 1955, 1956; Qlechta, 1955; Finn and Sfat, 1956; Gaden, 1956; Sikyta et al., 1958). All these measures concerning the method of sterilizing the medium, the influence of sterilization upon the quality of nutrient medium, construction of equipment, methods of sterilization of air necessary for the aeration of the culture, etc., are able to effectively reduce contamination of production cultures by all microorganisms with the exception of contamination by phages. To reduce the adverse effects of phage contamination, special phageresistant strains were developed, especially for the industrial production of streptomycin (Woodruff, 1952).

V. Application of Antimicrobial Agents for the Protection of Fermentation Processes

The application of antimicrobial substances for the protection of classic fermentation processes against contamination is in some instances a regular part of the technological procedure. The application of antimicrobial suhstances for the propagation of yeasts was suggested as far back as a quarter of a century ago (Hayduck, 1923) and in the production of ethanol even earlier (Day et al., 1954). In the production of ethanol, ammonium bifluoride is used as an antispetic, and in the production of fodder yeast, formaldehyde, and hydrofluoric, formic, salicylic, and picric acids are used (Underkofler and Hickey, 1954). From other agents the application of pentachlorophenol in the production of ethanol was lately suggested (Konovalov, 1955; Verbina, 1955; Drews, 1956). The possibility of the application of quaternary ammonium bases was studied by a number of workers (Kockov&-Kratochvilovit, 1950; Komarova, 1953; Konovalov, 1955; Verbina, 1955; Valentovh, 1957). It was especially proved that their toxicity against cultures of yeasts depends upon the type of medium, amount of inoculum used, and extent of agitation. By repeated subculturing on a medium containing 0.001 % of cetylpyridinium bromide or sodium pentachlorophenolate which by itself was not toxic, a decrease of brewing energy and especially of culture multiplication occurred (Verbina, 1955). Application of penicillin is probably more advantageous (Day et aE., 1954). Penicillin in a concentration of 0.75-2 units per milliliter suppresses multiplication of bacteria in yeast cultures very effectively. Suppression of contamination is manifested not only by an increased yield but also by an improved quality of ethanol

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due to the lowering of the formation of acrolein and glycerin. Penicillin increases also the number of live yeast cells present in the mashes at the end of the fermentation. Chlortetracycline, bacitracin, chloramphenicol, and oxytetracycline proved much less active compared to penicillin; polymyxin was inactive. The amounts of penicillin necessary for the suppression of the bacterial microflora are very small, and this method of protection of the fermentation is probably advantageous from the economic viewpoint. In the production of ethanol by continuous fermentation of blackstrap molasses, a similar positive effect of penicillin used for the protection of a fermentation process was claimed by Borzani and Aquarone (1957). In experiments on a laboratory and pilot-plant scale the presence of penicillin in a concentration of 1 unit per milliliter increased the yield of ethanol by 4.8-19.5 % and lowered the production of acids by 17.0-66.6 %. Penicillin did not influence the final number of yeasts and the fermentation time. For the suppression of Leuconostoc infection a relatively high concentration of 8 units per milliliter of penicillin was needed. The differences of activity of penicillin, chlortetracycline, and chloramphenicol against different strains of the contaminating microflora were studied by Protiva (1958). He found that for the successful application of these antibiotics it is necessary to test the sensitivity of the microflora in each production plant and to det'ermine the most suitable antibiotic for each specific case. Strandskov and Bockelmann (1953) compared the effects of usual antibacterial agents (tartaric and phosphoric acid) and of antibiotics upon contaminating gram-negative microflora in the production of beer. Polymyxin appeared to be the most active, the tetracyclines showing somewhat smaller activity. Polymyxin also showed a considerable activity against grampositive lactic bacteria, although somewhat kmaller than penicillin. The antibiotics used for the protection of the fermentation caused a simultaneous stimulation of the growth of the yeasts. This can be explained by the suppression of the contaminating microflora and by the elimination of its adverse effect upon the production culture. The activity of polymyxin against bacterial contaminations in the preparation of pure cultures of yeasts for the production of beer was verified by Grossmann (1958) who compared its effect with that of penicillin, oxytetracycline, and thiolutine. In the United States official permission was obtained for the application of polymyxin in a concentration of 0.0015 % for the protection of cultures of yeasts in the production of beer. In the tank its concentration has to be lowered to 0.05 p.p.m. (Barnes, 1957). For protection of cultures of filamentous fungi against bacterial contamination, chlorine dioxide (Woodward, 1948), 2,4-dichlorphenoxyaceticacid (Stevenson and Mitchell, 1948), and 2-substituted 5-nitrofuran derivatives

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(Ueno, 1951) were also suggested. Antibiotics were recommended as protection against bacterial contamination in the cultivation of fungi for the production of albumins and fats (Biosyn, 1945). The application of antibiotics was also suggested for the protection of bacterial fermentation processes (Froquet and Pichon, 1952). Interesting results and possibilities are offered by the application of antibiotics in the protection of industrial cultures of rhizobia (Vintika, 1955; Vintikovh, 1956), in the production of fodder yeast (Dyr and GrBgr, 1958), and in the production of acetone-butanol (Vintika, 1957). From the above examples of protection of fermentation processes which are not very sensitive to contamination, it follows that antimicrobial agents and antibiotics have become a valuable means of increasing the economy of the production process and of improving its technology, and that their range of application is steadily increasing. It can therefore be expected that their effect will be substantial also in the second group of fermentation processes, i.e., in those requiring the maintenance of asepsis. Experiments with the suppression of bacterial contamination in the biosynthesis of penicillin are almost as old as the industrial production of the antibiotic itself. In 1945 Knight and Frazier published their results showing that, of thirty-scvcn different agents, only borax and boric acid were able to inhibit multiplication of contaminating bacterial microflora without a simultaneous suppression of penicillin formation (Knight and Frazier, 1945). They found, however, that various penicillin producing strains differ in their resistance to borax. With the strain NRRL-1951-B 25 the production of penicillin in flasks on a shaker was stimulated with respect to the control by a concentration of borax of 0 . 2 4 3 % . At this concentration of borax the production of penicillin was not decreased even by experimental contamination of the flasks by gram-negative rods and micrococci in a total amount of approximately lo6bacteria per flask. The cultures used for the expcrimental infection were isolated from contaminated fermentations and were representative of the usual bacteria from air, dust, and cooling water. In fermentations with the above strain in pilot plant tanks of 80 g:illons vohime, the production of penicillin was in the first hours stimulated by the addition of borax, but appeared, however, somewhat lower than the control towards the end of the fermcntation. With an inoculum cultivated on media containing 0.2 % of borax no similar lowering of production was observed in fermentations on borax-containing media. These positive results were not applied on an industrial scale, probably because of the rapid and successful development of specialized fermentation equipment decreasing thc danger of contamination (Foster, 1949). The stimulating effect of neither borax nor boric acid (Koffler et uZ., 1945) was ever utilized on an industrial scale.

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Experiments with the suppression of bacterial contamination by antimicrobial agents in the production of penicillin were resumed later (Fernandez, 1953). It was found that multiplication of contamination penetrating from air into the flasks (which were on purpose not closed by cotton plugs) was inhibited by the addition of 0.1 % of benzalkonium chloride (alkyldimethylbenzylammonium chloride) or phenol, 0.3 % ethanol and 0.18 % toluene, benzene or xylene in 50-100% of cases. Benzalkonium chloride or phenol in a concentration of 0.1 % decreased appreciably the production of penicillin. A concentration of 0.18 % of toluene, benzene, or xylene did not decrease production compared with the control. Penicillin isolated from the fermentation medium with 0.18 % toluene did not differ in any respect from penicillin obtained from a control experiment with no toluene added. Another case of protection of a sensitive fermentation process against bacterial infection is the submerged production of amylase by a strain of Aspergillus niger. It was found that in this case bacterial contamination may be successfully suppressed by ammonium bifluoride or sodium pentachlorophenolate. The latter also stimulates production of amylase by suppressing sporulation of the production culture (Erb et al., 1948). Great possibilities are offered by the protection of fermentation processes in the production of some vitamins. In the biosynthesis of riboflavin by Eremothecium ashbyii, it was found that the addition of penicillin into the fermentation medium not only successfully eliminates bacterial contamination, but at higher levels (about 1000 units per milliliter) also slightly stimulates the production of the vitamin (Tozer and Speedie, 1951). In another case it was found that a concentration of chlortetracycline above 20 bg./ml, decreases the production of riboflavin (Hanus and Munk, 1958). Concentrations of 5-10 kg./ml., however, successfully suppressed contamination by sporulating bacteria occurring because of the difficult sterilizability of the suspension fermentation medium on a pilot plant scale. The application of some compounds for the protection of cultures against actinophages is in some respects similar to the use of antimicrobial agents for the protection of fermentation processes against bacterial contaminations. Multiplication of the actinophage in cultures of strains of Streptomyces griseus is suppressed by the presence of sodium, potassium, and ammonium ions; multiplication of the phage is, on the other hand, stimulated by calcium, barium, magnesium, and manganese ions (Walton, 1951), acting as adsorption cofactors. By the addition of sodium citrate or sodium oxalate, or other multibasic organic acids used as components of the fermentation medium in a concentration of about 0.01 M , calcium ions are especially bound, thus inhibiting multiplication of the actinophage (Perlman et al., 1951; Perlman and Langlykke, 1953). This method of protection is useful only with a fermentation medium which does not contain an excess

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of calcium ions. Probably for this reason the method did not find wider practical application, in spite of the fact that the application of phage-resistant strains is not completely reliable. A production strain, resistant towards some actinophage strains, may be sensitive to others.

VI. Biosynthesis of Chlortetracycline without Maintenance of Aseptic Conditions In the production of broad spectrum antibiotics the production strain may be, in a certain stage of the fermentation, protected against contamination by its own antibiotic. This is mentioned already by Lee (1950) and by Beesch and Shull (1955) in the paragraph on sterilization and aseptic techniques in their review articles on fermentation. The success of such protection depends upon the concentration of the antibiotic, on the kind and number of contaminating microorganisms, and upon the stage in which penetration or multiplication of foreign microflora has taken place. If contamination penetrates into the medium at a time when the antibiotic is present in the medium at a concentration of at least several tenths of a microgram per milliliter, it is very probable that the contaminating microbes will not be able to multiply and the adverse effect of the contamination will be negligible. If, however, contamination takes place at the very beginning of fermentation, foreign microorganisms multiply quickly and are able to influence very unfavorably even the biosynthesis of broad spectrum antibiotics. Some authors paid attention to these circumstances and studied systematically the conditions of biosynthesis of chlortetracycline without maintenance of aseptic conditions (BWk et al., 1955; Herold et at?., 1956; B6lfk et ul., 1957). When fermentation media in flasks on a shaker were inoculated with 5-10 % of a vegetative inoculum containing approximately 200 pg. of chlortetracycline per milliliter, no differences were observed between normally stoppered flasks and those that were open during the course of the whole fermentation. No differences in chlortetracycline production compared with the control were observed even in those cases when the medium in the flasks was experimentally contaminated by river water or soil. In experiments with artificial contamination by pure strains of microorganisms, production of chlortetracycline was lowered by 40% by only two strains of Torulopsis utilis. The production strains used in these experiments (Streptomyces aureofaciens Bg or BMK) produce, besides chlortetracycline, under the given experimental conditions a substance with antifungal activity. Examples of the results are given in Table 11. These results were later verified by fermentation in a pilot plant tank without the usual equipment for aseptic work (e.g., no tight cover, no steam isolation sections, etc.) and by 16 months of uninterrupted fermenta-

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TABLE I1 PRODUCTION OF CHLORTETRACYCLINE DURING FERMENTATION WITH STREPTOMYCES AUREOFACIENS

BG UNDER DIFFERENTCONDITIONS O F

Conditions of fermentation Fermentation under aseptic conditions Fermentation in open flasks Medium partly sterilized for 40 minutes a t 97-99°C. Contamination by 0.25 gm. of soil in each flask Contamination by 1 ml. of river water in each flask Contamination by a strain Torulopsis utilis var. maior Contamination by a strain Torulopsis utilis var. thermophila

AsEPsIS"

Chlortetracycline produced (pg./ml.)

1328 1336 1526 1382 1372 794 840

Each fermentation flask contained 80 ml. of medium and each was placed on a shaker. Q

tions in a very simplified equipment using wooden barrels of 1000 liters capacity (BBlik et al., 1958). No increase of resistance of the contaminating microflora towards chlortetracycline was observed during this whole period. Production of chlortetracycline without maintenance of aseptic conditions is one of .the methods used in Czechoslovakia for the production of antibiotics for animal nutrition (Herold and BBlik, 1958; Herold and Muller, 1958).

VII. Perspectives In order to obtain an optimum effect in protected fermentations, it is first of all necessary that the protection be economically advantageous and the protection method be as reliable as possible; the antimicrobial spectrum of the substances used must be sufficiently broad and its effect must not be limited by species and strain differences of the contaminating cultures. Similarly, as in the application of antimicrobial chemotherapeutics,it is necessary that the antimicrobial substance possess maximum toxicity for the Contaminating microflora and be at the same time practically nontoxic for the production culture and the corresponding metabolic processes. With respect to these requirements and to the favorable results obtained in the biosynthesis of chlortetracycline without maintenance of aseptic conditions, attention was turned to problems of biosynthesis of antibiotics (especially those with a narrow spectrum of activity) under the protection of broad-spectrum antibiotics. Antibiotics were selected as protecting agents

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because of their high activity against foreign microflora in concentrations which in some cases do not unfavorably influence the production culture. The results obtained up to now in the protection of fermentation processes by synthetic antimicrobial agents indicate that these substances may be utilized to a certain extent only in the protection of fermentations relatively insensitive towards contamination; they can practically not be utilized a t a11 in aseptic fermentation processes, due to their relatively low activity against the contaminating microflora and usually relatively high toxicity for the production strain. In preliminary experiments with the application of antibiotics for the protection of antibiotic fermentations, we obtained the following results (Lokvenc et al., 1959;Matelovit et al., 1959). In a study of the biosynthesis of streptomycin by the strain Streptomyces yriseus LS-1 (Alikhanian, 1957), it was found that an addition of oxytetracycline in a concentration of 10 pg./ml. of the fermentation medium suppresses considerably both the growth and the productivity of the culture. In a resistant strain obtained by the method of replacement cultures followed by successive passages a t an increasing concentration of oxytetracycline, and disregarding streptomycin productivity in selection, increased resistance to oxytetracyclinc was accompanied by decreased streptomycin productivity. An even larger decrease of productivity was observed in fermentations on media containing oxytetracycline. A t an oxytetracycline concentration of 100 pg./ml. of fermentation medium, production of streptomycin dropped to 29% of the control, with a strain resistant to the above concentration of oxytetracycline. Additions of penicillin and fungicidin, on the other hand, do not decrease the production of streptomycin even at a concentration of 50 units per milliliter. In the biosynthesis of erythromycin by the strain Streptomyces erythreus 5S/lOO production was inhibited by additions of chlortetracycline, oxytetracycline, streptomycin, or neomycin in a concentration of 10 pg./ml. and by an addition of bacitracin in a concentration of 1 unit per milliliter. With an addition of streptomycin in a concentration of 5 pg./ml. or bacitracin at a concentration of 0.5 unit per milliliter, production dropped to about 50 % of the control. The effect of oxytetracycline and chlortetracycline was studied in the biosynthesis of fungicidin by the strain Streptomyces noursei 2-9 (Popova, 1959). At a concentration of 500 pg./ml. of the fermentation medium, oxytetracycline made the production drop to 53 % of the control, chlortetracycline to 29 ?A of the control. Both these tetracycline antibiotics did not influence the production of furigicidin a t a concentration of 100 pg./ml.

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Production of streptomycin by the strain S. griseus LS-1, of chlortetracycline by the strain S. aureojaciens BMK (BBlik et al., 1957), and of oxytetracycline by the strain S. rimosus LS-T 118 (Alikhanian, 1957) with an addition of fungicidin in a concentration of 500 units per milliliter amounted to 98 %, 50 %, and 86 % of the control, respectively. The biosynthesis of the above antibiotics was not influenced by the presence of fungicidin a t a concentration of 100 units per milliliter. Production of penicillin by the strain Penicillium chrysogenum NG (Alikhanian, 1958) was decreased to 85-80 % of control by the presence of oxytetracycline a t a concentration of 5 0 4 0 0 pg./ml. Even the concentration of 400 pg./ml. was, however, not able to counteract the decrease of penicillin production caused by experimental contamination by unsterile soil. This is not surprising, due to the well-known property of penicillin which undergoes inactivation by penicillinase. Due to the encouraging results with the biosynthesis of fungicidin in the presence of tetracycline antibiotics and vice versa, the possibility of a parallel production of fungicidin and oxytetracycline on a medium inoculated simultaneously with both production strains was investigated. On a medium suitable for the biosynthesis of both these antibiotics separately, a considerable lowering of fungicidin production was observed after inoculation by both production strains. Simultaneous productivity of both antibiotics a t an acceptable level was obtained when the medium was inoculated by the culture producing oxytetracycline somewhat later than by the culture producing fungicidin. Similar results may be achieved by a modification of the ratio of the amount of both inocula in favor of the fungicidin inoculum. It is evident that in a number of cases where it could be advantageous to apply some antibiotics for the protection of fermentation processes in the biosynthesis of other antibiotics, the toxicity of the protecting antibiotics for the production strain suppresses its productivity or even its growth. The mutual antagonism of the actinomycetes caused by the cross-sensitivity toward the antibiotics produced by them seems, according to a recent review by Krasilnikov (1958) to be a considerably reliable criterion in the species identification of the actinomycetes. From the results of some other workers it follows, however, that the suppression of growth of a certain antibiotic-producing streptomyces by other antibiotics produced by streptomycetes is of no general validity. Thus, a strain of Streptomyces rimosus producing oxytetracycline was not inhibited by streptomycin, chlortetracycline, neomycin, viomycin, and actinomycin C in concentrations of 150750 pg./ml. when cultivated on solid media (Musilek, 1957). The different results obtained in the study of the influence of antibiotics from streptomycetes upon other atreptomycetes is most probably caused by strain

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specificity. Besides, the response of the microbe to the presence of an antimicrobial agent always depends on its concentration and is modified by extraneous factors influencing the fermentation. The mutual antagonism of antibiotics-producing actinomycetes is, however, not the only reason for the decrease of productivity and growth of the strains in the biosynthesis of antibiotics by actinomycetes. In a study of the transformation of progesterone by washed suspensions of cells of two strains of streptomyces it was, e.g., found that the formation of the enzymes performing the transformation is profoundly inhibited by antibiotics produced from both streptomycetes and bacteria, and by penicillin. An addition of the antibiotics influenced the transformation only slightly in the case that the strains were cultivated in the presence of a small amount of progesterone (Perlman et al., 1957). It can be expected that in those cases where the production cultures of streptomycetes are unfavorably affected by the antibiotics added into the nutrient media, a considerable effect could be reached by a purposeful strain improvement for resistance towards the protecting antibiotic at undiminished original productivity. It is probable that the methods of formation of heterocnryonts and recombinants will play an important part in such strain improvement (Sermonti and Spada-Sermonti, 1950; Bradley and Lederberg, 1956; Braendle and Szybalski, 1957). A possibility which is both theoretically and practically interesting is the simultaneous production of two antibiotics, or the production of an antibiotic together with some other product, in a mixed culture of both production strains, in which one or both substances produced protect the process as a whole against contamination. This principle of simultaneous cultivation is to a certain extent analogous to the process which had been suggested for the production of vitamin BI2by propionic bacteria with the help of a strain of Lactobacillus casei on substrates containing lactose. In this case the nutrient medium with lactose is first fermented by the strain L. casei. After a certain cultivation time, the culture of the propionibacterium is inoculated; this culture which produces the vitamin B12 can by itself not metabolize lactose (Leviton and Hargrove, 1952; Leviton, 1956). In a simultaneous production of two products the problems are complicated by the fact that the fermentation process has to be regulated in such a way that the two production cultures in simultaneous growth do not adversely affect the metabolic and synthetic abilities of each other. Greatest possibilities are offered by the application of antibiotics for the protection of such fermentation processes where the antibiotics do not affect the production strain at all. Relatively very good results were therefore obtained in the production of ethanol and in other cases where the production culture consists of yeasts or yeast-like organisms. Considerable possi-

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bilities may be expected for the protection by antibiotics in the biosynthesis 'of other products of the metabolism of fungi, e.g., the giberrellins. For a similar reason very favorable results were quickly obtained in the biosynthesis of chlortetracycline without maintenance of aseptic conditions, as the submerged culture of S. aureofwiens is relatively very resistant to its own antibiotic. Favorable results may also be expected in the biosynthesis of other broad-spectrum antibiotics.

VIII. Conclusions and Summary From the above review it is evident that protection of various fermentation processes against contamination by means of antimicrobial agents has been capable of improving and economizing the fermentation process in a number of cases. In fermentations relatively insensitive to contamination which are therefore not carried out under aseptic conditions, elimination of foreign microflora usually increased the yield of the metabolite produced. In aseptic fermentations, e.g., in the production of antibiotics and similar biological preparations, a lowering of the number of batches usually discarded because of contamination can be expected, besides a considerable simplification of the fermentation equipment. The present results have proved the practical possibility of the production of chlortetracycline without maintenance of aseptic conditions (BUk el al., 1958). Application of the protected fermentation of technical grade chlortetracycline proved especially advantageous in those cases where it was necessary to build up quickly a large production capacity, because it made possible the use of much simpler equipment than the usual stainless steel fermentation tanks with isolation sections, tightly fitting lids, perfect stuffing boxes on impeller shafts, etc. In connection with the biosynthesis of antibiotics also some further results of practical importance were obtained (Lokvenc et al., 1959). A basic prerequisite of the possibility of practical application of the protection of fermentation processes by antimicrobial agents is, besides the economy, the requirement of maximum toxicity of the protecting agent for the contaminating microflora and minimum toxicity for the production culture and the corresponding metabolic process. In addition, the spectrum of activity of the protecting agent should not, as far as possible, undergo variations due to species and strain differences of the contaminating microflora. The application of highly active broad-spectrum antibiotics, especially of the tetracycline group, is advantageous mainly with respect to this last requirement. In their application for the protection of Streptomyces fermentation processes, the possibility of the sensitivity of the production strains towards these antibiotics has to be kept in mind. It will probably be possible to overcome this difficulty by the application of selected strains resistant toward the antibiotics used for protection. This method seems

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advantageous with respect to the broad possibility of application in the hiosynthesis of a number of products, e.g., of some antibiotics without antibacterial activity, or with a very narrow spectrum. In fermentation processes using fungi as production organisms, the application of broad spectrum antibiotics for protection against contamination can be expected to bring considerable improvements quickly. Further studies should show to what extent simultaneous fermentations producing two antibiotics complementary in their protecting action, or an antibiotic together with another product, can be utilized. The mixture of substances thus obtained will naturally have to be isolated easily from the fcrmentation medium and thus offer a possibility of easy final adjustment to the combined prepar LZt'ion. It would be incorrect for surgeons to disregard aseptic techniques in the performance of surgical operations; in exactly the same way it would be very unprofessional for bioengineers to excuse insufficiencies of technology and fermentation equipment by the possibility of protected fermentations. Protected fermentations have to be used in the right place, for the lowering of the per cent of contaminated batches, for the purposeful simplification of t,he fermcritatiori cquipment arid its maintenance, and for the lowering of production costs.

REFERENCES Abe, Y . , Shiotsu, S., and Endo, T. (1952). J. Antibiotics (Japan) 6.84-91. Abraham, E. P., and Chain, E. (1940). Nature 146, 837. Alikhanian, S. I. (1957). Antibiotiki (Medgiz) 2, (5)) 31-35. Alikhanian, S. I . (1958). Bjulleten m . obshch. i s p . prirody, old. biologii 63, (3), 79-96. Barnes, E. M. (1957). Roy. SOC.Promotion Health 1.7 7 , 446-457. Beesch, S. C. (1952). Znd. Eng. Chem. 44, 1677-1682. Beesch, S. C., and Shull, G. M. (1955). Znd. Eng. Chem. 47, 1857-1875. Beesch, S. C., and Shrill, G. M. (1956). Znd. Ens. Chem. 48, 15861603. Beesch, S. C., and Shull, G. M. (1957). Ind. Eng. Chem. 49, 1491-1505. BElfk, IC., Herold, M., and UoskoEil, J. (1955). Czech Patent, 87,520. BBlik, E., Herold, M., and Dosko6i1, J. (1957). Feskoslov. mikrobiol. 2, 30-35. BEIik, E., Herold, M., Hudec, M., MiBe6ka, E., and Zelinka, J. (1958). Chem. zvesti 12, 121-127. RioRyn, G. m. b. H. (1945). Freiich Patent 905,075. Borzani, W . , and Aqriarone, E. (1957). J. Agr. Food Chem. 6, 612-616. Bradley, S. G., and Lederberg, J . (1956). J . Bacteriol. 72, 219-225. Braendle, D. H., and Szybalski, W . , (1957). Proc. Nall. Acad. S c i . U.S. 43, 947-955. Campbell, A. H. (1953). Research (London) 6, 42-50. Carvajal, F. (1953). Mycologia 46, 209-234. Chain, E. B., Paladino, S., Ugolini, F., Callow, S. D., and Van der Sluis, J. (1954a). Rend. ist. super. sanitd 17, 61-86. Chain, E. B., Paladino, S., Ugolini, F., and Callow, D. S. (1954b). Rend. i s t . super. sunit& 17, 133-144.

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Day, W. H., Serjak, W. C., Stratton, J. R., and Stone, L. (1954). J . Agr. Food C h e w 2, 252-258. Drews, W. (1956). Branntweinwirtschaft 78, 477-482. Dyr, J., and GrBgr, V. (1958). Private communication. Erb, N. M., Wisthoff, R. T., and Jacobs, W. L. (1948). J . Bacterial. 66, 813-821. Fernandez, H. (1953). Anales inst. farmacol. espafi. 2, 225-240. Finn, R. K., and Sfat, M. R. (1956). I n d . Eng. Chem. 48, 2172-2217. Florey, H. W . , Chain, E., Heatley, N. G., Jennings, M. A., Sanders, A. G., Abraham, E. P., and Florey, M. E. (1949). “Antibiotics,” Vol. 2, pp. 691-720. Oxford Univ. Press, London and New York. Foster, J. W. (1949). “Chemical Activities of Fungi,” pp. 593-599. Academic Press, New York. Froquet, L. and Pichon, P. (1952). French Patent 1,000.165. Gaden, E. L., Jr. (1956). Chem. Eng. 63, 159-174. Garibaldi, J. A,, Ijichi, K., Snell, N. S., and Lewis, J . C . (1953). I n d . E n g . Chem. 46, 838-846. Grossmann, H. (1958). Lebensmittelind. 3, 13&140. Hanson, C. T. (1937). U. S. Patent 2,085.428. Hanus, J., and Munk, V. (1958). Private communication. Hayduck, F. (1923). U. S. Patent 1,449.112. Herold, M., and BBlik, E. (1958). Antibiotiki (Medgiz) 3, ( l ) , 67-73. Herold, M., and Muller, Z. (1958). Intern. Congr. Biochem., 4th Congr., Vienna. Herold, M., and NeE&sek,J. (1952). Czech Patent 84,631. Herold, M., BBlik, E., and DoskoEil, J . (1956). Giorn. microbial. 2,302-311. Herold, M., VondrAEek, M., NeE&sek,J., and DoskoEil, J. (1957). “Antibiotika.” pp. 34-40, 72-73. Nakl. CSAV, Prague. Herold, M., Matelov4, V., NeE&sek,J., and HoBthlek, Z.(1958). Ceskoslov, mikrobiol. 3, 313-319. Katagiri, H., and Itagaki, K. (1950). B d . Znst. Chem. Research, Kyoto U n i v . 2 2 , 9 4 95; Chem. Abstr. 46,4399 (1951). Katznelson, H., and Lochhead, A. G. (1944). Can. J . Research C22.273-279. Knight, S. G., and Frazier, W. C . (1945). J. BacterioE. 60, 505-516. Kockov4-Kratochvilov4, A. (1950). Sbornik Ceskoslov. akad. zemdddl. vdd 23, 349-354. Koffler, H., Knight, S. G., Emerson, R. L., and Burris, R . H. (1945). J . Bacterial. 60, 549-559. Koffler, H., Knight, S. G., Frazier, W. C., and Burris, R. H. (1946). J. Bacterial. 61, 385-392. Komarova, L. I. (1953). Mikrobiologiya 22, 566-571. Konovalov, S. A. (1955). Mikrobiologiya 24, 199-207. Krasilnikov, N. A. (1958). Ceskoslou. mikrobiol. 3, 288-297. Lee, S. B. (1949). Znd. Eng. Chem. 41, 186&1877. Lee, S. B. (1950). Znd. Eng. Chem. 42, 167S1687. Lee, S. B. (1951). I n d . Eng. Chem. 43,1948-1969. Legg, D. A. (1928). U. S. Patent 1,668,814. Leopold, J. (1953). Ceskaslov. bid. 2, W 5 6 . Leviton, A. (1956). U. S. Patent 2,753,289. Leviton, A., and Hargrove, R. E. (1952). Znd. Eng. Chem. 44, 2651-2655. Lewis, J. C., Ijichi, K., Sagihara, T. F., and Garibaldi, J. A. (1953). J . Agr. Food Chem. 1, 897-899.

20

MILOB

HEROLD AND JAN

NEEASEK

Lokveno, F. A., Herold, M., Nekisek, J . , and Mustlkovh, M. (1959).Folia Microbiol. 4. I n press. McCoy, E. F. (1946).U. S.Patent 2,398,837. Mhlek, I. (Ed.) (1958). “Continuous Cultivation of Microorganisms, A Symposium.” Publ. House of the Czech. Acad. Sci., Prague. Matelovh, V., Herold, M., and Bkola, V. (1959).Folia. Microbiol. 4. In press. Musilek, V. (1957).ceskoslov. mikrobiol. 2, 183-184. Paladino, S.,Ugolini, F., and Chain, E. B. (1954).Rend. ist. super. sanitb 17,87-120. Perlman, D.(1950).Botan. Rev. 16,449-511. Perlman, D.,and Kroll, Ch. L. (1954).Ind. Eng. Chem. 46, 1809-1826. Perlman, D.,and Langlykke, A. F.(1953).U. S. Patent 2,638,436. Perlman, D., Langlykke, A. F., and Rothberg, H. D., Jr. (1951).J . Bacteriol. 61,135143. Perlman, D., Brown, W. E., and Lee, S. B. (1952).Znd. Eng. Chem. 44, 1996-2012. Perlman, D., Tempel, A. IC., and Brown, W. E. (1953).Ind. Eng. Chem. 46,1944-1969. Pcrlman, I)., Weinstein, M. J., and Peterson, G. E. (1957).Can. J . Microbiol. 3, 841846. Peterson, 1). H., Murray, H. C., Eppstein, S. H . , Reineke, 1,. M., Weintraub, A,, Meister, P. I)., Lcigh, H. M.(1952).J . A m . Chem. Soc. 74, 5933-5936. Pfeifer, V. F., Vojnovich, C., and Hegcr, E. N. (1952).Znd. Eng. Chem. 44,2975-2980. Popova, L.A. (1959).Antibioliki (Medgiz) 4. In press. Prescott, S.C., and Dunn, C. G. (1949).“Industrial Microbiology,” 2nd ed., pp. 729751.McGraw-Hill, New York. Protiva, J. (1958).Private communication. Rautenktejn, J. I. (1955).Antibiotiki 8, (4),1-11. Reilly, H.C., Harris, D. A., and Weksman, S. A. (1947).J . Bacteriol. 64, 451-456. Sakaguchi, K.,Asai, T., und Uriekata, H. (1942). Nippon Nbgei-kagaku Kaishi 18, 793. Saudek, E. C. (1956).Bacteriol. Revs. 20, 279-281. Saudek, E. C.,and Colingsworth, 2). R. (1947).J . Bacteriol. 64, 41. Sermonti, G.,and Spada-Sermonti, I. (1956).J. Gen. Microbiol. 16, 609-616. Sikyta, B., Herold, M., Slechta, J. and Zaji&ek,J. (1958).Kvasny przbmysl 4, 275-278. Silcox, H. E.,and Lee, 8 . B. (1948). Znd. Eng. Chem. 40, 1602-1610. gkoda, J., Hess, V. F., and Sorm, F. (1957).Collection Czechoslov. Chem. Comniun. 22, 1330-1333. glechta, J. (1955).Zn “Antibiotika” (M. Herold, ed.), pp. 43-49. Stht. zdrav. nakl., Prague. Smith, G. (1954). “An Introduction to Industrial Mycology,” 4th ed., p. 221. Edward Arnold, London. Stevenson, E.C., and Mitchell, J. W. (1948).U.S.Patent 2,437,766. Strandskov, F. B., and Bockelmann, J. B. (1953).J . Agr. Food Chem. 1, 1219-1223. Taira, T.,and Yamatodani, S. (1953).J . Antibiotics (Japan) Ser. A . 6, 43. Tozer, H., and Speedie, J. D. (1951).British Patent 650,000. Ueno, M. (1951).Japanese Patent 5899;Chem. Abstr. 47, 3529 (1953). Underkofler, L. A., and Hickey, R. J. (1954). “Industrial Fermentations,” Vol. 1, pp. 81,283;Vol. 2,pp. 16,245-251,274-280.Chemical Publ., New York. Valentovh, M. (1957).Thesis. Technical University, Prague. Verbina, N. M. (1955). Trudy Znst. Mikrobiol., Akad. Nauk. S.S.S.R. 4, 54-97, Vintika, J. (1955). Sbornik ceskoslov. akad. zemZdt.1. v6d, Rostlinnd vyroba 28, 693704.

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Vintika, J. (1957). Kvasng prZimysl3,109-110. Vintikov4, H. (1956). Sbornik ceskoslov. akad. zeme'de'l vkd, Rostlinnd vyroba. 29, 952953. Walton, R . B. (1951). Antibiotics & Chemotherapy 1, 518-522. Welsch, M. (1957). Rev. fermentations et i n d . aliment. 12, 22-28. Woodruff, H. B. (1952). U. S.Patent 2,585,713. Woodruff, H. B., Nunheimer, T. D., and Lee, S.B. (1947). J . Bacterial. 64,535-541. Woodward, E. R. (1948). U. S.Patent 2,443,172.

This Page Intentionally Left Blank

The Mechanism of Penicillin Biosynthesis ARNOLD L. DEMAIN Merck Sharp and Dohine Research Laboratories, Rahway, New Jersey

B. Incorporation of L-Cyateine. ...... C. Role of Penicillamine and Serine.. . . . . . . . . . . . . . . . . . . . . . .

A. Phenylacetate and the Initial Condensation Reaction.. . . . . .

V. Future Experimentation. ..................... References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction The golden era of antibiotics no doubt began with the discovery of penicillin by Alexander Fleming (1929). The tremendous successes attained in the battle against disease with this compound have not only led to the development of the new field of antibiotic research but in addition created an entire new industry. Today, penicillin is functioning in yet another role. Studies on its biosynthesis, although still in infancy, are leading the way into biosynthetic studies on other antimicrobial agents, and it is only a matter of time before results of these studies will revolutionize the field of industrial fermentation. The structure of penicillin and its three moieties are shown below. The p-lactam and thiazolidine rings are common to all forms of penicillin; the side chain determines the specific type of penicillin. Pictured above is the main penicillin of commerce-benzylpenicillin or penicillin G-whose side chain is derived from phenylacetic acid. Penicillin is produced by strains of Penicillium notatum or Penicillium chrysogenum when inoculated into a suitable nutrient medium. Although some penicillin can be produced in surface culture (Moyer and Coghill, 1946), maximum concentrations are obtained only with the more desirable 23

24

ARNOLD L. DEMAIN

‘S

/ \

C~H~-CHZ-CO--NH~CH-~CH

I

I

*C(CHJ 2

’CO-4N-aCH-C0

I

OH

Bensylpenicillin

SH C ~ H ~ - C H ~ - C o OH

/

H~N-CH-CH~

I

COOH Phenylacetic acid

L-Cysteine

H~N-CH-COOH D-Valine

submerged fermentation technique. The initial phase of the fermentation involves the growth of the fungus. When the stationary phase of growth is reached, penicillin production begins. The apparent rate of antibiotic production is the net result of synthesis and inactivation during fermentation (Demain, 1957a). The antibiotic appears to be elaborated into the medium as soon as it is formed (Demain, 1957b), so assay of culture filtrates is a valid measure of net synthesis. In commercial production the medium of choice is a complex one, usually composed of corn steep liquor, lactose, side-chain precursor, surface-active agent, and mineral salts. During the growth phase, the fungus utilizes the nitrogen compounds and lactic acid of the corn steep for growth. When the available carbon compounds of corn steep near depletion, the fungus ceases to grow and begins its phase of penicillin formation a t the expense of the slowly utilized lactose (Koffler et al., 1945). Although there have been many attempts to isolate a specific “stimulating factor” in corn-steep liquor, they have all failed. It is generally accepted today that the value of corn steep lies in its content of a balance of materials generally favorable for penicillin synthesis (Liggett and Koffler, 1948). The traditional use of lactose as carbon source for penicillin production came about as a result of its slow breakdown. More rapidly assimilable sugars, such as glucose, were considered inferior until it was discovered that such carbohydrates, added continuously so as to maintain a low level in the medium, would support effective penicillin production (Davey and Johnson, 1953). The role of surface-active agents in stimulation of penicillin synthesis still remains to be explained (Goldschmidt and Koffler, 1950). For a more detailed summary of the effect of environmental conditions on the production of penicillin, the reader is referred to the review of Arnstein and Grant (1956). Although Fleming’s original culture produced only traces of penicillin, strains in use today synthesize several grams per liter of broth. This remarkable increase has been brought about chiefly by intensive mutation

THE MECHANISM OF PENICILLIN BIOSYNTHESIS

25

programs. Summaries of this work can be found in the papers of Johnson (1952), Backus and Stauffer (1955), Farrell(1953), and Arnstein and Grant (1956). For assay of total penicillins in broth, the most common method is the cup-plate microbiological technique (Schmidt and Moyer, 1944; Foster and Woodruff, 1943). Other widely used methods include the hydroxylamine (Boxer and Everett, 1949) and iodometric (Alicino, 1946) techniques. An isotope dilution method has also been devised for total penicillins (Gordon et al., 1954). Assays of particular types of penicillin have been carried out by isotope dilution (Craig et al., 1951; Anonymous, 1951) and by chromatography (Higuchi and Peterson, 1949; Glister and Grainger, 1950).

II. Precursors of the Side Chain Research on the biosynthesis of the side chain of various penicillin types is a well documented story and will not be considered in detail here. The reader is referred to the reviews of Johnson (1952) and Arnstein and Grant (1956) for material greater in detail than the summary below. Moyer and Coghill (1947) f i s t reported that phenylacetic acid, a degradation product of benzylpenicillin, stimulated penicillin synthesis. Independently, Smith and Bide (1948) showed that phenyl acetate and its derivatives were capable of both stimulation of penicillin production and induction of benaylpenicillin formation a t the expense of other penicillins. Thus, this work demonstrated that a limiting factor in biosynthesis was the synthesis of the side-chain precursor and that one could control the type of penicillin produced by varying the precursor added to the medium. Since these early studies, a large number of compounds have been shown to be effective precursors of the phenylacetyl side chain of penicillin G (Behrens et al., 1948a;Singh and Johnson, 1948; Tabenkin et al., 1952;Perlman and O’Brien, 1954). Similarly, other acyl compounds have been shown to induce formation of other penicillins, both natural and new types (Behrens et al., 1948b, c; Corse et al., 1948; Jones et al., 1948; Soper et al., 1948; Thorn and Johnson, 1950;Mortimer and Johnson, 1952).From these studies one may conclude that side-chain synthesis is relatively nonspecific and that many acids can be used. The effectiveness of the side-chain precursor depends on its toxicity and its resistance to oxidation by the fungus. Many effective compounds are those which are slowly hydrolyzed to active but relatively toxic or to rapidly oxidized precursors. This maintenance of a low but steady level of the active precursor allows the fungus to convert a large part of it to penicillin. Direct proof of the incorporation of phenyl acetate into penicillin was provided by Behrens et d.(1948d) who showed that 92.5% of the penicillin formed in the presence of deuterophenylacetylvaline contained the side

26

ARNOLD L. DEMAIN

group derived from the isotope. Similar results have been obtained using C13-phenylacetamide(Craig et al., 1951) and C1*-phenylacetate (Gordon et al., 1953; Sebek, 1953; Halliday and Arnstein, 1956).

111. Precursors of the 0-Lactam-Thiazolidine Ring Nucleus Synthesis of the double-ring nucleus common to all penicillins will be the major subject of the remainder of this review. Investigations into this problem have shown it to be much more involved than the simple precursor situation encountered in the above-mentioned studies on the side chain. Because of this, description of the methods of study used by various investigators is warranted and will be given in the next few paragraphs. Further discussion can be found in several recent reviews (Arnstein and Grant, 1956; Hockenhull, 1956; Arnstein, 1956; Ganapathi, 1957). A. METHODS OF STUDY 1. Growing Cultures

The early work on the biosynthesis of the ring nucleus of penicillin involved the use of complete fermentations in complex media. These media were generally the same as those used for commercial production of penicillin which have been briefly described above. To simplify the studies on the mechanism of biosynthesis, several synthetic media have been used. The most popular of these was devised by Jarvis and Johnson (1947). Its main features include the use of glucose for rapid mycelial growth during phase 1, lactose for the penicillin production phase, and ammonium lactate and ammonium acetate to supply both nitrogen and a means of pH control. Also present are mineral salts and sidechain precursor. Both complex and synthetic media have been used to search for precursors and intermediates of penicillin biosynthesis. Generally, three techniques have been used in this endeavor: a. Stimulation Test. In this method, a compound is added to the fermentation medium and a stimulation in the amount of penicillin produced indicates its importance in biosynthesis. b, Isotope Incorporation Test. The additive in this case is a labeled compound. The penicillin fraction is then isolated and its radioactivity is determined. Radioactivity measurements can be carried out on the penicillin extract, the crystallized penicillin, or on specific fragments of the penicillin molecule after controlled degradation (Tome et al., 1953). c. Isotope Dilution Test. In this test a labeled compound, which is known to be incorporated into penicillin, is included in the medium. In addition, another compound containing no radioactivity is added. If the latter com-

THE MECHANISM OF PENICILLIN BIOSYNTHESIS

27

pound is a precursor or an intermediate, it should lower the radioactivity of the penicillin produced. All of the above methods suffer from certain limitations, the main one being the permeability of the mycelium to the added compound. In the isotope incorporation test, this can be circumvented by searching for the radioactivity in various fractions of the mycelium. If it is found, it can be assumed that the compound entered the cell. With the other two methods, however, it is difficult to prove that the compound in question gained entry. Stimulation tests are further limited by the fact that the compound being tested will only stimulate if it is involved in the rate-limiting step. Thus, it is clear, first, that with the stimulation and isotope dilution tests, only positive results should be accepted in the absence of permeability data; and second, that more reliable information can be obtained by isotope incorporation. 2. Washed Suspensions

A further simplification of the penicillin biosynthetic system has been provided by the use of washed “resting” mycelial suspensions instead of growing cultures. Rolinson (1954) harvested cells from a complex medium, washed them, and suspended them for a short time in a complex medium. The mycelium was able to produce penicillin at a uniform rate for a t least 3 hours. Halliday and Arnstein (1956) further developed the technique of Rolinson and noted that the washed mycelium required only oxygen and phenyl acetate for synthesis of penicillin. Independently, Demain (1Y56a) developed a resting cell system employing cells grown in a modified Jarvis and Johnson medium and harvested after growth cessation and during the phase of rapid pencillin production. If the cells were then washed and suspended in a phosphate buffer-phenyl acetate-mineral salts medium, the initial rate of penicillin production was almost as great in the absence of energy source (lactose) as in its presence, although the former rate did taper off sooner (Demain, 1955). To eliminate this high rate of endogenous synthesis, the washed cells were first starved for 24 hours in a phosphate buffer-minera1 salts-ammonium phosphate medium, then rewashed and resuspended in the production medium. Now the rate in the presence of lactose was considerably greater than the endogenous rate. Demain’s system thus involved growth in a synthetic medium, washing, starvation, washing, and resuspension in a synthetic medium. Generally, these cells showed a lag before the maximum rate was established, so net rates of penicillin synthesis were computed from the straight line portion of a curve drawn from assays done at %hour intervals for 4 4 4 8 hours. Thus, production was observed here for a longer time than in the short-time assay of Rolinson. Demain (1956a) further simplified his system by substituting low concentrations of cystine and

28

ARNOLD L. DEMAIN

valine for the highcr levels of lactose previously used. Deshpande and Ganapathi (1957) have also described experiments with washed suspensions in a system similar to Rolinson's with the exception that longer incubation times were used. The techniques used in conjunction with resting mycelium to detect precursors of penicillin include the three types of tests described above for growing cultures. I n addition, the technique of inhibition analysis has been used. This involves the addition of a compound to the production medium to determine whether it can inhibit the net rate of penicillin synthesis. If it does, other compounds are added along with the inhibitor in the hopes of reversing the inhibition. The successful reversal of the inhibitor's action indicates that the reversing compound or a derivative may be involved in penicillin synthesis. Inhibition analysis is also limited by permeability effects, and only positive results are significant.

B. INCORPORATION OF L-CYSTEINE The similarity in structure of cysteine with a portion of the ring nucleus of penicillin can be seen on page 24. With this in mind, stimulation tests with cysteine and cystine were performed with growing cultures in synthetic and complex media, but no increases were noted (Behrens, 1949; Hockenhull, 1948). It should be pointed out here that the inactivation of penicillin by cysteine (Wintersteiner et al., 1949) has limited its use to cases where low concentrations can be employed. Most of the biosynthetic studies to be discussed have been carried out with cystine, and it has thus been assumed that the fungus can convert it to the reduced form. Since stimulation tests gave no positive results, isotopic studies were initiated. Stevens et al. (1953) found that L-cysteine and L-cystine were able to dilute out the radioactivity of the penicillin formed by cultures growing in st synthetic medium to which P6-labeled inorganic sulfate had been added. The dilution obtained was much greater than that observed when an equivalent amount of unlabeled Na2S04was added, demonstrating a preferential use of the sulfur of cysteine. The stereochemical specificity of the reaction was indicated by the lack of dilution effects when u-cysteine was used, but the possibility of permeability effects was not eliminated. Arnstein and Grant (1954a) , however, demonstrated that incubation of growing cultures (in a synthetic medium to which yeast extract was added) with D-(P-C'4)cystine resulted in the production of labeled mycelium and labeled respiratory carbon dioxide, indicating that the u-isomer can enter the mycelium. They also noted that ~-(P-C14)cystinewas a much better precursor of penicillin than was the D-form. Addition of ~ ~ - ( @ - C l ~ ) c y sttoi n e the medium resulted in the formation of C-5-labeled penicillin. Further

29

THE MECHANISM O F PENICILLIN BIOSYNTHESIS

work by Arnstein and Grant (1954b) showed that ~-(p-C~~,N~~,S*~)cystine gives rise to penicillin which is labeled in a manner which would be expected from intact incorporation of the amino acid. This was further proven by the fact that the ratios of C14,N16, and S36 isotopes in the penicillin was simi-

I * '.1 1 HzN-CH-*CHz

/

LOOH

* * + C~H~-CH~-COO--NH-CEI-CH

I

CO-N-

Z]

r,-Cystine

*/"\ C(CHJ2

I

I

CH-COOH

Benzylpenicillin

lar to that of the added radioactive L-cystine. Arnstein and Crawhall (1957) next showed that both washed mycelium and growing cultures convert D L - ( ~ Hcystine ~ ) to C-6-labeled penicillin. In addition to the above studies, Stevens et al. (1956) showed that Sa6-~-cystine is immediately and linearly

i 1

* H zN-C H-C LOOH DL-Cystine

/"l Hz

Z]

+ CaH6-C H 2-C

0-NH-CH-C

*

I

CO-N-

/"\ C (CH I / H

3)

z

CH-COOH

Ben2ylpenicillin

incorporated into penicillin when added to a culture growing in a complex medium (see Fig. 1). The conclusion that cystine is a precursor of the penicillin molecule, obtained from these isotopic studies, was strengthened by the nonisotopic studies of Demain (1956a). Using starved resting cells, it was found that L-cystine stimulated the net rate of penicillin synthesis in a synthetic medium. Furthermore, 8-ethyl-DL-cysteine caused an inhibition of penicillin synthesis which could be reversed by L-cystine (see Table I). Recently, Arnstein and Margreiter (1958) and Grau and Halliday (1958) have obtained data confirming stimulation of resting cell synthesis by cystine and, in addition, the former workers found DL-cysteic acid capable of stimulation. Previously, Stevens et al. (1954a) had shown by the isotopic dilution technique that cysteic acid was utilized about equally in competitition with sulfate but less effectively than cystine. Arnstein and Margreiter (1958) have suggested that cysteic acid might be converted into cystine and thus show stimulating effects. These workers also demonstrated inhibition of penicillin formation by a-methyl-DL-cystineand p ,p-dimethy1-L- and D-cystine (L- and D-penicillamine disulfide), but no attempts to reverse these inhibitions were reported.

30

ARNOLD L. DEMAIN

TIME CHRS.)

FIG.1. Rate of formation of Sa6-penicillinfrom Ss6-labeledL-cystine (Stevens e l al., 1956). Labeled cystine was added after 60 hours. 0 ,determination of radioactivity in amyl acetate extract; 0 , determination of radioactivity by isolation of crystalline penicillin and conversion to benzidine sulfate before counting.

TABLE I INHIBITION OF PENICILLIN SYNTHESIS BY S-ETHYL-DL-CYSTEINE AND REVERSAL B Y L-CYSTINEO

I Experiment

No. 1 No. 2

Molar concentration of L-cystine

0 6 X 10-3

0 2.5 X 10-8

10-2 a

From Demain (1956a).

Net rate of penicillin synthesis

No S-ethylS-ethyl-DL~ ~ - c y s t e i n e cysteine a t

I (units/ml./hr.)

10-2

1 Inhibition

(%)

M

(units/ml./hr.)

6.7 10.6

4.6 9.3

31 11

9.2

4.9 5.2

47 49

11.5

1

10.1 11.6

31

THE MECHANISM O F PENICILLIN BIOSYNTHESIS AND SERINE C. ROLE OF PENICILLAMINE

The isolation of penicillamine from the hydrolytic products of penicillin (Peck and Folkers, 1949) led to the speculation that it might be involved in penicillin biosynthesis via condensation with serine. This postulated reaction would resemble the previously described condensation of serine with SH H~N-CH-CHZOH

I

COOH

\

C (CHd a

+ HzN-CH-CI

Serine

HzN-CH-CHz 3

0 OH

/s\

C(CHa)Z

I I COOH HzN-CH-COOH

p, p-Dimethyl-lanthionine

Penicillamine

S-CH2

/ I

SH-CHz HzN-CH-CHzOH

I

COOH Serine

+

I

H ZN-CH-C

CHz

I

HzN-CH-C

3

OOH

Homocysteine

H2

I

CHz

I

COOH HeN-CH-COOH Cyst a thi onine

homocysteine to produce cystathionine (Binkley, 1951) and would require serine to be a more immediate precursor of penicillin than cystine. After negative results were reported by Behrens (1949) for penicillamine and serine in stimulation tests, Stevens et al. (1953) tested the former by theisotope dilution method against S36-labeledNazSOl. Again, a negative result was obtained. Arnstein and Grant (1954a) next studied the incorporation of ~~-(p-C'~)serine into penicillin. They found that, although serine was incorporated, its specific radioactivity was diluted to a greater extent than that of ~~-(p-Cl~)cystine during biosynthesis. This indicated that serine incorporation occurred via cysteine rather than the reverse case. Further studies by these investigators (Arnstein and Grant, 1954b) demonstrated that, when labeled cystine was added to the fermentation, the specific radioactivity of mycelial serine was only a small per cent of that found in the mycelial cystine or penicillin. Similar results were obtained by Arnstein and Crawhall (1957) using DL-(a-Ha)cystine.Thus, it is evident that the postulated condensation of serine and penicillamine does not occur. Incorporation of serine into penicillin probably proceeds via cysteine in reactions similar to those observed by Binkley and du Vigneaud (1942) with rat liver.

D. INCORPORATION OF L-VALINE If we again refer to the penicillin molecule shown on page 24, it is evident that the part remaining to be discussed resembles valine (in the Dconfiguration). As in the case of cysteine, early attempts to stimulate peni-

32

ARNOLD L. DEMAIN

cillin synthesis by addition of DL-valine to growing cultures in complex media failed (Behrens, 1949). Arnstein and Grant (1954a), however, succeeded in incorporating D L - ( ~,~'-C'~)valine into penicillin and observed that the radioactivity was confined to that part of the molecule containing C-2 and C-3 and the accompanying carboxyl and methyl groups. The exact site of the C14was not determined. Stevens et al. (1954b) next showed S

/ AI

* CsH,-CHz-CO-NH-CH-CH

CH(CHs)z

I

--+

H2N-CH-COOH

I

I

C _ e - _ _ _ _ _ _

C(CHd2

I 1 CO-N-IrCH-COOH --

I

I I

_ _ - L _ _ _ - -

m-Valine

I

'

1

C14area

Benzylpenicillin

that addition of carboxy-labeled DL-valine resulted in the production of penicillin with C14exclusively in the carboxyl group attached to C-3. Finally,

CH(CHs)z

I

*

HZN-CH-COOH oL-Valine

C sH5-CHz-C

-+

/"\

0-NH-CH-CH

I

C (CHa)2

1

I

CO-N-CH-COOH Benzylpenicillin

*

data by Arnstein and Clubb (1957) using uniformly labeled C14-valine offered convincing proof that the intact carbon skeleton of valine was incorporated into penicillin. Nonisotopic evidence for the role of valine in penicillin biosynthesis was obtained by Demain (1956a). Starved resting cells were shown to be inhibited by the addition of a-methyl-DL-vahe to the lactose-phenyl acetate-minera1 salts medium. The inhibition was reversed by m-valine. Arnstein and Margreiter (1958) confirmed the inhibition by a-methyl-DL-valine and also showed that the related amino acid, m-isoleucine, was an inhibitor. Once it was established that valine carbon entered the penicillin molecule, the emphasis shifted to the amino group of valine. Although the valine moiety of penicillin was known to be of the D-configuration, evidence rapidly accumulated from three laboratories which indicated that L-valine was a more efficient precursor than D-valine. Arnstein and Clubb (1957) showed that washed mycelial suspensions synthesized C14-penicillinof a greater specific activity in the presence of ~-(l-C'~)valine than with D-( 1-C'")valine. Although they attributed this to a slower entry of the D-form into the mycelium, a later study by the same group (Arnstein and Margreiter, 1958) showed that this form is a poor precursor even in strains that are relatively more permeable to D-valine. Stevens et al. (1956) reported that, while CI4labeled L-valine was rapidly incorporated into penicillin, incorporation of

33

THE MECHANISM OF PENICILLIN BIOSYNTHESIS

TABLE II COMPARISON OF THE EFFECTIVENESS OF VALINEIBOMERS FOR PENICILLIN SYNTHESIS I N A N L-CYSTINE-VALINE-PHENYLACETATE-SALTS MEDIUMO Experiment

Valine isomer

No.1

DL L

Molar concentration

6X

L

No. 2

a

L

2

x

DL

2

x

Net rate of penicillin synthesis (units/ml. /hr .)

10-2

7.3

10-8

7.9 9.6 11.2

10-2 10-2

L

10-2 10-2

D

10-2

6.5 8.4 3.9

From Demain (1956a).

the D-isomer showed a lag period. Since Penicillium chrysogenum has the ability to convert added D-valine tomycelial L-valine (Stevens and De Long, 1958), it is probable that even the penicillin produced in the presence of D-valine arises via the L-form. Demain (1956a) showed that addition of DLvaline to the synthetic medium used for starved resting cells resulted in a decrease in the rate of penicillin synthesis. The inhibition was later confirmed by Arnstein and Margreiter (1958) and Grau and Halliday (1958). Demain also found that the inhibition was due to the D-isomer alone. Addition of L-valine to the lactose-phenyl acetate-salts medium produced no inhibition or stimulation. The lack of stimulation was evidently due to endogenous production of L-valine from the lactose since the stimulating nature of the L-form was quite evident when L-cystine plus valine was substituted for lactose in the resting cell system (Table 11). Although the above data tell us that the carbon skeleton of valine is used intact and that L-valine is a better precursor of penicillin carbon than D-valine, it does not give us any information as to whether the nitrogen atom of valine becomes the nitrogen common to the thiazolidine and /3lactam rings in penicillin. The first data on this point were supplied by Behto growing rens et al. (1948d) who added deuterophenylacetyl-NIK-DL-valine cells in a complex medium. These workers showed that, while 92.5 % of the penicillin formed arose from the phenylacetyl moiety, only 2.69 % was derived from the labeled valine moiety. More definitive studies were conducted by Arnstein and Clubb (1957). Upon addition of DL-(LU-C'~, N16)valine to cultures growing in Jarvis and Johnson medium supplemented with yeast extract, both penicillin and mycelial valine were produced with approximately a 10-fold dilution of G I 4 specific activity and a 50-fold dilution of NIK.

34

ARNOLD L. DEMAIN

Thus, both studies indicated that a large part of the DL-valine nitrogen was lost during biosynthesis. Arnstein and Clubb gave further proof of this loss by showing that the side chain nitrogen of penicillin generally contained about as much isotope as the ring nitrogen (N-4) and, furthermore, that other mycelial amino acids contained from 56 to 84 % as much N16 as did the mycelial valine. This loss of valine nitrogen is not surprising in view of the fact that P. chr3sagenumhas been shown to contain D- and L-valine oxidases (Knight, 1948; Emerson et al., 1950) and probably possesses transaminase(s) capable of acting upon valine. The poor incorporation of N”%L-valine observed by Behrens et al. could be explained on the basis of dilution by unlabeled valine in the corn-steep liquor, dilution by unlabeled endogenous valine produced during the fermentation, and high valine oxidase activity which is associated with cells grown on complex media (Knight, 1948; Emerson et al., 1950). The data of Arnstein and Clubb, however, cannot be explained on the basis of dilution by unlabeled valine since both C14 and N16would be diluted equally. However, the supplementation of the synthetic medium with yeast extract may have induced formation of high levels of valine oxidases leading to extensive deamination of DL-valine and poor “6 incorporation relative to that of 0 4 . Furthermore, the long-term fermentations employed by these workers may also have contributed to large-scale deamination. On the other hand, Stevens and De Long (1958) recently reported on experiments carried out with growing cultures in synthetic Jarvis and Johnson medium which would be expected to produce cells with a low degree of valine oxidase activity (Knight, 1948; Emerson el al., 1950). The study was conducted by adding ~ - ( l - C,N16)valine l~ to fermentations a t 72 hoursof age and isolating the mycelial valine and the valine moiety of penicillin a t 75 and 78 hours. The results obtained in these short-term experiments showed that N16 retention in both mycelial and penicillin valine was quite high, amounting to about 50 % of that observed with C1*.It thus appears that the amino group of L-valine is retained during biosynthesis despite the fact that inversion to the D-configuration must occur before the penicillin molecule is completely synthesized. These valine studies indicate that inversion of the amino group from the L- to the D-COnfiguratiOn occurs after condensation of valine with cysteine or a derivative thereof. Some further evidence on this point has been supplied by Stevens and De Long (1958). ~-(l-C14)Valinewas added to two fermentations a t GO hours. To one of the fermentations was also added an equimolar amount of unlabeled ketovaline (a-ketoisovaleric acid) and a t 83, 86, and 72 hours, the penicillin was analyzed for content. It was found that the ketovaline diluted the activity by only 31 to 41 % showing that the keto acid was not used preferentially. The fact that dilution OC-

35

THE MECHANISM OF PENICILLIN BIOSYNTHESIS

curred at all indicated that ketovaline was able to penetrate the mycelium. Since ketovaline is a known precursor of valine in other fungi (Abelson and Vogel, 1955), the dilution was probably caused by conversion of the keto acid to nonlabeled valine before incorporation into penicillin. Of course, it is possible that the poor dilution is a result of slower penetration of the keto compound into the cell relative to valine; however, this would be the less likely possibility. It thus appears from the limited amount of evidence on hand that the inversion on C-3 of L-valine to the D-form occurs after condensation of the amino acid into a larger structure.

E. ROLEOF P-HYDROXYVALINE On theoretical grounds, Hockenhull et al. (1949) suggested that penicillin might be synthesized from P-hydroxyvaline and cysteine (plus side-chain precursor) in a manner similar to the condensation of cysteine and homoserine to form cystathione in Neurospora (Horowitz, 1947). This mechanism

HaN-CH-CHz

/SH

I

+

COOH Cysteine

HOC(CHJ2

I HnN-CH-COOH

+

8-Hydroxyvaline S

HzN-C

I

H-CHa

/\

C (CHa)2

I

COOH HzN-CH-COOH 8,,%Dimethyl-lan thionine

HzN-CH-CH~

I COOH

Cysteine

/

SH

HOCHz

+

I

CHa

-4

I

HaN-CH-COOH Homoserine S-CH, HzN-CH-CHz

I

/

I

CHz

I

C 0 OH HzN-CH-C Cystathionine

0 OH

attracted quite a bit of attention for the next few years and prompted studies on P-hydroxyvaline. Stevens and Halpern (1949) reported that hydroxyvaline was not present in the natural products which they examined which included mycelium and culture filtrates of P. chrysogenum. Further-

36

ARNOLD L. DEMAIN

more, Stevens and De Long (1958) found hydroxyvaline incapable of competing with valine in isotopic competition experimknts. Demain (1956b), on the other hand, found it to stimulate penicillin production by washed, starved mycelial suspensions in his lactose-phenyl acetate-salts medium. However, when he substituted p-hydroxyvaline for valine in his amino acid suspension medium, the hydroxy acid was no better than valine. These puzzling results can now be explained on the basis of the results of Arnstein and Clubb (1957). These workers showed that ~~-P-hydroxy(carboxy-C~~) valine underwent a much greater isotopic dilution during incorporation into penicillin than that occurring with valine. Furthermore, most of the radioactivity was incorporated into C-7 of penicillin indicating that the hydroxy acid was serving not as a source of the valine moiety but as a source of cysteine. Since the radioactivity of the penicillin was of the same order as mycelial glycine, it indicated that p-hydroxyvaline was first converted to glycine, which could then serve as a precursor of the cysteine moiety of penicillin (see next Section). Thus, in Demain’s experiments, hydroxyvaline was evidently supplying cysteine which would stimulate in the lactose suspension medium but not in the amino acid system where sufficient cystine was present in the medium. It therefore is clear that penicillin synthesis does not proceed via a condensation involving p-hydroxyvaline. F. TESTSWITH OTHERCOMPOUNDS Several other compounds of biochemical interest have been shown to contribute to the biosynthesis of penicillin. In the following few paragraphs, experiments concerning these precursors will be discussed briefly along with the probable pathways by which they enter the penicillin molecule. 1. Cystathionine

Stevens et al. (1954a) found L-cystathionine to compete equally with labeled L-cystine. Its activity is probably due to its function as a precursor of cysteine, a pathway previously demonstrated in rat liver (Binkley, 1944). 2. Hornocysteine Preferential me of DL-homocysteinein the presence of labeled sulfate has also been demonstrated (Stevens el al., 1954a). Since studies with rat liver (Binkley, 1951) have shown that serine (see Section C) condenses with homocysteine to form cystathionine, it is probable that homocysteine also functions as a cysteine precursor. 3. Methionine Competitive utilization tests with DL-methionine have shown this amino acid to be active in supplying penicillin sulfur (Stevens et al., 1953). In ani-

THE MECHANISM O F PENICILLIN BIOSYNTHESIS

37

ma1 tissues, it is known that methionine undergoes demethylation to homocysteine (du Vigneaud, 1942). 4. Glycine Arnstein and Grant (1954a) demonstrated that (a-Cl4)glycine is converted into penicillin with the radioactivity contained in C-6. Apparently, glycine is converted into serine, which in turn serves as a cysteine precursor. Conversion of glycine to serine has been shown in rat liver (Winnick et al., 1948) and in yeast (Ehrensvard et al., 1947). Furthermore, Arnstein and Grant (1954a) isolated labeled serine from the mycelium when labeled glycine was added to the fermentation. 5. Formate The incorporation of (C14)formate into penicillin was demonstrated by Martin et al. (1953). Its activity apparently lies in its ability to participate with glycine in the formation of serine. Such a reaction has been shown to occur in Leuconostoc mesenteroides and Saccharomyces cerevisiae (Lascelles and Woods, 1950). 6. Glutathione Glutathione was found to be as active in isotopic dilution tests as L-cystine for penicillin biosynthesis (Stevens et al., 1953). Incorporation of the tripeptide probably occurs after hydrolysis to free cysteine.

7. Acetate Several investigators (Stone and Farrell, 1946; Calam and Hockenhull, 1949) have noted that acetate was beneficial for penicillin production when added to synthetic media. This prompted Martin et al. (1953) to study the utilization of acetate-1-04 and acetate-2-U4 for penicillin synthesis by growing cultures in synthetic medium. They found radioactivity from both types to enter the penicillin molecule, and by the isotopic dilution technique they noted that considerable amounts of acetate were formed from sugar and subsequently used by the mold. Independently, Hockenhull et al. (1954) also noted the production of acetate during the penicillin fermentation. Tome et al. (1953) attempted to localize the site of carbon incorporation from acetate. It was concluded that one molecule of acetate enters the valine moiety while another becomes the carbons of the p-lactam ring. In the latter case, the acetate carboxyl becomes C-7 of penicillin while C-2 of acetate is incorporated into the C-5 to C-6 region, the exact position not being determined. Stevens and De Long (1958) further demonstrated the incorporation of radioactivity from acetate-l-C14 into the carboxyl group of the d i n e moiety of penicillin. This incorporation was reduced when unlabeled

38

ARNOLD L. DEMAIN

L-valine was added, indicating that acetate is first converted to carboxyllabeled L-valine before being converted to penicillin. It was also shown that most of the radioactivity of the mycelial valine occurred in the carboxyl group. Thus, in Penicillium chrysogenum, acetate C-1 is a precursor of the valine carboxyl as in other microorganisms (McManus, 1954; Strassman et al., 1953; Ehrensvard et al., 1951). No data have been obtained yet on the exact site of incorporation of acetate C-2 into valine as carried out by Penicillium. In yeasts, radioactivity from C-2 of acetate is distributed between all the carbon atoms of valine except the carboxyl group (McManus, 1954; Strassman et aE., 1953). This suggests that, in yeasts, acetate first is converted to pyruvate via the citric acid cycle before being converted to valine, as suggested by Strassman et al. (1953). No further work has been done to explain the incorporation of acetate C-1 and C-2 into penicillin C-7 and C-5 to C-6, respectively. However, it has been found that acetate-grown P. chrysogenum contains enzymes which carry out the reaction sequence, citrate cis-aconitate d-isocitrate glyoxylate -+ glycine (Olson, 1954). Thus, in addition to the conventional citric acid cycle (Casida and Knight, 1954; Godzeski and Stone, 1955; GoldSchmidt et al., 1956), P. chrysogenum apparently can carry out the recently discovered glyoxylate cycle (Kornberg and Krebs, 1957), supplying glyoxylate which can then be converted to glycine, presumably by transamination (Tolbert and Cohan, 1953; Cammarata and Cohen, 1950; Wright, 1951). The remaining steps between glycine and cysteine have already been discussed.

IV. Condensation of the Precursor Moieties A. PHENYLACETATE AND THE INITIAL CONDENSATION REACTION Arnstein et al. (1954) considered the possibility that the initial reaction in penicillin formation was a condensation of phenyl acetate and cysteine. Upon adding (carboxy-C14)diphenylacetyl-L-(Sa6)cystine to a growing culturein the presence of unlabeled phenyl acetate, they found unequal dilution of CI4and SS6 in the isolated penicillin, indicating that there was no intact incorporation of the added compound. However, the similarity in 0 4 : S a 6 ratio between the diphenylacetylcystine isolated after the fermentation and the penicillin produced indicated that the diphenylacetylcystine had been extensively degraded and resynthesized, bringing forth the possibility that it could have been a precursor after resynthesis. Since the above experiment failed to provide information as to the importance of phenylacetylcysteine, another approach was tried. A fermentation was carried out with (carboxy-CI4)phenylacetate and, at various times, broth was removed, added to unlabeled diphenylacetylcystine, filtered, and

THE MECHANISM OF PENICILLIN BIOSYNTHESIS

39

diphenylacetylcystine was isolated. Since the isolated compound contained no radioactivity, it was concluded that the mold produced no detectable diphenylacetylcystine and that phenylacetylcysteine was not a likely intermediate. The authors conceded that, if the rate of formation of phenylacetylcysteine was slower than its rate of utilization, it might not be detected by their methods; however, other possibilities exist which further decrease the significance of the results. First, it is possible that, if labeled phenylacetylcysteine or diphenylacetylcystine were produced, their presence may have been restricted to the inside of the cell and thus would not equilibrate with the exogenous carrier. Secondly, even if radioactive phenylacetylcysteine were produced and liberated from the mycelium, are we sure that it would equilibrate with diphenylacetylcystine? Thus, the above experiments leave much to be desired concerning the role of phenyl acetate in the initial condensation reaction. Nevertheless, other data do exist which indicate that the side chain is incorporated only during the final stage of biosynthesis. Several years ago, Kato (1953a, b) reported on the accumulation in penicillin fermentation broths of a material which was formed only when the side chain precursor was omitted from the medium. What made this compound interesting was that, despite its lack of antibacterial action on penicillin sensitive microorganisms, it was a substrate for penicillinase, an inducer of Bacillus cereus penicillinase and gave a positive reaction in the hydroxylamine assay for penicillin (Boxer and Everett, 1949). Its lack of antibiotic activity differentiates it from cephalosporin N produced by Cephalosporiumspecies and other hydrophilic penicillins which are produced by Penin‘llium chrysogenum in the absence of side-chain precursor (Hale el al., 1953). Kato assayed this material by a differential iodometric assay involving butyl acetate extraction since the accumulated compound, unlike penicillin, was not extracted by the solvent at low pH. He has suggested that the compound is a precursor of penicillin and may represent the penicillin molecule without its side chain. The production of Kato’s compound has been confirmed recently by Demain (1955) who compared the increase in units of hydroxylamine reacting activity with bioactivity in fermentations run in a corn-steep medium. Table I11 shows that while addition of the side-chain precursors phenyl acetate or phenylacetylethanolamine yields only penicillin (hydroxy1amine:bioactivity ratio = l), the omission of the precursor results in broth possessing higher hydroxylamine activity than microbiological activity. Figure 2 shows the course of production of Kato’s material, the values being obtained by subtracting the biological values from the hydroxylamine units. When broth filtrate with a hydroxylamine: bioactivity ratio of 3: 1 was extracted with butyl acetate a t pH 2, the residue showed a ratio of 71.7, again confirming Kato’s results. Such material was tested by Demain

40

ARNOLD L. DEMAIN

TABLE I11 EFFECTOF ADDITION OF SIDECHAINPRECURSOR ON PRODUCTION O F KATO’SCOMPOUND^ ~

Phenylacetylethanolamineb

No precursor

Hours

H (units/ml.)

(ratio)

1

Phcnyl

1

B

/H/B

H

B

(units/ml.)

(ratio)

(units/ml.)

iH/B (ratio)

~

48 72 96 120 141

360 480 840 1050 1360

2.3 3.0 4.2 4.8 5.0

160 160 200 220 270

450 950 1460 2120 2680

1.2 1.1 1.0 1.1 1.0

380 860 1430 2000 2560

570 960 1800 2530 3140

510 890 1630 2660 3130

-

1.1 1.1 1.1 1 .o 1 .o

From Demain (1955). b Phenylacetylethanolaminewas added at a concentration of 0.25% (w./v.) at the start. Phenyl acetate was added at 0.1% (w./v.) daily after 24 hours. H, hydroxylamine assay; R , biological assay. a

c 4 t

a

1200

I

I

I

W

5 -

C

.

-

~1000

k

z

0 m

800

-

t

z

4

,

0

20

40

60

80

100

I20

140

TIME (HRS.1

FIQ.2. Formation of Kato’s compound in the absence of added side chain precursor (Uemain, 1955).

THE MECHANISM O F PENICILLIN BIOSYNTHESIS

41

for stimulation of penicillin formation by washed resting cells but it failed to show any activity. However, this may have been due to an inability to enter the mycelium. Although no further work has been reported on the isolation or characterization of this interesting compound, the above data strongly suggest that it is a very late intermediate and that the initial steps of biosynthesis do not involve the side chain precursor.

B. CONDENSATION OF CYSTEINE AND VALINE Although most investigators feel that the initial condensation occurs between cysteine and valine, no evidence has been brought forward as to the mechanism involved. The only reported data are negative in nature and will be described briefly in the following paragraphs. It was shown above that a Condensation of fi-hydroxyvaline and cysteine does not occur; however, it is possible that the product of such a reaction, p ,p-dimethyl-lanthionine might be produced via some other route. Stevens et al. (1954a) prepared mixed isomers of this compound as well as the form containing the L-configuration in the cysteine moiety and the D-configuration in the valine part. Both preparations, however, failed to compete with labeled L-cystine or sodium sulfate for penicillin production. Whether or not the compound penetrated the mycelium, however, is not known. Similar experiments were carried out recently by Arnstein and Clubb (1958) who tested dimethyl-lanthionine (I) as well as its acetyl (11) and phenylacetyl (111) derivatives in isotopic competition experiments against L-(carb~xy-C'~)valine. Again, no dilution of radioactivity was noted, and no information was obtained as to whether the compounds could enter the mycelium.

R-NH-CH--C

I; It

=

I1

/\ 1

Hz

C ( CH,) 2

I

COOH H2N-CH-COOH 11; R = CHs-CO 111; R = C6Ha-CH2-CO

Since no evidence has been obtained in favor of the initial linkage being one involving the @-carbonof valine and the sulfur of cysteine, we should now examine the data concerning formation of a peptide between these two amino acids, e.g. , cysteinylvaline. Again, the only available results are those based on isotopic competition experiments conducted by Arnstein and Clubb (1958). These workers synthesized a number of cyclic peptides based on the structure which would result from the intramolecular cycliza-

42

ARNOLD

L.

DEMAIN

tion of cysteinylvaline. However, none of these (IV, V, VI) competed with labeled valine although, again, we do not know whether they penetrated

R-NH-

CH-CH2

I

C(CHa)2

I

C 0-NH-CH-C V; R = CHs-CO VI; R

IV; R = H

OOH = CeHs-CH-CO

the mycelium. In the same experiments, phenylacetyl-L-cysteinyl-n-vahe also failed to compete. Thus, the limited amount of experimental work conducted to date has given us no firm data concerning the initial condensation reaction between cysteine and valine. However, before leaving this area, some further observations of Arnstein and Crawhall (1957) dealing with reactions subsequent to the initial condensation should be mentioned. These workers studied the incorporation of DL-((uH~)and ~ ~ - ( p H ~ ) c y s tinto i n e penicillin. They found that the a-hydrogen atom and one of the ,&hydrogen atoms were retained during biosynthesis, being incorporated into the C-5 to C-6 area, and that no migration of H3to the valine moiety of penicillin occurred. They were thus able to conclude that neither of the two mechanisms shown below could account for the formation of the link between C-5 and N-4.

7-

HzN-C H-C Ha

I

CO-NH-CH-

I

S-

2

~

H~N-CECH

/ -+

___f

CO-NH-CHS-

/ H2N-CH-CH

I

I

CO-N-CHS-

S-

/

/ HZN-C H-CHz

I

C 0-NH-CH(2)

I

I

-2H

H2N-C H-CH2

I

'

CO-N=C-

+

\ I S-

H2N-CH-CH

I

/

I I

CO-N-CH-

THE MECHANISM OF PENICILLIN BIOSYNTHESIS

43

Any proposed mechanism for the biosynthetic process will have to take these data into account.

V. Future Experimentation

It is clear that the future work on the mechanism of penicillin biosynthesis will be greatly enhanced by the discovery of the initial condensation reaction between cysteine and valine. However, the attempts to elucidate this reaction by isotopic competition experiments have yielded only negative data, which would be important only if it were known that the added compounds were capable of penetrating the mycelium. The permeability question could, of course, be easily answered if the additives themselves were labeled, but they are not. Since synthesis of every possible intermediate in labeled form would be a tremendous task, what other alternatives present themselves? The simplest approach would appear to be the use of cell-free enzyme preparations capable of carrying out the reactions of penicillin synthesis. If such preparations could be obtained, the value of isotopic competition would be increased manyfold. Although the penicillin-synthesizing ability of the mycelium appears to be quite sensitive to physical treatments such as grinding or freeze-drying (Halliday and Arnstein, 1956; Deshpande and Ganapathi, 1957), the rewards for obtaining an active preparation are so great that the problem requires a thorough and complete examination. In the absence of such cell-free preparations, what other avenues might be profitably followed? One that stands out is the study of the material found by Kato to accumulate in fermentations conducted without sidechain precursors. Identification of its chemical structure would be a valuable addition to our knowledge of the biosynthetic scheme. Another approach which might yield valuable data is that concerned with lysine inhibition of penicillin formation. Demain (1957~)observed that lysine at 5 X M could inhibit synthesis by starved resting cells in a cysteine-valine-phenyl acetate-salts system. In this same system, arginine was stimulatory but could not reverse the lysine effect, indicating that the two phenomena were not related. The lysine effect has been confirmed repeatedly and also occurs in corn-steep media (Somerson, 1957). Since lysine

HzN-C

Hz-C Hz- C H2-C Ha-C HNHa-C 00H

dimethylH~N-CH-CH~-S-C(CHa)zCHNHz-COOH 1anthionine I

COOH

lysine bears some structural relationship to dimethyl-lanthionine, elucidation of its mechanism of inhibition could possibly aid in efforts to determine the path of penicillin biosynthesis.

44

ARNOLD L. DEMAIN

VI. Summary The biosynthesis of benzylpenicillin occurs via the condensation of its precursors, phenylacetic acid, L-cysteine, and L-valine. The rate-limiting reaction is the synthesisof the side-chain precursor, and thus phenyl acetate is usually added to the fermentation medium. Acylation of the B-lactamthiazolidine ring appears to be one of the final steps in biosynthesis. A large number of compounds are capable of being incorporated into the cysteine moiety of penicillin. They apparently enter by the following pathways : acetate -+ citrate

-+

formate -+ glycine J-+

1 @-hydroxyvaline

cis-aconitate -+

d-isocitrate -+ glyoxylate

+

hoxnocysteixie + methionine serine

-

cystathionine + cysteine

'i

cysteic acid

glut athione

Added L-cystine (after conversion to L-cysteine) is used as the intact molecule for penicillin synthesis. The incorporation is stereospecific for the Lform which is the configuration present in penicillin. The remaining portion of the molecule is synthesized from t-valine, despite the fact that the valine moiety of penicillin is of the D-configuration. Results of several studies have indicated that the intact carbon skeleton of valine is used, but that the amino group is lost during biosynthesis. However, recent work done under more favorable conditions for amino group retention indicates that the entire valine molecule can enter penicillin. It is not known at which stage the inversion from the L- to the D-form takes place. Acetate is incorporated into the valine moiety of penicillin but its pathway has not yet been determined. Neither penicillamine nor p-hydroxyvaline can serve as precursors of this portion of the penicillin molecule. Very little is known about the mechanism of condensation of valine and cysteine. The two main possibilities are (a) the condensation of the sulfhydry1 group of cysteine and the p-carbon of valine to form dimethyl-lanthionine, and (b) the formation of the peptide, cysteinylvaline. Several possible intermediates related to both schemes have been tested by the isotopic competition technique with negative results. However, since permeability data are not available, neither of the above pathways can be eliminated from consideration. The only facts known about the biosynthesis of the ring system are that during the formation of the C-5 to N-4 bond a dehydrocysteine intermediate is not formed, and that the one hydrogen atom that is given up from C-5 does not enter the valine moiety of penicillin.

THE MECHANISM OF PENICILLIN BIOSYNTHESIS

45

ADDENDUM Kato’s compound has been isolated and identified as 6-aminopenicillanic acid, i.e., the penicillin nucleus minus its side chain (Batchelor et al., 1959). This excellent piece of work suggests strongly that the last step in penicillin synthesis is the acylation of the nucleus. Another recent development was the demonstration that there is a greater utilization of ~-cystinyl-~-(carboxy-C~~)valine than ~-(carboxy-C~~)valine for penicillin synthesis while the amino acid is better utilized than the peptide for protein synthesis (Arnstein and Morris, 1959). Since the calculations included corrections for permeability differences, the work provides the first piece of experimental evidence pointing to the peptide as the initial condensation product. REFERENCES Abelson, P. H., and Vogel, H. J. (1955). J. Biol. Chem. 213, 355-364. Alicino, J. F. (1946). Ind. Eng. Chem., Anal. Ed. 18, 619-620. Anonymous. (1951). Antibiotics & Chemotherapy 1,418. Amstein, H. R. V. (1956). Giorn. microbial. 2,268-284. Arnstein, H. R. V., and Clubb, M. E. (1957). Biochem. J. 66, 618-627. Arnstein, H. R. V., and Clubb, M. E. (1958). Biochem. J . 68,528.535. Arnstein, H. R. V., and Crawhall, J. C. (1957). Biochem. J. 67, 180-187. Arnstein, H. R. V., and Grant, P. T. (1954a). Biochem. J. 67, 353359, Arnstein, H. R. V., and Grant, P. T. (1954b). Biochem. J . 67, 360-368. Arnstein, H. R.V ., and Grant, P. T. (1956). BacterioZ. Revs. 20, 133-147. Amstein, H. R. V., and Margreiter, H. (1958). Biochem. J . 68, 339-348. Arnstein, H. R. V., and Morris, D. (1959). Biochem. J . 71, 8P. Amstein, H. R. V., Clubb, M., and Grant, P. T. (1954). Proc. Radioisotope Conf., 2nd Conf., Oxford, 1954. 306-312. Backus, M. P., and Stauffer, J. F. (1955). Mycologia 47, 429-463. Batchelor, F. R., Doyle, F. P., Nayler, J. H. C., and Rolinson, G. N. (1959). Nature 183, 257-258. Behrens, 0. K. (1949). I n “The Chemistry of Penicillin” (H. T. Clarke, J. R. Johnson, and R. Robinson, eds.), pp. 657-679. Princeton Univ. Press, Princeton, New Jersey. Behrens, 0. K., Come, J., Jones, R. G., Mann, M. J., Soper, Q . F., Van Abeele, F. R., and Chiang, M. C. (1948a). J. Biol. Chem. 176, 751-764. Behrens, 0. K., Come, J., Huff, D. E., Jones, R. G., Soper, Q . F., and Whitehead, C. W. (1948b). J . Biol. Chem. 176, 771-792. Behrens, 0. K., Come, J., Edwards, J. P., Garrison, L., Jones, R. G., Soper, Q. F., Van Abeele, F. R., and Whitehead, C. W. (1948~).J. Biol. Chem. 176, 793-809. Behrens, 0. K., Come, J., Jones, R. G., Kleiderer, E. C., Soper, Q. F., Van Abeele, F. R., Larson, L. M., Sylvester, J. C., Haines, W. J., and Carter, H. E. (1948d). J. Biol. Chem. 176, 765-769. Binkley, F. (1944). J. Biol. Chem. 166, 39-43. Binkley, F. (1951).J. Biol. Chem. 191, 531-534. Binkley, F., and du Vigneaud, V. (1942). J. Biol. Chem. 144, 507-511. Boxer, G. E., and Everett, P. M. (1949). Anal. Chem. 21, 670-673.

46

ARNOLD L. DEMAIN

Calam, C.T., and Hockenhull, L). J. D. (1949). J . Gen. Microbiot. 3, 19-31. Cammarata, P. S., and Cohen, P. P. (1950). J . Bio2. Chem. 187, 439-452. Casida, L. E., Jr., and Knight, S. G. (1954). J . Bacteriol. 67,658-661. Corse, J., Jones, R. G., Soper, Q . F., Whitehead, C. W., and Behrens, 0. K. (1948). J. Am. Chem. Sac. 70, 2837-2843. Craig, J . T., Tindall, J. B., and Senkus, M. (1951). Anal. Chem. 23, 332-333. Davey, V. F., and Johnson, M. J . (1953). Appl. Microbiol. 1, 208211. Demain, A. L. (1955). Unpublished experiments. Demain, A. L. (1956a). Arch. Biochem. Biophys. 64, 74-79. Demain, A. L. (1956b). Bacteriol. Proc. SOC.Am. Bacteriologists, pp. 119-120. Demain, A. L. (1957a). Antibiotics & Chemotherapy 7, 361-362. Demain, A. L. (1957b). Antibiotics & Chemotherapy 7, 359360, Demain, A. L. (1957~).Arch. Biochem. Biophys. 67, 244246. Deshpande, V. N., and Ganapathi, K. (1957). Experientia 13, 475-476. du Vigneaud, V. (1942). Harvey Lectures, Ser. 58, 39-62. Ehrensviird, G., Sperber, E., Saluste, E., Reio, L., and Stjernholm, R. (1947). J . Biol. Chem. 169, 759-760. Ehrensviird, G., Reio, L., Saluste, E., and Stjernholm, R. (1951). J.Biol. Chem. 189, 93-108. Emerson, R. L., Pueiss, M., and Knight, S. G. (1950). Arch. Biochem. 26, 299-308. Farrell, L. (1953). Can. J . Med. Sci. 32, 512-522. Fleming, A. (1929). Brit. J . Exptl. Pathol. 10, 22G-236. Foster, J. W., and Woodruff, H . B. (1943). J. Biol. Chem. 148, 723. Ganapathi, K. (1957). Experientia 15, 172-175. Glister, G. A., and Grainger, A. (1950). Analyst 76, 31G-314. Godzeski, C., and Stone, R . W. (1955). Arch. Biochem. Biophys. 69. 132-144. Goldschmidt, E. P., Yall, I., and Koffler, H. (1956). J. Bacteriol. 72, 43fM46. Goldschmidt, M. C., and Koffler, H. (1950). Znd. Eng. Chem. 42, 1819-1823. Gordon, M., Pan, S. C., Virgona, A., and Numerof, P. (1953). Science 118, 43. Gordon, M., Virgona, A. J., and Numerof, P. (1954). Anal. Chem. 26, 1208-1210. Grau, F . H., and Halliday, W. J. (1958). Biochem. J.69,205-209. Hale, C. W., Miller, G. A., and Kelly, B. K . (1953). Nature 172, 545-546. Halliday, W. J., and Amstein, H. R. V. (1956). Biochem. J. 64,38&384. Higuchi, K., and Peterson, W. H. (1949). Anal. Chem. 21, 659-664. Hockenhull, D. J . D. (1948). Biochem. J . 43, 498-504. Hockenhull, D. J. D. (1956). Giorn. microbiol. 2, 25S269. Hockenhull, D. J . D., Ramachandran, K., and Walker, T. K . (1049). Arch. Biochem. 23, 160-161. Hockenhull, D. J. D., Herbert, M., Walker, A. D., Wilkin, G. D., and Winder, F. G. (1954). Biochem. J. 66, 73-82. Horowite, N. H. (1947). J . Biol. Chem. 171, 255-264. Jarvis, F. G., and Johnson, M. J. (1947). J. Am. Chem. Sac. 69,3010-3017. Johnson, M. J. (1952). Bull. World Health Organization 6, 99-121. Jones, R. G., Soper, Q . F., Behrens, 0. K., and Corse, J. W. (1948). J.Am. Chem. Soc. 70, 2843-2848. Kato, K. (1953a). J. Antibiotics (Japan) Ser. A . 6 , 130-136. Kato, K. (1953b). J. Antibiotics (Japan) Ser. A . 6, 184-185. Knight, S. G. (1948). d . Baclerid. 66, 401-407. Koffler, H., Emerson, R. L., Perlman, D., and Burris, R. H. (1945). J. Bacteriol. 60, 517-548.

THE MECHANISM OF PENICILLIN BIOSYNTHESIS

47

Kornberg, H. L., and Krebs, H. A. (1957). Nature 174,696696. Lascelles, J., and Woods, D. D. (1950). Nature 166,649-650. Liggett, R . W., and Koffler, H. (1948). Bacteriol. Revs. 12, 297-311. McManus, I. R. (1954). J . Biol. Chem. 208, 639-644. Martin, E., Berky, J., Godzesky, C., Miller, P., Tome, J., and Stone, R. W. (1953). J . Biol. Chem. 203,239-250. Mortimer, D. C., and Johnson, M. J. (1952). J . Am. Chem. SOC.74,4098-4102. Moyer, A. J., and Coghill, R. D. (1946). J . Bacteriol. 61, 57-78. Moyer, A. J., and Coghill, R. D. (1947). J . Bacteriol. 63, 329-341. Olson, J. A. (1954). Nature 174,695-696. Peck, R. L., and Folkers, K. (1949). In “The Chemistry of Penicillin” (H. T. Clarke, J. R. Johnson, and R. Robinson, eds.), pp. 52-75. Princeton Univ. Press, Princeton, New Jersey. Perlman, D., and O’Brien, E. (1954). Arch. Biochem. Biophys. 61, 266-270. Rolinson, G. N . (1954). J . Gen. Microbiol. 11, 412419. Schmidt, W. H., and Moyer, A. J. (1944). J . Bacteriol. 47, 199-208. Sebek, 0. K. (1953). Proc. SOC.Exptl. Biol. Med. 84, 170-172. Singh, K., and Johnson, M. J. (1948). J . Bacteriol. 66, 339-355. Smith, E. L., and Bide, A. E. (1948). Biochem. J . 42, xvii-xviii. Somerson, N. L. (1957). Personal communication. Soper, Q. F., Whitehead, C. W., Behrens, 0. K., Corse, J. W., and Jones, R. G. (1948). J . A m . Chem. SOC.70,2849-2855. Stevens, C. M., and De Long, C. W. (1958). J . Biol. Chem. 230,991-999. Stevens, C. M., and Halpern, P. E. (1949). J . Biol. Chem. 179, 389-394. Stevens, C. M., Vohra, P., Inamine, E., and Roholt, 0. A. (1953). J . B i d . Chem. 206, 1001-1006. Stevens, C. M., Vohra, P., Moore, J. E., and De Long, C. W. (1954a). J . Biol. Chem. 210, 713-718. Stevens, C. M., Vohra, P., and De Long, C. W. (1954b). J . Biol. Chem. 211, 297-300. Stevens, C. M., Inamine, E., and De Long, C. W. (1956). J . Biol. Chem. 219,405-409. Stone, R. W., and Farrell, M. A. (1946). Science 104,445-446. Strassman, M., Thomas, A. J., and Weinhouse, S. (1953). J . A m . Chem. SOC.76,5135. Tabenkin, B., Lehr, H., Wayman, A. C., and Goldberg, M. W. (1952). Arch. Biochem. Biophys. 38, 4348. Thorn, J. A,, and Johnson, M. J. (1950). J . A m . Chem. SOC.72,2052-2058. Tolbert, N. E., and Cohan, M. S. (1953). J . Biol. Chem. 204, 649-654. Tome, J., Zook, H. D., Wagner, R. B., and Stone, R. W. (1953). J . Biol. Chem. 203, 251-255. Winnick, T., Moring-Claesson, I., and Greenberg, U. M. (1948). J . Biol. Chem. 176, 127-132. Wintersteiner, O., Stavely, H. E., Dutcher, J. I)., and Spielman, M. A. (1949). In “The Chemistry of Penicillin” (H. T. Clarke, J. R. Johnson, and R. Robinson, eds.) pp. 207-242. Princeton Univ. Press, Princeton, New Jersey. Wright, B. E. (1951). Arch. Biochem. Biophys. 31, 332-333.

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Preservation of Foods and Drugs by Ionizing Radiations

W. DEXTERBELLAMY General Electric Research Laboratory, Scheneclady, New York

............ 49 60

V. VI. VII.

X.

. . . . . . . . . . . . 50 51 54 Dosimetry.. . . . . . . . . . .................................... Physical and Chemical ........................... 54 Microbiological Effects ........................... 60 63 ................. . . . . . . . . . . . . . . 65 A. Insect Eradication., . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65 65 66 D. Pasteurization. . . . . . .................................... 66 66 .................................... 67 67 Containers.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .................................... 68 69 70 References. . . . . . . ............................

I. Introduction and Scope Interest and research in irradiation preservation of foods have grown to the point where about 18 tons of food per month will be processed in 1959. This food will be used for a variety of studies ranging from basic chemistry on the isolation and identification of products in irradiated proteins, carbohydrates, and fats to human volunteer feeding experiments. This paper will cover some of the more important aspects of the subject and is not intended to be a comprehensive review. Several hundred technical papers have been published in this field in the past ten years. There have been a number of reviews (Hannan, 1955; Bellamy et al., 1955; Kuprianoff, 1955; Hannan, 1957; Morgan, 1958; Niven, 1958; Siu, 1958) to mention a few. Because of the limited space available only a few references have been given for each subject discussed. A complete listing would leave space for very little else. The interested reader should consult the references and the more comprehensive reviews. Popular interest in the subject has been great, and the number of popular articles probably exceeds that of the technical papers. At first “cold sterili49

50

W. DEXTER BELLAMY

zation” was touted as the panacea for all preservation problems. When some of its limitations were more fully realized, the process was rejected by many as worthless. It is now emerging as a process that will find application in specific areas, depending upon many parameters.

II. History Shortly after the discovery of radioactivity it was found that living cells could be penetrated and killed by this new type of energy. Any practical application had to await the development of sources capable of treating large volumes of material and the accumulation of knowledge of the physical and chemical effects on exposed materials. Coolidge (1925) and Coolidge and Moore (1932) were the first to report on the biological and chemical effects of an intense electron beam. However, little more was done until after World War 11. The present period of activity may be considered to have begun with the work of Brasch and Huber (1947) and that of the M.I.T. group (Dunn et al., 1948; Trump and Van de Graaff, 1948; Proctor and Goldblith, 1948). A large fraction of the present study is supported by the Quartermaster Corps of the United States Armed Forces and by The Office of the Surgeon General. The United States Army Ionizing Radiation Center a t Stockton, California, is expected to be in operation in 1960. The plans call for a 24 m.e.v. linear accelerator as an electron source and two million curies of cobalt60 as a y source. This pilot plant will have a capacity of 1,000 tons per month, and Irradiated Products, Inc. was awarded the production and planning contract .

111. Nature of High-Energy Radiation The term high-energy radiation is applied to types of radiation, both electromagnetic and particulate, in which the energy greatly exceeds that of the chemical bond. Although ultraviolet radiation shorter than 2,000 A should be included, it is of little practical interest because of its limited penetration. Only P- or y-rays from natural sources and X-rays and highvelocity electrons from machines are of interest for sterilization purposes. Other types of high-energy radiation have too little penetration or are likely to produce residual radioactivity. Contrary to visible or ultraviolet light, the absorption of high-energy radiation is dependent upon the atomic number of the target and is almost independent of the chemical structure. The absorbed energy is dissipated by ionization and excitation. The excitation energy may appear as light but will eventually appear as heat. The average ionization energy in air is about 32 electron volts (e.v.). While those occurring in a condensed medium such as biological material are much more difficult to measure,

RADIATION PRESERVATION

51

they are assumed to be about the same.' Thus the available energy is far greater than is necessary for the breaking of any chemical bond. The details of energy absorption are complex and only partially understood. They will be dealt with in later sections. The reader is referred to sections by Lea (1955), Nickson (1952), and Hollaender (1954) for more detailed discussion of this complex subject. Although X-rays and y-rays are electromagnetic rays without mass and p-rays are high-velocity electrons, their effect on biological materials is quite similar. Both interact to a large extent by ejection of an orbital electron, e.g., HzO

HIOf

+e

(1)

The ejected electron may have enough energy to repeat the above process but is eventually captured to form a negative ion, e.g., HzO

+e

+ HzO-

(2)

The excited ions dissociate into radicals, e.g.,

+ OH. HzO- -+H. + OH-

H20+ -+ Hf

(3) (4)

The original molecule may dissociate after excitation rather than eject an electron, e.g., He0 + H -

+ OH.

(5)

The fate of the radicals, ions, and excited molecules formed by the interaction of the high-energy radiation and biological matter depends upon chemical structure of the ionized molecule, although the original interaction was almost independent of chemical structure. The electron because of its mass and charge interacts more strongly with matter than do X- or y-rays. Therefore, the penetration of an electron beam is much less than that of an X- or y-ray of equal energy. Examples of penetration are presented in Fig. 1.

IV. Sources of High-Energy Radiation Radioactive elements both natural and artificial that emit only 8- or y-rays of suitable energies are potential sources. Of all such elements only cobalt-60 and cesium-137 may become available in sufficient quantities to be considered for commercial use. Mixed radiation from spent fuel plugs from atomic reactors may be used (Ceran et al., 1953). It seems improbable The energy of a C-3 bond is about 4 e.v. (1 e.v. per molecule = 23.05 kg. calories per mole). Because the ionic yields are unknown, the products are frequently reported as G values, i.e., the yield per 100 e.v. absorbed.

52

W. DEXTER BELLAMY

DEPTH OF WATER (cml

FIQ.1A. Distribution of ionization in water by X-rays and cathode rays (Trump and Van de Graaff, 1948).

that the @-radiationwill be of much use because of the low initial energy and the great amount of self absorption in these materials. Some of the pertinent data relating to these sources are the following: Cobalt-60 has a half-life of 5.2 years and average energy of y = 1.2 m.e.v.; cesium-137 has a half-life of 30 years and average energy of y = 0.66 m.e.v.; spent fuel rods’ half-life and average energy is variable, depending upon composition, but may be considered the order of 6 months and 1 m.e.v. All radioactive sources have the disadvantage of continuous decay in activity, thus requiring periodic replacement as well as continuous changes in the dose rate. They cannot be turned off by pushing a button or throwing a switch, thereby necessitating more complex shielding and protection systems. The accelerators can be used for the production of X-rays or electrons. As previously mentioned, X-rays and y-rays are similar and much more penetrating than electrons of the same energy. The accelerators are generally used for the production of electrons because of the much higher efficiency of production and absorption. The types of accelerators now considered for sterilization are: (1) Resonant transformers (Knowlton et al., 1953). (2) Van de Graaff (Foster et al., 1953). (3) Linear accelerator (Dewey et al., 1954). The details of the operation of these machines cannot be included in this article because of lack of space. The interested reader is referred to the

RADIATION PRESERVATION

53

RANGE ( p m l c 3 1

FIG.1B. Distribution of ionization in depth of aluminum produced by cathode rays of different energies (Trump and Van de Graaff, 1948).

specific papers. But, in general, these machines operate by accelerating electrons within a vacuum tube to high velocities. The velocity is directly related to the accelerating voltage. The electron beam can be brought out into the air through a thin window of aluminum, titanium, or stainless steel. If the electron beam is made to impinge on a heavy metal target in place of the thin window at the end of the vacuum tube, X-rays are generated. The conversion of the electron energy into X-rays is inefficient, and the X-rays generated are not as directional as the electron beam. Furthermore, the fraction of the X-ray beam which can be usefully absorbed in the product is much less. Therefore, for those applications where the penetration of the electron beam is sufficient, it is to be preferred. The resonant transformer and the Van de Graaff are direct linear accelerators, and the practical limit is about 5 m.e.v. maximum because the machines become very bulky and unwieldly due to the necessary insulation. A 4-m.e.v. resonant transformer, for instance, is a tank about 9 feet in diameter and 15 feet long with an 18-foot accelerating tube. An indirect accelerator such as the r-f linear type can be built to operate up to several hundred m.e.v. without the dimensional limitations of the direct accelerators. The upper limit is that range which will produce SUEcient nuclear side effects to result in undesirable residual radioactivity. The present evidence seems to indicate that the upper practical limit is

54

W. DEXTER BELLAMY

greater than 25 m.e.v., particularly if there is a storage period of several days following cxposure (Baldwin and Clark, 1953; Hannan, 1955).

V. Dosimetry The control of dosage in radiation processing is a t least as important as the control of time and temperature in thermal processing. The intense beams of high energy have introduced new problems in dosimetry. A search is in progress for a material which can be placed on or in the container of food and which will, with a minimum of processing, indicate the integrated dose received. The indicator should be simple and unaffected by time, temperature, light, etc., and reproducible within a few per cent. Silver phosphate glass fills some of the requirements (Davidson et al., 1956), and a polyvinyl chloride film has been developed as a dosimeter (Anonymous, 1957). Machine sources can be controlled by monitoring the beam current, but radioactive sources with their continuous decay require other methods (e.g., Davidson et al., 1953). The unit of X-radiation was the roentgen which was defined in terms of ionizations in air. The units now used in this work are the roentgen equivalent physical (rep) which is equal to 93 ergs per gram in most biological tissues and the rad which by definition is equal to 100 ergs per gram.

VI. Physical and Chemical Change Knowledge of irradiation chemistry has not developed to the point where it can predict all the changes that will be produced in a material as complex as food. It is still necessary to treat each food as a separate problem. There are, however, a few general observations that can be made which have wide application. Studies on the radiosensitivity of dilute aqueous solutions of an organic molecule may have little relation to its behavior within a living cell or in food products. In dilute solutions most of the radiation is absorbed by the water, resulting in numerous ions and/or radicals which are free to migrate. Because of the low probability of reaction, these ions and radicals may have a relatively long life and migrate correspondingly long distances. Any specific molecule or system in a cell is protected by the high probability that a water radical will react with any of the other more numerous components present. In a cell a large fraction of the water is in an ordered state and is therefore not free to migrate. The probability that a radical will interact with a protein or other large organic molecule is large : E 1 ;therefore, the lifetime of a radical may be only 10-9 seconds. The distance of migration is the order of 30-100 A (Hutchinson, 1957). Estimates of the fraction of indirect

55

RADIATION PRESERVATION

effect in living cells have been made by Hutchinson (1958) as not more than 50 %. This will vary, depending upon the system under study. There is ample evidence that energy can be transformed intra- and intermolecularly in solids. (The scintillation counter is based upon intramolecular transfer of excitation.) Under proper circumstances, however, radicals formed in solids by radiation will have a long lifetime (Lawton et al., 1958; Wolfrom et al., 1958). A considerable amount of work on the radiation of synthetic polymers has been reported in the past few years (c.f. Bovey, 1958). Some of these results can be applied to the much more complex biological systems. It was found that all vinyl polymers in which degradation predominates over crosslinking have the structure:

i.e., both the hydrogens on one of the carbons have been replaced. If either R or R' is an H, the polymer will crosslink (Miller et al., 1954). It appears that a simultaneous expulsion of hydrogens from adjacent carbons occurs about as frequently as the formation of a radical by ejection of a single hydrogen [see Eqs. (6a) and (6b)l. -C Hz-CH2-

-

-CH=CH-+Hz -CH2-CH-

(64

+ H.

(6b)

The G value (yield per 100 e.v. absorbed) for each process in high density polyethylene is about 2 (Miller et al., 1956). Crosslinking has been proposed as due to hydrogen abstraction from an adjacent polymer.

-CHz-C-

H

Lauryl methyl methacrylate was found to crosslink (Shultz, 1958). This

56

W. DEXTER BELLAMY

is probably due to the long hydrocarbon side chain. This process can be pictured as shown in Ey. (8) CHa CHs CHZ-C-CH

CHa C-

-4

I *- 1 c=o c=o I I ( C i J L J (C i i H 4 I I CHg

-CH=C

CHs

c=o

I I c=o

CH3

CHs

CH3

C=CH-

I I I (CiIHzi)-(CiiH21) I I

(8)

where the main chain is broken but the long hydrocarbon side chain crosslinks. The backbone of a peptide chain more closely resembles the doubly substituted polyethylene R

H O R

H

N

N

H

H

O

\ / It \ / II -C c-c C\ / \ /

and therefore might be expected to degrade upon irradiation. The longer side chains such as in leucine, lysine, etc. would be expected to crosslink because they are aliphatic hydrocarbons. One might predict that proteins would degrade in the backbone but crosslink on the side chains. The over-all result would be a more highly branched polymer. Whether the size went up or down would depend upon the conditions of radiation, as well as the nature of the side chains. Sodium caseinate shows a decrease in particle size as determined by viscosity after the material was irradiated while dry in air with 1.86 X 107 rad. The same material irradiated as a 20% solution set up as a gel which was insoluble in water, 1 N HCl, and 1 N NaOH. The exact nature of the crosslink was not determined (Bellamy et al., 1955). A mechanism for the direct action on peptides and proteins was proposed (Caputo and Dose, 1957; Rajewsky and Dose, 1957) as shown in Eq. (9). Thus the products are an aliphatic imide Hz. The imide is unstable in a-amino acid iaqueous solution and decomposes to an a-keto acid ammonia with resulting chain cleavage. The production of carbonyls from amino acids was also reported. This agrees with the earlier work on leucine (Bellamy and Lawton, 1954). Garrison and associates (1958) irradiated dilute solutions of pepsin and gelatin. They found an amide carbonyl (imino-intermediate) as shown in Eq. (10).

+

+

+

57

RADIATION PRESERVATION

H O

O R

I1

RIH 0

\ / 1I I / c-c \ / \ \ /c-c N H

0

\ / NH

\ / NH

N

H

H

I

H

\ /

c-c \ /

\ / 4N H

N

0

0

7-

N

0 Rz

II R’ c-c

II

He

N H H

O

I It R-C-NH-C-C-RI I

1I

Ra

10..

The G value for this reaction was found to be about 1. There is a fundamental difference between radiolysis of a peptide bond and the conventional acid or enzyme hydrolysis in that an imide must be formed between the N and the a-carbon, rather than the carboxyl carbon. Therefore, the subsequent hydrolysis results in an a-keto acid plus an amide or plus ammonia and acid. If this reaction occurs by a molecular process as in Eq. (64, there seems little possibility of a crosslinking in the peptide backbone. It is also very unlikely that any protective agent or inhibitor can be added

58

W. DEXTER BELLAMY

which will alter the process. However, if the reaction proceeds by Eq. (Bb), which seems more probable for indirect action, then a radical termination would prevent the abstraction of the second hydrogen. Because aliphatic amides are quite resistant to hydrolysis at ordinary temperatures, the high yield of ammonia from both peptides and amino acids does not support their formation. However, many amides are readily hydrolyzed by enzymes so that the significance of these reactions in foods is in odor and flavor changes because irradiated foods have not been found materially less wholesome (see Section VIII). The production of volatile amines from irradiated beef has been confirmed by Burks et al. (1958). Methyl amine and ethyl amine and a t least four other unidentified amines were found. The largest fraction of the volatile bases was ammonia in both the unirradiated control (E 99.9%) and the 2.3-3.7 megarad irradiated sample (E 93.0%). There was an increase in ammonia as well as volatile amine due to irradiation. The source of these amines is not known. The authors assume that the source of these amines is the nonprotein fraction of the meat because it contains about thirty-eight times as many end amino groups as the protein fraction, but no experiments to check this point have been done. Several papers on the production of carbonyls in irradiated foods have been published. Day et al. (1957a, b) found acetaldehyde, acetone, and butanone in the volatile fraction of irradiated skim milk. They did not search for nonvolatile carbonyls. Batzer et al. (1957) found that the carbonyls produced in irradiated meat could be divided into two groups, depending upon the solubility of their 2,4-dinitrophenyl hydrazine derivatives. The benzene soluble group A was obtained mostly from fat, while the metaphosphoric acid-sodium chloride-soluble group B was obtained chiefly from lean ground beef, The carbonyl compounds in both meat and fat vary directly with dose up to 9.3 megarad, the maximum dose used. The color changes in irradiated meats are largely due to the effect on the heme pigments. Anaerobic irradiation of precooked meats may result in the conversion of the normal brown or grey hematin to red pigments. The red pigment was characterized as denatured globin hemochrome (Tappel, 1957). Fresh meats irradiated aerobically at low doses develop a brownish discoloration due to formation of metmyoglobin. Fresh meats irradiated in nitrogen develop a brighter color due to regeneration of oxymyoglobin. A later study indicates that anaerobic irradiation converts methemoglobin to oxyhemoglobin (Tappel, 1958). Oxygen has a profound effect on both the direct and indirect effects of radiation. In water the H. from Eq. (4)will react with 0 2 to form Hoe. or other oxidizing radical so that instead of an equal number of oxidizing and reducing events, there are chiefly oxidations.

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The radicals produced by the direct effect can react with oxygen to produce organic peroxides, carbonyls, or other oxidized products. The radiation-produced radicals may persist for long periods in solids, including foods which have been irradiated and stored frozen. Such stored radicals can react with oxygen or other compounds to cause postirradiation effects. The irradiation of lipids produces crosslinks similar to those in polyethylene and, in addition, if irradiated in air, produces saturated and unsaturated carbonyls and peroxides. The flavor and odor changes could not be correlated with peroxides or carbonyl formation. Ozone and nitrogen oxide may be of importance in flavor and odor change. Carbonyls are also produced under anaerobic irradiation. The radicals produced by irradiation may be involved in autoxidation. The radical concentration is so high, however, that antioxidants are much less effective than in ordinary oxidative changes (Chipault et al., 1957). Transisomerization was found in oleic acid (Pan et ul., 1958). Presumably, transunsaturation occurs as it does in polyethylene, although it was not reported. When irradiated dry, carbohydrates degrade with the production of reducing groups and acid groups. Crosslinking also occurs to some extent (Saeman et al., 1952; Price et al., 1954; Kertesz et ul., 1956). The nature of the crosslink is not known. Carbohydrate degradation is most important in the texture of foods, particularly fruits and vegetables because they do not contribute to off-odors. The products from irradiated mono- and polysaccharides have been implicated in the nonenzymatic “browning” of foods. It appears that mono- and disaccharides are not important in odor or flavor changes at sterilization doses. Sulfur-containing amino acids and proteins have been shown to be a major source of the “irradiated beef” odor, although the exact causes of this odor are not known. Aqueous solutions of cysteine liberate hydrogen sulfide rather than ammonia when irradiated as do other amino acids. No hydrogen sulfide was detected upon irradiation of aqueous solutions of cystine (Swallow, 1952). Irradiation of aqueous solutions of methionine has been found to yield both hydrogen sulfide and methyl mercaptan. Aldehydes and ketones will combine with mercaptans under conditions existing in foods to form mercaptol and mercaptals. Littman et al. (1957) proposed the protection of -SH groups by the addition of carbonyl compounds during irradiation. It also seems possible that some of the irradiation odors and flavors are due to this type of compound formed from condensed carbonyls and mercaptans produced by irradiation of foods. Whether the mercaptal or mercaptol would be volatile and malodorous depends upon its size and other functional groups. Dilute solutions of many enzymes are quite sensitive to ionizing radia-

60

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tion. However, they are much more resistant in cells, tissues, and in the presence of high concentrations of other organic material. Most natural products retain some enzyme activity after exposure to pasteurization or sterilization doses. The phosphatase of milk was not completely destroyed by ten times the dose necessary to sterilize raw milk (Proctor and Goldblith, 1957). Several workers have found tyrosine crystals in radiationsterilized beef and pork after storage at 72”-100°F. for several months (e.g. Drake et al., 1957) as well as increased amounts of soluble amino acids. From 10 to 90 % of the original activity of other enzyme systems has been found in radiation-sterilized meats. Heating to 160°F. for a few minutes during the processing cycle has been found effective as an enzyme inhibitor. This amount of thermal processing will result in a cooked product with the accompanying physical and chemical changes. It may have beneficial side effects by decreasing the sterilization dose necessary, by inactivation of some viruses, and by inactivation of any toxins (q.v.). Other methods of enzyme inhibition have been sought such as chemical additives, pre- and postirradiation treatment, etc. One method which may have application in irradiation pasteurization and result in a “fresh” meat is that described by Radouco-Thomas et al. (1958)and Zender et al. (1958). These authors found that ante-mortem injection of adrenaline (epinephrine) resulted in muscles with greatly reduced post-mortem autolysis. Because normal muscle is sterile, a combination of epinephrine injection and surface sterilization with electrons resulted in a product that had greatly increased storage tolerance at all temperatures up to 38°C. The process was studied in pork, beef, and rabbit. In addition to greatly decreased autolysis, there was increased fluid retention, better color retention, and increased tenderness. These physical changes in the muscle, as well as the decreased activity of cathepsins, is due to the higher pH of the postmortem meat. The pH of normal muscle drops to about 5.5 a few hours after slaughter, while the adrenaline-injected muscle remains close to 7.0. The injection process does not, however, alter the irradiation odor produced in beef or pork. Some bacterial toxins are molecules about the same size and complexity of enzymes, and one has been found to have the same order of radio resistance. The toxin of Clostridium botulinum was found to have a DWof about 8 megarad (Dack and Wagenaar, 1955).

VII. Microbiological Effects The factors affecting the radiosensitivity of microorganisms have been reviewed by Kelner et al. (less), Hollaender and Stapleton (1953), and Bellamy and Lawton (1955).Discussions of the mechanism of action can be found in the above reviews as well as in Lea (1947),Pollard (1954),and

RADIATION PRESERVATION

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Bacq and Alexander (1955). Inactivation by radiation usually means inability to continue indefinite growth. Inactivated cells may undergo one or more divisions and may have a large fraction of their original enzyme activity immediately following a lethal dose of radiation, but the organism or its progeny cannot reproduce properly. For this reason the lethal effect is thought to be due to genetic damage. It can be easily estimated that within a bacteria such as Escherichia coli less than one bond in lo8is broken by a lethal dose of radiation. Therefore, the damaged structure must be essential. Attempts to identify this structure have led to the target theory. The original target theory has been modified and refined by many, including Lea (1947) and Pollard (1954). Under suitable conditions it has been found that the survival of many bacteria fits a first order decay curve of the type N = N @ - D ‘ D o , where N = number of organisms surviving dose D,N o = original number, and Do = mean lethal dose (dose at which N = Noe-l). This type of survival has been interpreted to mean that a single ionization will be effective if occurring in the proper structure. It is now known that this interpretation is limited to aerobic conditions and within a narrow range of temperature (Howard-Flanders and Alper, 1957). Survival curves of a higher order have been obtained for many organisms and have caused many authors to reject the target theory in favor of the “indirect effect.” Here the lethal action is due to the activated or ionized water, e.g., H., OH., HOz., etc. Because most living systems are largely water, the indirect effect seemed most probable. More recent studies, however, indicate that the indirect effect accounts for not more than one-half of irradiation effect in microorganisms (Hutchinson, 1957). This apparent paradox is probably due to the short life of many of the mobile radicals and also to the fact that much of the water within a cell is not free to migrate but is bound in an ordered system. Most organisms are more radiosensitive under aerobic conditions. Recent work has shown that removal of oxygen 0.02second after aerobic irradiation will not prevent the increased sensitivity. Conversely, addition of oxygen 0.02 second after anaerobic irradiation does not increase the sensitivity (Howard-Flanders and Moore, 1958). Much of the work on the oxygen effect will have to be re-examined in the light of the above findings that very small concentrations are almost immediately effective. Most organisms are more sensitive when irradiated in distilled water than in nutrient broth. The addition of reducing compounds such as ascorbic acid and hydrosulfite, 8-mercaptoethylamine, and other sulfhydryl compounds (Stapleton and Woodbury, 1957; Doudney, 1957) during irradiation decrease sensitivity. In general these compounds do not increase the anaerobic resistance. Pseudomonas geniculata, a very sensitive psychrophilic aerobe, was found to be unaffected by oxygen during irradiation with y-

62

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rays (Wolin et al., 1957). A threefold increase in resistance was obtained by increasing the number from lo8per milliliter to 8 X los or greater per milliliter. High populations of Micrococcus sp. did not alter the radiation resistance of low numbers of Pseudommas cultures. Recent work (Alper and Gillies, 1958) seems to indicate that many postirradiation restorations of irradiation damage previously reported may act by a common mechanism, viz. the imposition of suboptimal conditions of growth. The type of damage which is susceptible to postirradiation influence is less influenced by oxygen than other forms of damage leading to lethal injury. The whole problem of pre- and postirradiation conditioning is an extremely complex one and undoubtedly differs with different species. Of interest in the field of food preservation are the findings of Morgan and Reed (1954) that the thermal resistance of spores of strain PA 3679 was reduced by prior exposure to sublethal doses of ionizing radiation. The radiation resistance of preheated spores was not changed, however. The spores of C. botulinum were found to respond in a similar manner (Kempe, 1955). Kempe et al. (1957) obtained less spectacular results when they tried pre-irradiation on C. botulinum spores in ground meat. Kan et al. (1957) have confirmed the decreased thermal resistance of pre-irradiated spores of PA 3679 and Bacillus cereus in canned ham. The effect of the temperature during irradiation received considerable attention. In general vegetative cells irradiated aerobically are more resistant when exposed in the frozen state. The sensitivity of anaerobically exposed cells is less altered by freezing. Freezing immobilizes the water radicals and thereby considerably reduces the indirect effect. Most microorganisms are more resistant dry than in aqueous suspension. This again is probably due to the reduction of the indirect effect and decrease of target size. Much less work has been published on the effect of radiation a t elevated temperatures. Denny et al. (1954) reported that spores of C. botulinum suspended in water were only slightly more resistant a t 150°F. (64°C.) than a t 72", 23", and 0°F. when exposed to y-rays from cobalt-60. Kempe et al. (1956) reported that C. botulinum 213B was more resistant at 27'C. by an order of magnitude than a t any other temperature tried between -70" and 95°C. PA 3679 was more resistant by a factor of 25 in the range of 85-95°C. Because of the importance of these observations, for both practical and theoretical reasons further studies on the effect of temperature during irradiation should be undertaken. In 1956 Anderson et al. reported the isolation of a pigmented Micrococcus resembling M. roseus or M . rubens tetragenus from ground beef following 7-doses of 2-3 megarad. Pure cultures on agar slants survived 6 megarad. This non-sporeforming organism is thus the most radioresistant bacteria yet reported. Further studies by Anderson et al. (1956) and Niven (1958)

RADIATION PRESERVATION

63

have shown that the organism is a strict aerobe, sensitive to sodium chloride above 2%, and appears to obtain energy by oxidation of amino acids. Other red pigments were found to be relatively radioresistant, although none approached the resistance of Anderson’s original isolate. Kilburn et al. (1958)were unable to grow this organism without its carotenoid pigment. They were not able to prove conclusively that the great resistance is due to the pigment, although radioresistance varied directly with the pigment content of cells. Most viruses are much more resistant to ionizing radiation than bacteria and would not be inactivated in irradiated foods if present in very large numbers. Because of their smaller size and simpler structure the radiosensitivity of viruses is much more dependent upon their environment than is that of bacteria. In terms of the target theory the increased resistance is due to the smaller size (cf. Pollard, 1954 and Benyesh et al., 1958). Although the radiosensitivity of all viruses is not simply related to their size, it is related to the content and organization of their genetic material. The importance of active virus in preserved foods would have to be evaluated in each product. Polio virus in irradiation-pasteurized milk would not be permitted. However, the presence of virus in fresh meats is not considered a problem now, and their presence in irradiation-pasteurized meats which will be cooked should be no greater problem.

VIII. Wholesomeness The effect of sterilization doses on the vitamin content of foods has been examined. As stated previously, studies on the radiosensitivity of dilute solutions of pure vitamins provide very little information about the same material in foods. They may provide useful information concerning the mechanism of radiolysis. The radiosensitivity varies among the foods and depends upon the conditions during exposure, as well as the pre- and postirradiation treatment. In general, vitamins niacin, riboflavin, folic acid, B12, D, and K are relatively stable to radiation. The stability of vitamins A, E, C, and thiamine is poor while the pyridoximers are intermediate. The vitamin loss in irradiated foods has been found to be about the same as in heat processed foods with the possible exception of thiamine which is more sensitive. In certain long-term animal feeding tests with diets high in individual foods, it has been found desirable to auppIement with vitamins E and K (cf. Groninger et al., 1956). The question of irradiation production of toxic products must be thoroughly investigated before large-scale use is permitted. Experiments have been under way for several years using thousands of animals including mice, rats, chickens, pigs, dogs, monkeys, as well as human volunteers. The

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results of these tests give no reason to believe that irradiation processed food is less safe than food processed by other means. A search for radiation produced carcinogenic materials has been completely negative so far. For a thorough discussion of this problem the reader should consult Siu (1958), Vorhes and Lehman (1956), and Lehman and Laug (1954). Residual radioactivity in irradiated foods and containers is popularly considered to be the most serious problem in irradiated foods. A more thorough examination indicates that the induced radioactivity is low and TABLE I APPROXIMATEDOSERANGE REaUrRED FOR SSVERAL FOOD PRESERVATION PROCESSES Process

Rad

Inhibition of sprouting carrots, onions, potatoes Inactivation of trichina: sterilization of trichina female Insect deinfestatioo of grains and cereals “Irradiation pasteurization” Sterilization of foods Enzyme inactivation

4,000-10,OOO 20,000-50, OOO 100,000-5OO,OOO 1OO,o0o-1,ooo,OOO 2,000,~5,OOO,000 2,000,000-10,OOO, 000

TABLE I1 THETHRESHOLD ENERGY LEVELSFOR THE PRODUCTION OF INDUCED RADIOACTIVITY Element Carbon-12 Oxygen-16 Nitrogen-14 Potassium-39 Sulfer-32 Calcium-40 Iron-54 Magnesium-24 Magnesium-25 Copper-65 Iodine-127 Aluminum-27 Silicon-28 Zinc43 Tin-119 Sodium-22

Threshold (m.e.v.) 18.7 16.3 10.6 13.2 14.8 15.9 13.8 16.2 11.5 10.2 9.3 14.0 16.8 11.6 6.6 12.1

Half-life of product 21 minutes 2.1 minutes 10 minutes 7.5 seconds 3.2 seconds 1 second 8.9 minutes 11.6 seconds 14.8 hours 12.8 hours 13 days 7 seconds 5 seconds 39 minutes 275 days 2.6 years

RADIATION PRESERVATION

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in nearly all cases the half-life so short that there is no hazard. A possible exception might be tin-119 in the containers or sodium-22 in the food if the initial radiation energy is greater than 12 m,e.v, Table I1 gives some of the possible radioactive products, their half-life, and the threshold energy of activation. Measurements on foods exposed to 30 m.e.v. electrons showed that the induced activity was several orders of magnitude less than the established tolerance levels (Skaggs, 1956).

IX. Applications Table I presents the dose range required for some preservation processes which are discussed in more detail below.

A. INSECT ERADICATION The control of such insects as Sitophilus granum'us (granary weevil) and Tribolium confusum (confused flour beetle) in grains and cereal products has been suggested. The dose necessary to bring about rapid death of the insects and larvae was found to be about 300,000 rad, while that necessary to prevent reproduction was the order of 15,000 to 30,000 rad (Hassett and Jenkins, 1952). Similar results were reported by Proctor et a,?. (1954) for five species of insects normally found in infected cereal and fruit products. Plans have been suggested for the design of facilities for deinfestation of bulk grain (Hassett and Jenkins, 1952; Brownell et al., 1955, 1957), but its use in prepackaged cereal and cereal products seems more feasible because of the possibility of recontamination of bulk materials. B. SPROUTINHIBITION Because of the low dose required (4,00040,000 rad) sprout inhibition of vegetables such as potatoes, onions, carrots, etc. has been examined (Sparrow and Christensen, 1954; Brownell et al., 1955; Brownell and Nehemias, 1955). The increased storage life without sprouting and the accompanying softening and loss of water is very clear-cut and may increase the usable fraction from 0 to over 90% after prolonged storage. It was found that doses higher than 14 kilorad caused a permanent increase in respiration during storage, while doses of 4.7 and 14.0 kilorad caused a temporary increase in respiration that dropped to approximateIy normal after the seventh week of postirradiation storage (Gustafson et al., 1957). Irradiated potatoes have been found more sensitive to bruises and less resistant to rot. Potatoes exposed to 2.3 kilorep or more did not exhibit periderm formation after wounding, although there was distinct soberization (Brownell el at., 1957; Burton and Hannan, 1957).

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C. TRICHINA The use of ionizing radiation to break the trichinosis cycle has been suggested (Gomberg et al., 1954), e.g., because of the relatively low dose (20,000-50,000 rad). It has been pointed out, however, that in this country other approaches to this problem appear more practical (Urbain, 1958). Its application in other countries with different technical and economical parameters merits study.

D. PASTEURIZATION (‘Irradiationpasteurization” has been suggested as a means of increasing storage l i e at low temperatures. Wolin et al. (1957) reported that the psychrophilic pseudomads, the principal cause of spoilage of fresh meats at low temperature (2”C.), were very sensitive to radiation, and doses of lob rad or less were found to extend the low temperature life severalfold. However, the meats eventually spoil due to growth of more radio resistant microorganisms such as Microbacterium thermosphactum. This increased low-temperature life may make prepackaging of fresh meat a commercial possibility. Physical changes due to nonmicrobial spoilage in certain meats may be a serious problem (Urbain, 1958). Minced chicken meat refrigerated anaerobically was wholesome after 80 days if pre-irradiated with 250,000 rad. Spoilage was due to M . thermosphactum and Streptococcus faecium (Thornley, 1957). Irradiation at 1 megarad was found to inactivate all microorganisms except endospores and has been proposed as a “pasteurization dose.” Brownell and Purohit (1956), Brownell et al. (1955), and Proctor et al. (1955) found that certain meats and vegetables had longer low temperature storage life after doses ranging from 250 to 1 X lo6 rad. Beef products developed undesirable off flavors at the higher doses, however.

E. STERILIZATION The fact that C. botulinum spores and toxin will not be destroyed by these pasteurization doses means that these foods will have t o be treated as possibly contaminated and uncooked. As previously mentioned the spores of C . botulinum are among the most radiation-resistant of microorganisms and any radiation-sterilized product must be free of this organism. If the criterion used in thermal processing is used, i.e., the dose necessary to reduce 1 X 10l2 spores to less than one survivor, then the sterilization dose has been calculated to be 4.5 megarad (Niven, 1958). Much of the earlier work based on a sterilization dose of 2 megarad must be re-examined in the light of the increased dose.

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F. PHARMACEUTICALS Studies on the properties of irradiated pharmaceuticals have been reported by several workers. Colovos and Churchill (1957) examined a series of parenteral products that had been sterilized by cathode rays. Stability and toxicity were used to determine the influence of exposure to 2 megarep (1.86 megarad). The pharmaceuticals fell into the following categories: antibiotics, hormones and steroids, multivitamin preparations, anticoagulants, proteins, alkaloids, and sulfonamides. Storage times up to 4 years a t 4",2 5 O , and 40°F. were reported, They concluded that irradiated products used clinically showed no unfavorable reactions and that with few exceptions drugs under the proper conditions are stable to sterilizing doses of cathode rays. However, each product must be treated as an individual problem. Ethicon has announced the commercial sterilization of sutures by a 7 m.e.v. linear acceIerator (Anonymous, 1957). Some of the advantages claimed are: (1) increased tensile strength, (2) sterilization in their final sealed containers, and (3) substantial increase in the safety margin. The sterilization by ionizing radiation of human blood vessels and bones for transplants has proved successful and is being used in several places (e.g. DeVries et aE., 1955). The use of ionizing radiation to prepare vaccines has been suggested (Traub et al., 1951) because of the differential sensitivity of the Virus components. The properties of irradiated virus have been examined (Bellamy et al., 1957; Benyesh et al., 1958), but further knowledge of the factors involved is needed.

X. Containers Radiation does not impart some miraculous self-sterilizing property to foods; therefore, treated foods must be protected against recontamination. Several studies are under way to determine the effect of sterilization doses on containers, of the interaction between irradiated foods and irradiated containers, and of storage of irradiated foods in irradiated containers. Both conventional rigid metal cans and Aexible plastic films are under consideration. Most films have been found to be permeable to oxygen and/or water. Some irradiated films have been found to interact with the foods to produce off odors or flavors. Mylar, nylon, polystyrene, and polyethylene alone or in combination with each other or several mills of metal film have shown promise. Plastic film is more permeable to electrons than metal cans, but so far problems of oxidation, dehydration, and light destruction have not been satisfactorily solved. Some of the standard can enamels appear to be satisfactory for irradiation processing of foods (cf. Morgan, 1958; Siu, 1958).

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XI. Economic Considerations Many papers written on this subject have not considered many of the factors that a business must deal with such as taxes, capital write-off, and profit on the investment. Cook (1958a, b) has examined some of these parameters. For instance, much has been made of the fact that cesium-137 has a half-life of 30 years and is essentially free as a by-product of reactors; however, there are few companies that do not expect to liquidate their investment in a much shorter time, usually in not more than 10 years. A medium-risk business requires 10-15 % net earnings before taxes to attract capital. Such considerations make cesium-137 an expensive source. Estimates ranging from $ 3 4 per curie to a “rock bottom” of $0.30 per curie have been made. At $1.00 per curie cesium-137 costs $240,000 per initial kilowatt of 7-radiation. Cobalt-60 is made by absorption of neutrons in a pile. The neutrons thus used are not available for other uses. Therefore, the minimum price for

I I I I l l

O.\

I

I 1 1 1 1 1 1

1.0 COST OF IRRADIATION

I

4

I IIllll1

I

I 1 1 1 1 1

10.0

PER POUND

FIG.2. The relatioiiship between the cost of radiation production (kilowatt hours absorbed) and the cost per pound of irradiated product; e.g., if radiation costing $0.60 per kilowatt hour is used for sterilizing meat at 4 megarep, the cost per pound will be $0.028.

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cobalt-60 cannot be less than the neutrons could bring in other uses, e.g., production of power. Neutron cost is estimated a t about $58,000 per mole (Cook, 1958b). Because 1 mole of neutrons is required to make 1 mole of cobalt-60, $58,000 per mole must be a minimum amount. This is equivalent to $60,000 per initial kilowatt of y-radiation. Present costs of cobalt-60 are two t o ten times this number with a “rock bottom” of $4,000 per initial kilowatt suggested by W. F. Libby of the Atomic Energy Commission. Linear accelerators now cost $35,000 to $40,000 per kilowatt of electron beam. A sufficiently large market to allow quantity production could reduce this amount to $10,000 or even $5,000 per kilowatt of electron beam. Resonant transformers now cost $10,000-$12,000 per kilowatt, and this cost might drop to $5,000 per kilowatt of electron beam. Machines will have servicing and replacement of parts costs but can be shut off when not in use, whereas isotope sources radiate continuously whether used or not. It is obvious that only that radiation which is absorbed in the food is useful. That part lost in the air, in the container, or in the shielding is wasted. The fraction of the beam used in overdosing part of the product in order that all the product receives a minimum dose is also wasted. Utilization may vary from 10 to 90 %, depending upon the product and the engineering. In general, electron beams can be utilized more completely than the undirected radiation from radioactive sources. Figure 2 presents the relation between the cost of radiation absorbed and the cost per pound of irradiated product. These numbers were calculated from the relationship: cost of radiation (cents per pound) = cost of beam absorbed (dollars per kilowatt hour) X dose in megarad X 1.18.2 Products which are highly unique can absorb a higher cost per pound than irradiated products which are little different from materials produced by other methods. High-cost items such as drugs can absorb a few cents per pound, whereas low-cost large-volume materials such as fruits and vegetables may be overpriced by addition of a fraction of a cent per pound, These and other problems make the financial considerations as important as the technical studies.

XII. Summary and Conclusions Irradiation preservation offers a new and different process to the food and drug industry. While many of the problems are formidable, there is no evidence at present which definitely precludes their solution. The production of irradiation odors and flavors is probably the major obstacle to its use in some meat products. Research now in progress may lead to methods 2 1 kw.-hr. = 3.6 X 101s ergs; 1 megarad = 1 X 108 ergs gram-’; 1 kw.-hr. 106 gram megarad, or % 840 lb. megarad.

3.6 X

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W. DEXTER BELLAMY

of preventing or masking them. It is clear that radiation will not replace the conventional methods of thermal processing, freezing, and drying but will supplement them in restricted areas, depending upon many parameters. Sterilization of sutures, of heat-sensitive medicinals, and of army field rations are examples of special applications which are now in use. It appears unlikely that irradiation costs can compete with present methods where the conventional product is satisfactory. Entirely new products with new properties seem more probable. Irradiation pasteurization in combination with enzyme inhibition may have wide application in countries where household refrigerators are not common. The potentials of irradiation preservation of foods and drugs cannot be overlooked by a world beset by an inadequate food supply for an exploding population.

REFERENCES Alper. T., and Gillies, N. E. (1958). Radiation Research 9, 86. Anderson, A. W., Nordon, H. C., Cain, R. F., Pornish, G., and Duggan, D. (1956). Food Technol. 10, 575-578. Anonymous. (1957). Hospital Topics 36 ( l l ) , 22-25. Bacq, 2. M., and Alexander, P. (1955). “Fundamentals of Radiobiology.” Academic Press, New York. Baldwin, G. C., and Clark, L. B. (1953). Science 117,9-10. Batzer, 0 . F., Sribney, M., Doty, D. M., and Schweigert, B. 6. (1957). J . Agr. Food Chem. 6 , 700-703. Bellamy, W. D., and Lawton, E. J. (1954). Nucleonics 12, 54-57. Bellamy, W. D., and Lawton, E. J. (1955). Ann. N . Y . Acad. Sci. 69, 595-603. Bellamy, W. D., Niven, C. F., Goldblith, S. A., and Colovos, G. C. (1955). Bacterial. Revs. 19, 266-269. Bellamy, W . D., Lawton, E. J., and Gordon, I. (1957). Gen. EEec. Research Lab. Rept. 67-RL-1846. Benyesh, M., Pollard, E. C., Opton, E. M., Black, F. L., Bellamy, W. D., and Melnick, J. L. (1958). Virology 6, 266-274. Bovey, F. A. (1958). “The Effect of Ionizing Radiation on Natural and Synthetic High Polymers.” Interscience, New York. Brasch, A., and Huber, W. (1947). Science 106,112-117. Brownell, L. E. and Nehemias, J. V. (1955). Nutl. Potato Council News 1 (10). Brownell, L. E., and Purohit, 8. N. (1956). Refrig. Eng. 64, 3949. Brownell, L. E., Kempe, L. L., Dennis, R. C., Graikoski, J. T. (1955). Refrig. Eng. 63, 43-47. Brownell, L. E., Nehemias, J. V., and Purohit, S. N. (1957). “Atomic Energy and Agriculture,” pp. 367-389. American Association for the Advancement of Science, Washington, D. C. Burks, R. E., Baker, E. B., Clark, P., Esslinger, J., and Lacey, J. C. (1958). In press. Burton, W. G., and Hannan, R. 5. (1957). J . Sci. Food Agr. 12, 707-716. Caputo, V. A., and Dose, K. (1957). 2. Naturjorsch. lab, 172-180. Ceran, L. E., Isaacs, P. J., Weiss, G. J., and Fahnoe, F. (1953). Nucleonics 11, 32-37. Chipault, J. R., Privett, 0. S., Mizumo, 0. R., Niokell, E. C., and Lundberg, W. 0. (1957). Ind. Eng. Chem. 49, 1713-17‘20.

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Colovos, G. C., and Churchill, B.W. (1957).J . Am. Pharm. ASSOC., Sci. Ed. 46,580-583. Cook, L. G. (1958a). Chem. Processing 21, 31-35. Cook, L. G. (1958b). Gen. Elec. Rev. 61, 33-35. Coolidge, W. D. (1925).Science 62,441442. Coolidge, W. D., and Moore, C. N. (1932). Gen. Elec. Rev. 36,413-417. Dack, G. M. and Wagenaar, R. 0. (1955). Proc. 16th Ann. Meeting, Inst. Food Technol., p. 28. Davidson, S., Goldblith, S. A., Proctor, B. E., Karel, M., Kay, B., and Bates, C. J. (1953). Nucleonics 11, 22-26. Davidson, S., Goldblith, S. A., and Proctor, B. E. (1956).Nucleonics 14, 34-39. Day, E.A.,Forss, D. A., and Patton, S. (1957).J . Dairy Sci. 40, 922-931. Day, E.A.,Forss, D. A , , and Patton, S. (195733).J . Dairy Sci. 40,932-941. Denny, C. B., Bohrer, C. W., and Reed, J. M. (1954).Natl. Canners’ Assoc. Research Rept. 3, 54. DeVries, P. H., Kempe, L. L., and Brinker, W. 0. (1955). Univ. Mich. Med. Bull. 21, 29-33. Dewey, D. R., Nygard, J. C., and Kelliher, M.G. (1954).Nucleonics 12,4041. Doudney, C. 0.(1957). Bacteriol. Proc. (Soc. A m . Bacteriologists) 67, 49. Drake, M. P.,Giffee, J. W., Jr., Ryer, R., 3rd, and Harriman, H. (1957).Science 126, 23. Dunn, C. G., Campbell, W. L., Fram, H., and Hutchins, A. (1948).J . Appl. Phys. 19. 605-616. Foster, F.L.,Jr., Dewey, D. R., 11,and Gale, A. J. (1953).Nucleonics 11, 14-17. Garrison, W. M., Jayko, M. E., and Weeks, B. M. (1958). April 7. Chem. Eng. News pp. 50-51;Jayko, M. E.,and Garrison, W. M. (1958). Radiation Research 9, 134135;Weeks, B. M., and Garrison, W. M. (1958).Zbid. 9, 202.213. Gomberg, H. J., Could, S. E., Nehemias, J. V., and Brownell, L. E. (1954). Food Eng. (September), pp. 78-80, 154-156. Groninger, H. S.,Tappel, A. L., and Knapp, F. W.(1956).Food Research 21,555-564. Gustafson, F. G., Brownell, L. E., and Martens, R. A. (1957). A m . Potato J . 34 (6), 177-182. Hannan, R. S. (1955). “Scientific and Technological Problems Involved in Using Ionizing Radiations for the Preservation of Food.” Her Majesty’s Stationery Office, London. Hannan, R.S. (1957). Bull. inst. intern. froid 37 (l), 179-209. Hassett, C. C.,and Jenkins, D. W.(1952). Nucleonics 10, 42-46. Hollaender, A. (ed.) (1954). “Radiation Biology.” McGraw-Hill, New York. Hollaender, A., and Stapleton, G. E. (1953). Physiol. Revs. 33, 77-84. Howard-Flanders, P.,and Alper, T. (1967).Radiation Research 7, 518-540. Howard-Flanders, P., and Moore, D. (1958).Radiation Research 9, 422-437. Hutchinson, F. (1957). Radiation Research 7, 473-483. Hutchinson, F. (1958). Radiation Research 9, 133. Kan, B., Goldblith, S. A., and Proctor, B. E. (1957).Food Research 22,509-518. Kelner, A,, Bellamy, W. D., Stapleton, G. E., and Zelle, M. R. (1955).Bacteriol. Revs. 19, 22-44. Kempe, L. L. (1955).Appl. Microbial. 3, 346-352. Kempe, L. L., Bonventre, P. F., Graikoski, J. T., and Williams, N. J. (1956). “A Conference on Radioactive Isotopes in Agriculture,” Supt. of pp. 253-263. Documents, Washington, D. C. Kempe, L. L., Graikoski, J. T., and Bonventre, P. F. (1957). Appl. Microbiol. 6, 292-295.

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Kertesz, Z. I., Morgan, B. H., Tuttle, L. W., and Louin, M. (1956). Radiation Research 6 , 372-381. Kilburn, R. E., Bellamy, W. D., and Terni, S. A. (1958).Radiation Research 9,207215. Knowlton, J. A.,Mahn, G. R., and Ranftl, J. W.(1953).Nucleonics 1 1 , 6 6 6 . Kuprianoff, J. (1955).2.Lebensm.-Untersuch. u.-Forsch. 100 (4),276303. Lawton, E.J., Balwit, J. S., and Powell, R. S. (1958).J . Polymer Sci. 32,257-274. Lea, U. E. (1947). “Actions of Radiations on Living Cells.” Cambridge Univ. Press, New York and London. Lea, D. E. (1955). “Actions of Radiations on Living Cells.” Cambridge Univ. Press. New York and London. Lehman, A. J. and Laug, E. P. (1954).Nucleonics 12,52-54. Littman, F.E.,Carr, E. M., and Clauss, J. R. (1957).Science 126, 737-738. Miller, A. A., Lawton, E. J., and Balwit, J. S. (1954).J . Polymer Sci. 14, 503-504. Miller, A. A.,Lawton, E. J., and Balwit, J. S. (1956).J . Phys. Chem. 60,699-604. Morgan, B. H. (1958/69).Intern. Conf. for the Peaceful Uses of Atomic Energy, 2nd Conf. Geneva. 22, in press. Morgan, B. H., and Reed, J. M. (1954). Food Research lB, 367-366. Nickson, J. J. (ed.) (1962). Symposium on Radiobiol., Oberlin College, 1960. Niven, C. F. (1958). Ann. Rev. Microbiol. 12, 507-524. Pan, H., Goldblith, S. A., and Proctor, B. E. (1958).J . A m . Oil Chemists’ Soc. 36, 1-5. Pollard, E. (1954).Advances i n Virus Research 2 , 109-161. Price, F. P., Bellamy, W. D., and Lawton, E. J. (1954). J . Phys. Chem. 68, 821-824. Proctor, B. E.,and Goldblith, S. A. (1948).Nucleonics 3, 32-43. Proctor, B. E.,and Goldblith, S. A. (1957). Am. J . Public Health. 47,439445. Proctor, B. E.,Lockhart, E. E., Goldblith, S. A,, Grundy, A. V., Tripp, G. E., Karel, M., and Brogle, R. C. (1964).Food Technol. 8, 536-640. Proctor, B. E., Nickerson, J. T. R., Licciardello, J. J . , Goldblith, S. A., and Lockhart, E. E. (1955).Food Technol. B, 523-627. Radouco-Thomas, C., Lataste-Dorolle, C., Zender, R., Busset, R., Mouton, R. F., and Bellamy, W.D. (1598/59).Intern. Conf. for the Peaceful Uses of Atomic Energy, 2nd Conf., Ueneva. 22, in press. Rajewsky, V. B., and Dose, K.(1967). 2.Naturforsch. lab, 384-393. Saeman, J. F., Millett, M. A., and Lawton, E.J. (1952). Ind. Eng. Chem. 44, 28482852. Shultz, A. R. (1958). I n “The Effect of Ionizing Radiation on Natural and Synthetic High Polymers” (F.A. Bovey, ed.), p. 138.Interscience, New York. Siu, R. G. H.(ed.) (1958). “Radiation Preservation of Food.” Supt. of Documents, Washington, D. C. Skaggs, L. S. (1956). Rept. U . S. Quartermaster Corps, For the Armed Forces Contract N o . 49-106-134-55(8-541). Sparrow, A. H., and Christensen, E. (1964).Nucteonics 12, 16-17. Stapleton, G. E., and Woodbury, D. H. (1957). Bacteriol. Proc. (SOC.Am. Bacteriologists) 67, 50. Swallow, A. J. (1952). J . Chem. SOC.pp. 1334-1339. Tappel, A. L. (1957).Food Research 22, 408-411. Tappel, A. L. (1958).Food Research 23,206212. Thornley, M. J. (1957).J . Appl. Bacteriol. 20, 286-298.

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Traub, F. B., Friedemann, U., Brasch, A., and Huber, W. (1951). J. Immunol. 67, 379-384. Trump, J. G., and Van de Graaff, H. J. (1948). J. Appl. Phys. 19,599-604. Urbain, W . M. (1958). Proc. Am. Meat Inst. Foundation, 10th Research Conf., Chicago. Vorhes, F . A. and Lehman, A. J. (1956). Public Health Repts. (U.S.) 71, 571-576. Wolfrom, M. L., Binkley, W. W., and McCabe, A. B. (1958). J. Am. Chem. SOC.In press. Wolin, E. F., Evans, J. B., and Niven, C. F. (1957). Food Research 22, 682-686. Zender, R., Lataste-Dorolle, C., Collet, R. A., Rawinski, P., Radouco-Thomas, C., and Mouton, R. F. (1958/59). Intern. Conf. for the Peaceful Uses of Atomic Energy, 8nd Conf ., Geneva. 22, in press.

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The Status of Antibiotics in Plant Disease Control' DAVIDPRAMER Department of Agricultural Microbiology, Rutgers, The State University, New Brunswick, New Jersey

I. Introduction. . . ................... 11. Selective Toxicity.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. Absorption and Transloca ..................................... IV. Mode of Action. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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I. Introduction The substances used most commonly for the control of plant diseases are compounds of sulfur, copper, and mercury. Since they are general poisons toxic to host tissue as well as to the pathogen, it is necessary that they be applied as protective coatings to the plant surface before exposure to infection. The use of surface protectants has enabled the phytopathologist to combat successfully many forms of disease. However, the limitations are obvious and the plant pathologist, having at last embraced the concept of selective toxicity (Albert, 1951), is now seeking chemicals that will destroy specific bacteria or fungi without injuring plant tissue. Selective toxicants may be absorbed by the plant and act systemically to control virus and vascular wilt diseases which by their nature are beyond reach of surface protectants. Likewise, systemically active compounds may: (a) decrease spray coverage requirements and make possible the use of lighter and less expensive field equipment; (b) reduce losses due to leaching; (c) protect new growth as it develops; (d) reduce the number of necessary treatments; and (e) permit destruction of pathogens without harm to beneficial microorganisms. Antibiotics are a group of organic chemicals distinguished by their selective toxicity to microorganisms and by their method of manufacture. They appeared on the commercial market a t approximately the same time that the systemic herbicides (2,4-D and 2,4,5-T) and the systemic insecticides (schradan and Systox) were ushering in a modern era of agricultural chem1 Paper of the Journal Series, New Jersey Agricultural Experiment Station, Rutgers, The State University of New Jersey, Department of Agricultural Microbiology, New Brunswick, New Jersey.

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icals. Although antibiotics were produced primarily for the medical profession and their use was limited by cost, some experiments were conducted soon after they were first produced commercially to determine their effectiveness in the control of plant diseases. Brown and Boyle (1944a, b) reported that penicillin controlled crown gall. Brian and Hemming (1945) showed that gliotoxin was able to limit various seed-borne diseases of cereals, and Brown and Heep (1946) eradicated Xanthomonas pruni from budwood infections using streptomycin. These and other early reports of the effectiveness of antibiotics in plant disease control were reviewed by Anderson and Gottlieb (1952). The results of more recent laboratory, greenhouse, and field studies have been summarized by Dunegan (1954), Leben and Keitt (1954), Tanner and Beesch (1958), and Zaumeyer (1955, 1956, 1958). Reviews by Darpoux (1952), Kohler (1953), Brian (1954), Dekker (1955), Schumann (1956), and Krasilnikov (1958) are indicative that interest in the use of antibiotics for plant disease control is widespread and international. These references provide an exhaustive summary of the literature, cataloguing most of the relevant papers published to date. In the present review no attempt was made at a comprehensive survey. Instead, discussion is limited to selected topics having fundamental and applied implications. Choice of material has been conditioned by the author’s interests and by his conviction that an understanding of the biochemical and physiological aspects of the subject is of great importance if antibiotics are to be employed effectively for the control of plant diseases.

It. Selective Toxicity Antibiotics are substances produced by microorganisms that have the capacity to inhibit or destroy other microorganisms. The antimicrobial spectrum of an antibiotic of value for plant disease control must include plant pathogenic bacteria, fungi, or viruses. Moreover, the substance must demonstrate little or no phytotoxicity when applied a t concentrations that inhibit or destroy the pathogen. Various investigators have demonstrated that antibiotics are active in vitro against many plant pathogens (Katznelson and Sutton, 1951; Mackay and Friend, 1953; Stessel et al., 1953a; Morgan and Goodman, 1955; Koaze et al., 1956; Napier et al., 1956a; Rangaswami, 1956; Thirumalachar et al., 1956; Muller, 1958). In some cases the substance tested was developed for use in the treatment of animal infections. However, there is no obvious correlation between activity against animal pathogens and plant pathogens. Moreover, there is no predictable correlation between in vitro and in vivo activity. In recent years screening procedures orientated toward plant pathological rather than medical requirements have been described (Stessel et al., 195313; Pridham et al., 1956a, b; Gray, 1957; Hirai et al., 1957; Shimo-

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mura et al., 1957). They were designed for the detection and isolation of compounds active against plant pathogenic bacteria, fungi, and viruses and have yielded a number of new antibiotics of agricultural interest that require further characterization and careful evaluation. It is apparent that antibiotics active against plant pathogenic microorganisms are not unique and may be obtained with comparative ease. The phytotoxicity of antibiotics was assessed by a number of procedures, including cytological techniques (Brown, 1948; Dufrenoy et aE., 1949)) a simple seed germination test (Wright, 1951; Gray, 1955a; Pramer and Wright, 1955), cell elongation of Avena coleoptile sections (Rosen, 1954a), nutrient solutions (Anderson and Nienow, 1947; Pramer, 1953; Rosen, 1954a; Norman, 1955), and spray applications (Altman and Bachelder, 1956). The extent and type of injury varied with the nature, concentration, and mode of application of the antibiotic tested. Likewise, different plants varied in their susceptibility to injury by any one antibiotic a t a given concentration. Usually root and shoot growth were inhibited more severely than seed germination. Streptomycin was of particular interest, for treated seedlings were stunted and partially devoid of chlorophyll (von Euler, 1948; Bogorad, 1950; Wright, 1951; Rosen, 195413; Pramer and Wright, 1955). Whether the bleaching effect of streptomycin was due to destruction of chloroplasts or to failure of colorless tissue to synthesize chlorophyll, or both, is not certain. However, it is not possible to obtain bleaching without toxic effects. Detailed discussions of the influence of antibiotics on plants were prepared by Brian (1957a, b). Most antibiotics that have been examined were found to be phytotoxic to some extent. Nevertheless, microorganisms are more sensitive than higher plants, and even the more toxic antibiotics can be used systemically at concentrations adequate for disease control without visibly affecting plant growth.

111. Absorption and Translocation The selective toxicity of antibiotics is exploited in full only when they act systemically and not when they are used as surface protectants. The distinction between surface and systemic action may appear obvious. However, it is possible that some compounds will penetrate the plant surface but fail to be translocated to any significant extent. They will not act systemically or behave as surface protectants, but assume an intermediate position of considerable importance (Davis and Rothrock, 1956). For antibiotics to act systemically it is necessary that they be absorbed and translocated by plants. Our knowledge of the movement of antibiotics in higher plants was reviewed by Crowdy and Pramer (1955). They indicated the ease with which antibiotics may be detected by biological tests but emphasized the importance of using highly specific bioassays and, when

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possible, providing chemical evidence for the presence of a translocated antibiotic in plant tissue. The latter recommendation was fulfilled when pure chloramphenicol and griseofulvin were recrystallized from extracts of the tops of root-treated broad bean plants. Estimates of these antibiotics in the tissues by bioassay agreed well with the chemical determinations (Crowdy et al., 1955,1956). The literature reviewed by Crowdy and Pramer (1955) showed that a number of antibiotics were absorbed and translocated, though some (gramicidin, neomycin, and subtilin) were not. Of the antibiotics that were translocated, some (chloramphenicol, griseofulvin, and penicillin) moved readily in all plants tested. Others (chlortetracycline, oxytetracycline, and streptomycin) appeared to move more freely in some plants than in others. In conclusion, it was noted that the readily translocated antibiotics were either neutral or acidic substances (chloramphenicol, griseofulvin, and penicillin), whereas anomalous results were reported for the basic (neomycin and streptomycin) and amphoteric (chlortetracycline and oxytetracycline) antibiotics. Little is known of the factors affecting antibiotic uptake. Stokes (1954) observed that respiratory inhibitors prevented the appearance of griseofulvin in guttation fluid of wheat and that the concentration of antibiotic measured in guttation fluid varied directly with the rate of transpiration. More recent studies (Crowdy et al., 1956) showed that the entry of griseofulvin into roots of broad bean plants was rapid and was inhibited by sodium azide and dinitrophenol at concentrations that did not reduce transpiration. Initial entry was followed by prolonged uptake, linearly related to transpiration and not affected by the inhibitors. In bean plants the accumulation of griseofulvin was most rapid in the lower leaves, whereas in tomato, the greatest accumulation was in the roots. The movement of chloramphenicol and streptomycin in these same two plants and in cucumber was investigated by Pramer (1953, 1954). A comparison of the results indicates that absorption and translocation are affected markedly by the nature of both the antibiotic and the plant tested. Temperature influenced the uptake of antibiotics by higher plants (Schrodter, 1956), and in many tests the concentration of antibiotic absorbed varied directly with that in the treating solution (Anderson and Nienow, 1947; Blanchard and Diller, 1951; Winter and Willeke, 1951a, b; Pramer, 1954; Stokes, 1954; Crowdy et al., 1956; Wallen and Millar, 1957). The rate and extent of antibiotic uptake was greater in cuttings than in rooted plants (Pramer, 1954; Robinson et al., 1954). Alcorn and Ark (1955, 1956) reported that movement of chlortetracycline, oxytetracycline, and tetracycline was more rapid than streptomycin in fire-thorn cuttings and that tetracycline moved more rapidly than neomycin or streptomycin in carnation cuttings. The rate of movement of streptomycin in carnation

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cuttings was enhanced by 1% dipotassium phosphate. Charles (1953) found the translocation of penicillin in cut petioles to be more rapid than that of streptomycin. Studies in which antibiotics were detected in the leaves of root-treated plants provide unequivocal evidence of uptake and translocation but afford little information regarding the mechanism of absorption involved. Pramer (1955, 1956, 1958a) and Litwack and Pramer (1957) employed cells of the alga Nitella clavata to investigate absorption without complication due to translocation. Streptomycin was absorbed rapidly and accumulated by a mechanism requiring an expenditure of energy on the part of the cell. Chloramphenicol penetrated slowly by simple diffusion, and the concentration of antibiotic in cell sap tended to equilibrate with that in the treating solution. No evidence of penicillin uptake was obtained, and the authors cautioned that observations on algae cannot be generalized or applied to higher plants without reservation. Furthermore, there is no justification for concluding that an antibiotic absorbed by roots and translocated to leaves will be absorbed by leaves and distributed throughout the plant. Root uptake is of great interest and importance, but the roots of agricultural crops are seldom accessible for treatment. The use of soil drenches containing antibiotics appears unsound technically and not admissible economically since our present knowledge (Pramer, 1958b) indicates that some antibiotics in soil are unstable chemically and that many, if not all, are degraded microbiologically. The more practical method of application of antibiotics is in foliar sprays. The absorption and translocation of antibiotics by leaves are receiving increased attention. Gray (195513) applied sprays containing the antiviral antibiotic noformicin to leaves and reduced infection. Evidence was obtained that the antibiotic was absorbed and translocated from the base to the tip of bean leaves and to a less extent, in the reverse direction. In more recent studies Gray (1958) showed that pleocidin and streptothricin were absorbed by intermediate leaves of bean plants and moved upward and downward to younger and older growth respectively. When primary leaves were immersed in solutions containing 1000 p.p.m. of streptothricin the antibiotic was translocated downward to the roots and upward to younger leaves. Downward movement took place through steam-killed sections of petioles and stems indicating that translocation occurred principally in the xylem. Streptomycin, dihydrostreptomycin, neomycin, oxamycin, bacitracin, and actinomycin did not move out of sprayed leaves, but several of these substances were absorbed and translocated by leaves immersed 2 to 5 days in a concentrated solution of the antibiotic. Cycloheximide applied as a spray to the leaves of wheat was absorbed and recovered from plant tissue for 5 weeks after application (Wallen and

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Millar, 1957). Acetate, anhydro, isomer, oxime, and semicarbazone derivatives moved more readily than cycloheximide itself (Hamilton et al., 1956). The oxime was absorbed by sprayed leaves and translocated to new growth in concentrations adequate to control cherry leaf-spot. Streptomycin was absorbed by stems of bean (Mitchell et al., 1953,1954) and tobacco (Hidaka and Murano, 1956b), and phytotoxic effects that developed after spraying suggested that the antibiotic penetrated leaves of pear seedlings (Dunegan and Wilson, 1953). However, the ease with which streptomycin is absorbed and translocated from foliar sprays appears to depend on the nature of the plant treated. Streptomycin did not enter leaves of pepper plants (Crossan and Krupka, 1955). Goodman’s observation (1955) that streptomycin sprays were relatively ineffective against lateseason twig-infection indicates that the antibiotic was not acting systemically, and although Dye (1956) noted that streptomycin entered peach leaves, he obtained no evidence that it became truly systemic. Similar results were obtained by Crosse and Garrett (1958) who concluded that streptomycin was not distributed systemically from foliar sprays on cherry trees, but that localized activity was an important factor in the control of bacterial canker. In contrast to the foregoing reports which indicate that streptomycin is a penetrant rather than a systemic, there are papers describing movement of the antibiotic by sprayed foliage of bean (Crossan and Krupka, 1955; Napier et al., 1956b), maize (Sabet, 1956), tobacco (Hidaka and Murano, 1956a, b), broccoli (Natti, 1957); and Coleus sp. (Dowler and Goodman, 1958). Absorption was greater in those cases where the antibiotic was applied to lower leaf surfaces (Hidaka and Murano, 1956a; Dowler and Goodman, 1958). A method for studying the absorption of streptomycin by leaves was developed and described by Lockwood (1958). There is little information concerning factors influencing the foliar absorption of antibiotics. Goodman (1954) increased the effectiveness of antibiotic sprays by supplementing them with a cuticle solvent (methyl Cellosolve) and a wetting agent (Carbowax 400~).In later studies Goodman and Hemphill (1954) reported that indole-3-acetic acid markedly increased the ability of antibiotic spray formulations to control fire blight on apple shoots. The ethyl ester was as effective as indole-%acetic acid, and similar results were obtained using a number of different plant growth regulators (Hemphill and Goodman, 1955). Gray (1956) found that the addition of glycerol to streptomycin sprays increased absorption of streptomycin by leaves of bean seedlings. The absorption of chloramphenicol and streptothricin was also influenced by glycerol, but that of neomycin and oxytetracycline was not. A number of polyhydroxy alcohols exhibited activity comparable to

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that of glycerol. The mode of action of these materials in increasing antibiotic absorption remains to be defined. The interest of Russian investigators in the subject of the present review dates from 1946. They have shown that a number of antibiotics were absorbed by plants through their roots, but not all were translocated, and the distribution of those that moved varied with the antibiotic and plant species examined (Krasilnikov, 1952a, b, 1953, 1954; Mirzabekian and Menkova, 1955). The uptake of penicillin and streptomycin by foliage was demonstrated using maize, pea, and tomato plants. Chlortetracycline was not absorbed by leaves. In some cases antibiotics were detected in the roots of leaf-treated plants. However, it was necessary to maintain the plants in humidity chambers and even then downward movement was not rapid. The results of Russian studies to evaluate the usefulness of antibiotics for plant disease control were summarized by Mirzabekian (1953) and Krasilnikov (1958).

IV. Mode of Action Antibiotics may control plant diseases by (a) acting directly on the pathogen; (b) neutralizing toxins secreted by the pathogen; (c) acting on the host; (d) being transformed within the plant to a substance having greater or different activity; or (e) a combination of two or more of these effects. Pramer et al. (1956) presented convincing evidence that the control of bacterial wilt of chrysanthemums by streptomycin was due to direct action of the antibiotic on the pathogen. Nevertheless, it is not possible to generalize this result since recent studies (Bonde, 1953; Grosso, 1954; Muller et al., 1954) describing control of potato and tomato late blight and tobacco blue mold in vivo by antibiotics that have no antifungal activity in vitro, can be explained only by an indirect mode of action. The ability of streptomycin-treated potato plants to resist infection by Phytophthora infestans is of particular interest, for evidence was presented (Voros et al., 1957) that the antibiotic acted indirectly by stimulating the plant polyphenol oxidase system to produce quinones a t tissue concentrations that inhibited the pathogen. The mode of action of an antibiotic is not necessarily a fixed characteristic of the compound. The plant itself may exert a controlling influence. This point was emphasized in a study by Hilborn (1953) who treated different plant species with the same antibiotics and inoculated with the same pathogen. Rhizoctonia infection of lettuce was controlled completely by magnamycin. It was reduced somewhat in potato and unaffected in tomato plants treated similarly with magnamycin. Likewise, chloramphenicol reduced the severity of Verticillium infection of potato but not of tomato.

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These different effects produced by the same antibiotic on the same pathogen in different hosts indicate the complex interrelationships that may be expected in studies of the mode of action of antibiotics for plant disease control. It is important to know whether systemically active compounds are transformed by the plant and whether the product of any such transformation is more or less active than the original substance. Fusaric acid, which is both a wilt toxin and an antibiotic, produced by species of Fusarium, was decarboxylated and transformed into neutral and basic substances by tomato cuttings (Sanwal, 1956). The degradation of griseofulvin in bean plants was observed by Crowdy et at!. (1956) to occur logarithmically. Prescott et al. (1956) observed that the rate of inactivation of cycloheximide was much greater on ripe cherry fruit than in aqueous solutions of similar pH and suggested that enzymatic reactions were involved. Streptomycin amine and streptomycin oxime were converted in plants to compounds of an unknown nature that demonstrated greater antibacterial activity than the original materials (Gray, 1958). In no case has unequivocal evidence been presented that disease control results from a degradation or transformation product of the antibiotic tested. However, in considering the mode of action of substances distributed within plants it is a possibility that cannot be ignored. Furthermore, the fact that an antibiotic may have two or more modes of action is worthy of consideration. The literature provides no clear example of an antibiotic demonstrating a multiple mode of action in plant disease control, but it is not difficult to visualize a situation in which this would be the case. Evidence for a unique mode of action was presented by Klemmer et al. (1955) who tested eight antibiotics for their effects on the crown gall organism as well as the growth of gall and normal plant tissue. Although the pathogen was sensitive in uitro to the same antibiotics that reduced gall formation in vivo, living cells of the bacterium were abundant in treated plants where disease was suppressed completely. This apparent paradox was resolved by further studies in which tissue culture techniques and antibiotic-resistant strains of Agrobacterium tumefaciens were employed. The results showed clearly that gall inhibition by chloramphenicol, chlortetracycline, and oxytetracycline was not due to their antibacterial activity. The mode of action was indirect and depended on the selective toxicity of the antibiotics to gall cells. V. Summary Information derived from numerous sources clarifies the status of antibiotics as chemicals for the control of plant diseases. It is not difficult to isolate antibiotics which are active against plant pathogenic microorgan-

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isms and demonstrate little or no phytotoxicity. Nevertheless, most of the materials available were developed for medical rather than plant pathological use and serve only as an indication of what may be obtained from a properly orientated search. The selective toxicity of antibiotics is not exploited in full when they are employed as surface protectants. Many antibiotics are absorbed by the roots of plants, translocated to stems and leaves, and act systemically to control infection. Root uptake is of greater academic than applied interest since in practical agriculture roots are seldom available for treatment, whereas foliar sprays are assuming increasing importance. Some antibiotics penetrate foliage but many do not. Those that are absorbed are not translocated readily and act as penetrants rather than as systemics. The results obtained vary with the nature of the antibiotic and plant tested and are influenced by a number of factors including antibiotic concentration, method of application, temperature, and humidity. It is not surprising that our knowledge of the mode of action of antibiotics in plant disease control is obscure since the possibilities are legion and almost completely unexplored. Despite the difficulties involved and the relatively short time antibiotics have been studied in this regard, some of the most interesting examples of systemic protection of plants against microbial attack have involved the use of antibiotics.

REFERENCES Albert, A. (1951). “Selective Toxicity,” Wiley, New York. Alcorn, S. M., and Ark, P. A. (1955). Phytopathology 46,692. Alcorn, S. M., and Ark, P. A. (1956). Appl. Microbiol. 4,126-130. Altman, J . , and Bachelder, S. (1956). Plant Disease Reptr. 40, 1081-1083. Anderson, H . W., and Gottlieb, D. (1952). Econ. Botany 6,294-308. Anderson, H . W., and Nienow, I . (1947). Phytopatholoyg 37, 1. Blanchard, F. A., and Diller, V. M. (1951). Am. J . Botany 38,111-112. Bogorad, L. (1950). A m . J . Botany 37,676. Bonde, R. (1953). Phytopathology 43,463-464. Brian, P . W. (1954). J. Appl. Bacteriol. 17, 142-151. Brian, P. W. (1957a). Ann. Rev. Plant Physiol. 8, 413-423. Brian, P . W. (1957b). Symposia Soe. Exptl. Biol. No. 11, 166-182. Brian, P. W., and Hemming, H. G. (1945). Ann. Appl. Biol. 32,214-220. Brown, J. G. (1948). Phytopathology 38, 3. Brown, J. G., and Boyle, A. M. (1944a). Science 100, 528. Brown, J. G., and Boyle, A. M. (1944b). Phytopathology 34, 760-761. Brown, J. G., and Heep, D. M. (1946). Science 104, 208. Charles, A. (1953). Nature 171,43H36. Crossan, D. F., and Krupka, L. R. (1955). Plant Disease Reptr. 39,480-483. Crosse, J. E., and Garrett, M. E. (1958). Ann. Appl. Biol. 46, 310-320. Crowdy, S. H., and Pramer, D. (1956). Chem. & Ind. (London) 160-162. Crowdy, S. H., Gardner, D., Grove, J. F., and Pramer, D. (1955). J. Exptt. Botany 7, 371-383.

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Crowdy, S. H., Grove, J. F., Hemming, H. G., and Robinson, K. C. (1956). J. Ezptl. Botany 6, 42-64. Darpoux, H. (1952). Congr. intern. phytophurm., Compt. rend. 36 Congr., Paris, 1962, pp. 125-128. Davis, D., and Rothrock, J. W. (1956). Plant Disease Reptr. 40, 328-329. Dekker, J. (1955). Mededel. Directeur Tuimboun 18, 507-576. Dowler, W. M., and Goodman, R. N. (1958). Phytopatholoyg 48,392. Dufrenoy, J. R., Pratt, R., and Pickering, V. L. (1949). Phytopathology 39, 859. Dunegan, J. C. (1954). J . Agr. Food Chem. 2, 1020-1022. Dunegan, J. C., and Wilson, R. A. (1953). Phytopathology 43, 405. Dye, M. H. (1956). Nature 178,551-552. Goodman, R. N. (1954). Missouri Univ., Agr. Expt. Sta., Research Bull. No. 640. Goodman, R. N. (1955). Plant Disease Reptr. 39, 922-925. Goodman, R. N., and Hemphill, D. D. (1954). Science 119, 347-348. Gray, R. A. (1955a). Am. J . Botany 42,327-331. Gray, R. A. (1955b). Phytopathology 46,281-285. Gray, R. A. (1956). Phytopathology 46, 106111. Gray, R. A. (1957). Plant Disease Reptr. 41,576-578. Gray, R. A. (1958).Phytopathology 48, 71-78. Grosso, J. J. (1954). Plant Disease Reptr. 38, 333. Hamilton, J. M., Sekolnik, M., and Sondheimer, E. (1956). Science 123, 1175-1176. Hemphill, D. D., and Goodman, R. N. (1955). Science 122,122. Hidaka, Z., and Murano, H. (1956a). Ann. Phytopathol. Soc. Japan 20, 143-147. Hidaka, Z., and Murano, H. (1956b). Ann. Phytopathol. Soc. Japan 21,49-54. Hilborn, M. T. (1953). Phytopathology 43, 475. Hirai, T., Shimomura, T., Yamaguchi, A., Matsui, C . , and Nishikawa, Y., (1957). Ann. Phytopathol. Soc. Japan 22, 70-74. Katznelson, H., and Sutton, M. D. (1951). Can. J . Botany 29, 270-278. Klemmer, H. W., Riker, A. J., and Allen, 0. N. (1955). Phytopathology 46,618-625. Koaze, Y., Sakai, H., Yonehara, Y., Asakawa, M., and Misato, T. (1956). J. Antibiotics (Japan),Ser. A , 9,89-96. Kohler, H. (1953). Nachrbl. deut. Pjlanzenschutzdienst (Berlin) 7, 108-113. Krasilnikov, N. A. (1952a). Priroda 41, 17-27. Krasilnikov, N. A. (1952b). Uspekhi Sovremennoi Biol. 33,325-327. Krasilnikov, N. A. (1953). Izvest. Akad. Nauk S. S . S . R. Ser. Biol. 2.49-66. Krasilnikov, N. A. (1954). Doklady Akad. Nauk S . S . S . R . 94, 1177-1180. Krasilnikov, H. A. (1958). “Soil Microorganisms and Higher Plants,” Academy of Sciences, Moscow. Leben, C., and Keitt, G. W. (1954). J. Agr. Food Chem. 2, 234-239. Litwack, G., and Pramer, D. (1957). Arch. Biochem. Biophys. 68, 39t3402. Lockwood, J. L. (1958). Phytopathology 48,150-155. Mackay, J. H. E., and Friend, J. N. (1953). Australian J . B i d . Sci. 6,481-484. Mirzabekian, R. 0. (1953). Zzvest. Akad. Nauk S. S . S . R . Ser. Biol. 2, 67-88. Mirzabekian, R. O., and Menkova, K. A. (1955). Izvest. Akad. Nauk S . S . S . R . Ser. Biol. 6, 10-19. Mitchell, J. W., Zaumeyer, W. J., and Preston, W. H., Jr. (1953). Phytopathology 43,480. Mitchell, J. W., Zaumeyer, W. J., and Preston, W. H., Jr. (1954). Phytopathology 44, 2530. Morgan, B. S., and Goodman, R. N. (1955). Plant Disease Reptr. 39,487490. Muller, K. O., Mackey, J. H. E., and Friend, J. N. (1954). Nature 174, 878-879.

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Muller, W. H. (1958). Am. J . Botany 46, 183-189. Napier, E. J., Turner, D. I., and Rhodes, A. (1956a). Ann. Botany (London) 20,461467. Napier, E . J., Turner, D . I., Rhodes, A., and Tootill, J. P. R. (1956b). Ann. A p p l . B i d . 44, 145-151. Natti, V . J. (1957). Phytopathology 4 w 5 . Norman, A. G. (1955). Arch. Biochem. Biophys. 68, 461-477. Pramer, D. (1953). Ann. A p p l . Biol.40, 617-622. Pramer, D . (1954). Ann. Botany (London) 18,463470. Pramer, D. (1955). Science 121, 507-508. Pramer, D. (1956). Arch. Biochem. Biophys. 62, 265-273. Pramer, D. (1958a). E z p t l . Cell Research 16, 70-74. Pramer, D. (1958b). A p p l . Microbiol. 6, 221-224. Pramer, D., and Wright, J. M. (1955). Plant Disease Reptr. 39, 118-119. Pramer, D., Robison, R. S., and Starkey, R. L. (1956). Phytopathology 46,341-342. Prescott G. C., Emerson, H., and Ford, J. H. (1956). J . A g r . Food Chem. 4,343-345. Pridham, T. G., Lindenfelser, L. A., Shotwell, 0. L., Stodola, F. H., Benedict, R . G., Foley, C., Jackson, R . W., Zaumeyer, W. J., Preston, W. H., Jr., and Mitchell, J. W. (1956a). Phytopathology 46, 568-575. Pridham, T. G., Shotwell, 0. L., Stodola, F. H., Lindenfelser, L. A., Benedict, R . G., and Jackson, R. W. (195613). Phytopathology 46,575-581. Rangaswami, G. (1956). Mycologia 48, 8W804. Robison, R. S., Starkey, R. L., and Davidson, 0. W. (1954). Phytopathology 44, 646-650. Rosen, W. G. (1954a). Proc. SOC.E z p t l . Biol.Med. 86,385-388. Rosen, W . G. (1954b). Ohio J . Sci. 64,73-78. Sabet, K. A. (1956). Ann. A p p l . Biol.44, 152-160. Sanwal, B. D. (1956). Phytopathol. 2.26, 333-384. Schrodter, H. (1956). Umschau Fortschr. Wiss. u. Tech. 66,114-115. Schumann, G. (1956). Nachrbl. deut. Pflanzenschutzdienst (Berlin) 8,73-78. Shimomura, T., Nishikawa, Y., and Hirai, T. (1957). Ann. Phytopathot. Soc. J a p a n 22,260-264. Stessel, G. J., Leben, C., and Keitt, G. W. (1953a). Phytopathology 43, 23-26. Stessel, G. J., Leben, C., and Keitt, G. W., (195313). MycoZogia 46, 325-334. Stokes, A. (1954). Plant and Soil 2, 132-142. Tanner, F. W., Jr., and Beesch, S. C. (1958). Advances in Enzymol. 20, 383-406. Thirumalacher, M. J., Patel, M. K., Kulkarni, N. B., and Dhande, G. E. (1956). Phytopathology 46, 486-488. von Euler, H. (1948). Arkiv. K e m i , Mineral. Geol. 26A, 1-9. Voros, J., Kirhly, Z., and Farkas, G. L. (1957). Science 126, 1178-1179. Wallen, V. R., and Millar, R . L. (1957). Phytopathology 47, 291-294. Winter, A. G., and Willeke, L. (1951a). Naturwissenschaften 38, 262-264. Winter, A. G., and Willeke, L. (1951b). Naturwissenschaften 38, 457458. Wright, J. M. (1951). Ann. Botany (London) 16,493-499. Zaumeyer, W. J. (1955). J . Agr. Food Chem. 3, 112-116. Zaumeyer, W. J. (1956). Intern. Conf. Antibiotics Agr., f s t Conf., Proc. pp. 171-187. Zaumeyer, W. J. (1958). Ann. Rev. Microbiol. 12, 415-440.

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Microbial Synthesis of Cobamides’ D. PERLMAN Squibb Institute for Medical Research, New Brunswick, New Jersey

I. Introduction.

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B. Nomenclature.

A. Microbiological Metho

111. Microbial Processes for Synthesis of Naturally Occurring Cobamides.. . . . . A. Selection of Microorganism.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Media Used in the Production of Cobamides.. . . . . . . . C . Precursors for the Biosynthesis of Cobamides.. ....................... D. Preparation of Radioactive Cob E. Occurrence of 5,6-dimethyl-a Microbial Fermentations F. Vitamin BIZand Cobami G. Cobamide Peptides from IV. Microbial Synthesis of “Unnatural Cobamides” .......................... A. Total Synthesis.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Partial Synthesis.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . obamides.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ...............................................

97 97 102

106 106 110 110 112

1. Introduction A. HISTORICAL BACKGROUND The first evidence that vitamin BIZ (later named cobamide) could be a fermentation product was presented in July, 1948, by Stokstad et al. Their bacterium, later classified as Fluuobmterium solare (Petty and Matrishin, 1949), produced a product which was active in the animal protein factor assay in chicks and was effective in the treatment of pernicious anemia in human patients. In a report appearing in December, 1948, Rickes et d. (1948~)gave comparative data on crystalline vitamin Blt isolated from liver and from media fermented by a grisein-producing strain of Streptomyces griseus. In this communication Rickes et al. mentioned that culture broths from fermentations by Mycobmterium smgmatis, Lactobacillus 1 Presented in part in lectures before School of Pharmacy, University of Wisconsin, March, 1958.

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arabinosus, Bacillus subtilis, Streptomyces roseochromogenus, and Streptomyces antibioticus contained significant amounts of this vitamin as measured by the microbial assay based on the growth response of Lactobacillus lactis (Dorner strain) devised by Shorb (1947a, 1948). These experimental programs were related to a continuing study over a 20-year period of the anti-anemia factor present in liver (Schindler and Reichstein, 1952; Smith, 1948a, 1949, 1952a, 1955; SubbaRow et al., 1945; Wijmenga, 1952). The isolation in pure crystalline form of the anti-anemia factor present in liver had been announced in April, 1948, by Rickes et al. (1948a) and achieved shortly thereafter in other laboratories (Ellis et al., 1949a; Fantes et al., 1950a; Smith, 1948a, o; Wijmenga et al., 1949). The activity of the crystalline material (isolated from both the liver source and the microbial fermentations) in treatment of pernicious anemia was demonstrated by West (1948) and Rickes et al. (194%) and in the animal protein factor test (Ott et al., 1948; Rickes et al., 1948~).As this review will be primarily concerned with a summary of the investigations studying the problems involved in microbial synthesis of the vitamin BIZgroup, a discussion of the biological properties is not considered pertinent to this objective, and for information on these properties the reader is advised to consult the reviews and publications dealing directly with these attributes (Briggs and Daft, 1955; Coates and Kon, 1957; Coates et al., 1951a; Hausmann, 1953; Heinrich and Lahann, 1954; Jukes and Stokstad, 1951; Smith, 1957a, c; Stokstad et al., 1949, 1950; Wijmenga, 1952). Barker et al. (1958) have found that pseudovitamin BIZis a portion of the coenzyme in the enzymatic conversion of glutamate to P-methylaspartate by cell extracts of Clostridium tetanomorphum. The elucidation of the chemical structure of vitamin BIZis one of the more outstanding examples of the successful collaboration of the chemist, the X-ray crystallographer, and the biologist. The chemistry of this complex molecule has been reviewed by those actively connected with the research and their interpretation of the X-ray crystallography (Brink et al., 1954; Hodgkin et al., 1955, 1957) and the degradative studies leading to the confirmation of the structural formula proposed by Dr. Hodgkin and associates should be consulted for a guide to the literature (Bonnett et al., 1955; Folkers and Wolf, 1954; Folkers et al., 1957; Smith, 1955, 1957a; Wijmenga and Veer, 1952; Wolf and Folkers, 1954). However, some aspects of the chemistry of the vitamin are pertinent to our discussion of microbial synthesis of the vitamin. These include the discovery of the presence in the vitamin of the elements cobalt (Rickes et al., 1948b; Smith, 1948b) and phosphorus (Brink et al., 1949; Buchanan et d.,1950a, b; Ellis et al., 1949a, b; Rickes et al., 1948a), the isolation from the degradation mixtures of 5,6dimethylbenzimidazole (Armitage et al., 1953; Beaven et al., 1949) and

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the fragments which suggested the pyrrole nucleus (Brink et d.,1949), as well as the projection of vitamin BIZ as a cyanide complex (Brink et d., 1950; Veer et al., 1950; Wijmenga et al., 1950). Degradation of the pseudovitamin B12group showed the presence of adenine and 2-methyladenine instead of the benzimidazole derivative (Dion et al., 1952,1954; Smith and Brown, 1954), and similar treatment of other vitamin Blz-like factors showed the presence of hypoxanthine and 2-methylhypoxanthine (Brown and Smith, 1954; Smith, 1957b). The recent studies by Barker (1959) have shown that vitamin BIZexists in nature as a group of coenzymes containing an adenine moiety attached to the cobalt. The importance of microbial synthesis of this group of vitamins has been summarized as follows by Smith (1950-51) : “It seems probable that the only primary source of vitamin Blz in nature is the metabolic activity of microorganisms; there is no convincing evidence for its elaboration in tissues of higher plants or animals. It is synthesized by a wide range of bacteria and actinomycetes, though apparently not to any extent by yeasts or fungi.” Robbins et al. (1950b) have concurred and stated, “It appears probable that the synthetic activity of microorganisms especially bacteria and actinomycetes is the original source of vitamin BIZ in nature.” Pure crystalline material has been isolated from the residues of a number of antibiotic-producing fermentations including those used for the production of grisein (8.griseus), (Rickes and Wood, 1951; Rickes et al., 1948c), of streptomycin (8.griseus) (Fricke el al., 1950; Janicki et al., 1953a; Rickes and Wood, 1951, 1954, 1955b), of chlortetracycline (Streptomyces aureofuciens) (Pierce et al., 1950), and of neomycin (Streptomyces fradiae) (Jackson et al., 1951). Microbial synthesis was also implicated in the early studies of the occurrence of the animal protein factor in manures and feces (Cary et al., 1946; Combs et al., 1948; Hartman and Cary, 1946; Lillie et al., 1948; Rubin and Bird, 1946). Subsequent investigations which showed that the potency of incubated feces and manures was higher than the freshly voided material confirmed the earlier hypotheses of the importance of microorganisms in this synthesis (Groschke et al., 1950a, b; McGinnis et al., 1947; Sahashi et al., 1953). With this background of information the finding of vitamin BIZin sewage was not unexpected (Hoover et al., 1951), and some efforts have been made to exploit this source (Bernhauer and Friedrich, 1954; Friedrich et al., 1957; Stevens et al., 1955; Wolnak and Zinn, 1958). While consideration of the commercial aspects of the production of vitamin BUis beyond the scope of this review, some mention of the development of the commercial processes is of interest as representative of the application of the results of laboratory research studying microbial synthesis of the vitamin. At first the vitamin Blz used for human therapy and

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as a supplement for animal feeds was produced as a by-product of antibioticproducing fermentations. These included processes producing streptomycin (Brunings et al., 1953; Janicki et al., 1953a, b; Schindler and Reichstein, 1952; Smith and Ball, 1953), neomycin (Jackson et al., 1951), chlortetracycline (Pierce et al., 1950), tyrothricin (Lugones and Mundel, 1952), and trichomycin (Matsuda, 1955; Matsuda and Nunome, 1955). Waste-liquors from a fermentation plant used for production of acetone and butanol were also utilized (Oguni, 1955). As the demand for the vitamin grew, it became economically feasible to use processes in which the vitamin Bl2 was the product of major interest. A number of processes were tested and apparently operated on a commercial scale. These included the processes using the following organisms: Bacillus megaterium (Anonymous, 1952; Lewis et al., 1949; McGinnis et al., 1949); Streptomyces olivaceus (Hester and Ward, 1954); Streptomyces sp. (ATCC 11072) (Pagano and Greenspan, 1954); Propimibacterium freudenreichii (Sudarsky and Fisher, 1957); and a mixed culture fermentation containing Pseudommm species, Streptococcus bovis, Proteus vulgaris, and Clostridium putrifium (Hanson and Hodge, 1957; Hodge et al., 1952). The products of some of these fermentations were used mainly for enrichment of feeds low in animal protein factor (a use promoted by Ansbacher and Hill, 1949; Ansbacher et al.,1949) while the pure material was isolated for use in human therapy. Further information concerning aspects of the commercial-scale operations is found in the discussions by Takata (1957) and Wuest (1954). At present there are fermentation manufacturing operations for the production of vitamin B12 located in at least twelve countries.

B. NOMENCLATURE As the investigations of the structure and biological properties of the vitamin Bla group of substances isolated from microbial cultures and fermented materials proceeded, it became evident that several different entities were under study. Among these were the materials called vitamin BI2a (Kaczka et d.,1951; Lichtman et al., 1949; Pierce et al., 1949; 1950), vitamin B120 (Buchanan et al., 1950b; Smith, 1950a, b), vitamin B12r (Lewis et al., 1952a, b), pseudovitamin B12 (Pfiffner et aZ., 1951, 1952), pseudovitamin BIZd(Dion et al., 1954; Pfiffner et al., 1954), factor I11 (Bernhauer and Friedrich, 1954), factor A (Brown et al., 1955), factor B (Armitage et al., 1953; Ford et al., 1951, 1952a), and factor H (Brown et d.,1955). With the perfection of analytical techniques, especially those of paper chromatography and paper ionophoresis (Holdsworth, 1953; Kon, 1955; Smith, 1952d) and elucidation of the structural formulas of the vitamin B I ~ group, it became possible to devise more descriptive systems of nomenclature. One of the systems proposed at a meeting in Hamburg in 1956 (and

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NH

':CN

FIG.1 . Relationship of nomenclature to structure of vitamin BIZmolecule

summarized in part by Smith, 1957d) proved particularly useful in designating the group of factors produced by microbial systems. A summary of this scheme is presented in Fig. 1.2 In this scheme the cobalt porphyrin nucleus (called factor B by Ford et d.,1951) is called cobinamide, and that portion of the molecule containing the cobalt, the porphyrin, the ribose, and the phosphate is called cobamide. The purine, benzimidazole, or other base found in the nucleotide-like portion of the molecule is specified, and all members of the vitamin Bl2 group are considered as salts (Kacaka et al., 1950). Thus, cyanocobalamin (Brink et al., 1950) becomes 5,6-dimethyIa-benzimidazolylcobamide cyanide; vitamin BIZ^ and BIZ,, are 5,6-dimethyIa-benzimidazolylcobamide hydroxide; vitamin Bizo is 5,6-dimethyl-abenzimidazolylcobamide nitrite; vitamin B1aIII (or factor 111) is 5-hydroxy-c~-benzimidazolylcobamide cyanide; pseudovitamin BIZ is a2 The recent report by Barker (1959) of the isolation of coenzymes containing a-adenylcobamide, a-benzimidazolylcobamide and 5,0-dimethyl-~-benzimidazolylcobamide moieties will necessitate some revision of this system of nomenclature.

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adenylcobamide cyanide and pseudovitamin B12d is 2-methyl-a-adenylcobamide cyanide. The reviews by Ford and Hutner (1955), Kon (1955), and Porter (1957) contain discussions of the identities of many of the members of the vitamin Blz group isolated from microbial sources.

II. Analytical Methods for the Determination of Cobamides To provide a perspective in evaluating the technical literature, it is advisable to give some consideration to those methods which have been useful in the detection of cobamides produced by microorganisms. The recent reviews by Coates and Ford (1955), Coates and Kon (1957), Ford and Hutner (1955), Hutner et al. (1958), and Jukes and Williams (1954) all include information on the technical aspects of the various assay methods for the determination of cobamides in various solutions and cellular materials. The combined use of a bioassay method and paper chromatography or paper ionophoresis is capable of yielding sufficient information for most purposes.

A. MICROBIOLOQICAL METHODS The usefulness and value of microbiological assays in studying the biosynthesis of cobamides and for detection of cobamides in natural materials cannot be overemphasized. Smith (1955) commented, “In retrospect it may seem less surprising that the isolation of vitamin Blz was so long delayed than that it was ever accomplished with clinical tests as the only guide. My colleagues and I at Glaxo Laboratories were denied the microbiological assay that helped forward the prior iso1atio.nin America by Rickes and colleagues of Merck.” The microbiological assay mentioned was that based on the growth response of Lactobacillus lactis (Dorner strain), as first described by Shorb (1947a, b). In this assay the amount of material needed to produce half-maximal growth was determined, and experimental tests showed that the pure crystalline 5,6-dimethyl-a-benzimidazolylcobamide cyanide had a potency of 11 million “LLD units” per milligram (Shorb, 1948). The growth response of this strain was found to be somewhat variable with changes in aeration of the culture (Greene et al., 1949), the presence of ribosides in the samples (Shive et al., 1948), and dissociation of the culture (Shorb and Briggs, 1948). In some instances the values obtained with the chick test were much lower than those obtained with the microbial assay (Coates et al., 1951b, 1953; Menge et al., 1952). When only one variety of cobamide was present in the samples, agar diffusion assays were relatively satisfactory, but when fermentation samples containing mixtures were analyzed, the zones of growth of the L. lactis culture were often “fuzzy-edged” and difficult to measure accurately (Cuthbertson et al.,

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1951; Foster et al., 1949; Larkin and Stuckey, 1951; Smith and Cuthbertson, 1949). Substitution of strains of Lactobacillus leichmannii for the L. lactis resulted in more reproducible assays (Jukes and Williams, 1954; Skeggs et al., 1948). Although a number of problems were encountered, this turbidimetric assay found general acceptance for the analysis of fermentation samples and other materials (Emery et al., 1951; Jukes and Williams, 1954; Peeler et al., 1949; Pritchard, 1951; Sakai and Tsunoda, 1951; Southcott and Tam, 1953). I n some instances the presence in the samples of traces of physiologically active substances such as antibiotics, inorganic salts, purines, and degradation products from cobamides lead to inaccurate assay results (Cuthbertson et al., 1956; Hendlin and Wall, 1954; Jukes and Williams, 1954;Princivalle and Lorch, 1957; Tarr, 1952; Weygand and Wacker, 1950; Weygand et al., 195413). Further investigations showed that all of the vitamin BIZ-requiring lactobacilli respond to varying degrees to all of the cobamides but not to cobinamide (Ford, 1953a, b; Peterson et al., 1956). These differences in response have been used as the basis for differential-type assays, but unsatisfactory results have been obtained when samples contained more than two types of cobamides (Berman et al., 1956). Davis and Mingioli (1950) reported that certain mutants derived by treatment of E. coli (ATCC 9637) with ultraviolet light required either vitamin BIZ or methionine for growth. One of these, strain 113-3, has been widely used as a test organism in bioassays for the determination of the cobamide content of fermentation materials and other natural products (Bessell et al., 1950; Burkholder, 1951; Chiao and Peterson, 1953; Hine et al., 1958). As this strain responds to all of the cobamides as well as to cobinamide (Coates and Ford, 1955; Ford and Hutner, 1955)) it has been very useful for detection of all of these factors in bioautographs of paper chromatograms of such solutions (Ford and Holdsworth, 1952). In another bacterial assay, a “wild-type” culture of E. coli was used, This assay was based on the ability of cobamides to reverse the growthinhibitory effects of sulfanilamide on this strain, and all cobamides were active in this assay (Shive, 1949). The usefulness of the growth response of certain protozoa as a method of measuring the vitamin B I content ~ of samples of natural products was first reported by Hutner et al. (1949), who introduced a sensitive assay technique based on the vitamin BIZrequirement of Euglena gracilis var. bacillaris. This method has been examined by Robbins et al. (1950a; 1951; 1952; 1953) who measured the vitamin BIZpotency of many natural materials. All of the cobamides stimulated the growth of this organism, but

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some problems in assaying fermentation samples were encountered as a result of the sensitivity of this strain of E . gracilis to antibiotics often present in fermentation samples (Robbins et al., 1953; Sakai et al., 1953). Other protozoal species found useful in bioassay for cobamides include Poteriochrommas stipitata (Barber et al., 1953) and several strains of Ochrommaa malhamensis (Ford, 1953a; Hutner et al., 1953). Assays based on the response of 0. malhamensis have been widely used since Ford’s finding (1953a) that only 5,6-dimethyl-a-benzimidazolylcobamidestimulated the growth of this culture, and that other cobamides and substances stimulating growth of vitamin BIZ-requiring lactobacilli had no effect on the growth of 0. malhamensis. More recent experiments have shown that a number of cobamides including 5-hydroxy-a-benzimidazolylcobamide (Bernhauer and Friedrich, 1954; Bernhauer el d.,1955), cobamide analogs such as benzimidazolylcobamides and 5 ,6 - dichloro - a - benzimidazolylcobamides (Ford and Hutner, 1955)) and cobamide peptides (Heathcote and Mooney, 1958) stimulated the growth of strains of this organism. However, as all of the cobamides tested which stimulated the growth of 0. malhamensis have also been found to stimulate the growth of chicks, the 0. mulhamensis assay despite its lack of specificity has remained in popular favor (Coates and Ford, 1955; Coates and Kon, 1957). The chief advantages of the above bioassays are derived from their sensitivity, and less from their specificity. In the turbidimetric assays the L. leichmunnii cultures are significantly stimulated by about 100 mpg. per milliliter of cobamide, the L. Zactis (Dorner strain) by about 20 mpg. per milliliter, the E. coli mutant (113-3) by about 0.5 mpg. per milliliter, the E. gracilis by about 0.1 mpg. and the 0. malhamensis cultures by about 0.05 mpg. per milliliter. While all of these cultures respond to more than one type of cobamide, none are stimulated by cobamides which have been chemically altered to replace some of the arnide groups on the side chains, a minor change in such a large molecule (Smith, 1957a; Smith et al., 1956). The L. leichmannii, 0. malhamensis, and E. coli (mutant 111-3) respond to cobamides present in coenzyme form (Barker, 1959) (without change in specificity). B. CHEMICAL METHODS A number of chemical methods applicable to the estimation of the cobamide content of fermentation samples have been developed, but only a few have apparently been used for this purpose. These chemical methods have been based on either separation of the cobamides from other substances in the fermentation samples and subsequent measurement of the absorption of the extract a t certain wavelengths, e.g., 361 mp (Fisher, 1953), or determination of a specific portion of the molecule such as the 5,6-

MICROBIAL SYNTHESIS OF COBAMIDES

95

dimethylbenzimidazole (Boxer and Rickards, 1950) or the red acid fragment formed by acid hydrolysis of cobamides (Fantes et al., 1950b). The ability of cobamides to form colored dicyanides has been the basis of a very useful method (Rudkin and Taylor, 1952) which in one series of experiments gave results in good agreement with bio-assays using E. gracilis as test organism (Janicki et al., 1953c, 1954). Boxer and Rickards (1951a, b, c) have taken advantage of the photolability of the cyanide in the cobamide cyanides (also noted by Wijmenga and Hurenkamp, 1951) and have perfected a microtechnique for the determination of the cyanide released from the cobamide cyanide salts. The minimum amount of cyanide detectable by their methods is of the order of 0.2 pg., corresponding to about 15 pg. of cobamide cyanide. An equally sensitive method, which has much more specificity (all of the other chemical methods do not distinguish between cobinamide and cobamide) is based on an isotope dilution assay using Coao-labeledcobamide (Bacher et al., 1954). C. FILTER PAPER CHROMATOGRAPHIC AND IONOPHORETIC TECHNIQUES The usefulness of filter paper chromatography in separating mixtures of cobamides and related factors was first demonstrated by Smith and Cuthbertson (1949) who used these methods to show the presence in liver and fermentation extracts of ribosides and several cobamides stimulating the growth of vitamin Biz-requiring microorganisms. In later investigations (Smith, 1952b; Smith et al., 1951) it was found that under certain conditions a pure cobamide would give two or more zones of growth stimulation due to decomposition of the cyanide salts during the development of the chromatogram (and conversion to the hydroxide) (Woodruff and Foster, 1950). Ford et al. (1951) utilized and improved the techniques described by Smith et al. (1950) and reported (Kon, 1955) that mixtures containing cobinamide, 2-methyl-a-adenylcobamide cyanide, a-adenylcobamide cyanide, and 5,6-dimethyl-cr-benzimidazolylcobamidecyanide were easily separated. Patte (1953) adapted their technique to the study of extracts from streptomycete cultures which also contained antibiotics, and found that by suitable choice of solvents it was possible to separate the antibiotics from the cobamides on the developed chromatograms and obtain valid bioautographs. Friedrich and Bernhauer (1955) and Friedrich et al. (1956) found that the resolving power of Ford’s sec-butanol-water-acetic acid solvent was improved by addition of potassium perchlorate, trichloracetate, sodium tetraphenylborate, or sodium camphor-sulfonate. They reported that mixtures of the vitamin Blz-species which are contaminated with interfering salts and cannot be separated by paper chromatography, can be effectively resolved into the individual constituents when the solvent system contains higher concentrations of sodium trichloracetate.

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The methods devised by Ford et al. (1955; Kon, 1955; Porter, 1957) and Friedrich et al. (1956) have been useful in the characterization of mixtures of “naturally occurring” cobamides produced by propionibacteria (Pawelkiewicz, 1954) and corynebacteria (Pawelkiewicz and Zodrow, 1956b) as well as the “unnatural” cobamides formed by these and other genera when grown in suitable media (DiMarco et al., 1957a; Ford and Kutner, 1955; Pawelkiewicz and Nowakowska, 1955; Pawelkiewicz and Zodrow, 1956a; Perlman and Barrett, 1958). Paper chromatographic methods have also been useful in studying the cobamides present in mushrooms, fermented fish products, and related materials (Ericson and Sjostrom, 1953; Tarr, 1951a, b, 1952; Tarr et al., 1950). The advantages of filter paper ionophoresis as a tool in separating mixtures of cobamides were first shown by Holdsworth (1953). He was able to separate satisfactorily mixtures containing cobinamide, a-adenyl-cobamide cyanide, 2-methyl-a-adenylcobamide cyanide, and 5,6-dimethyla-benzimidazolylcobamide cyanide. His experiments (Ford et al., 1953) showed that vitamin Blzr was a mixture of a-adenylcobamide cyanide and 2-methyl-a-adenylcobamide cyanide, and this method was subsequently used by Dion et al. (1954) to provide material for degradative studies. Paper ionophoresis was also investigated by Ericson (1953; Ericson et al., 1952; Ericson and Lewis, 1953) who demonstrated the presence of several cobamides in samples taken from S. griseus and from 8. aureofaciens fermentations. Examination of extracts of sewage sludge showed the presence of as many as eleven cobamides or cobamide-like materials according to Neujahr (1956). D. RELEASE OF COBAMIDES FROM MICROBIAL CELLS

As investigations of the biosynthesis of cobamides progressed, it became evident that most of the vitamin BIZ activity formed during the growth of the cells was retained in the cells until autolysis started. These observations were utilized in several processes for the recovery of cobamides from fermented media where the first step was to recover the cells and treat them by various procedures. Microorganisms whose cells were found to contain practically all of the vitamin synthesized included : Streptomyces species (Distillers Co. Ltd. and McCombie, 1953; Farbenfabriken Bayer, 1952; Janicki et al., 1954; Matsuda and Nunome, 1955; Meyer and DeVries, 1952; Pagano snd Greenspan, 1954); Bacillus subtilis (Tanaka et al., 1952); Bacillus coagulans (Guida and Correa, 1957); Bacillus megaterium (Garibaldi et al., 1951, 1953; Lewis et al., 1949); P. freudenreichii (Leviton, 1956b); Corynebacterium diphtheriae (Clarke, 1958); Mycobacteria species (Kocher and Sorkin, 1952); E. coli (Robinson, 1957); Nocardia species (Burton and Lochhead, 1951). Cells may be collected by filtration, cen-

MICROBIAL SYNTHESIS OF COBAMIDES

97

trifugation, or coagulation or by precipitation by specific antiserum (Allen, 1958). A number of procedures have been found to release the cobamides retained in the microbial cells. These include breaking the cell walls by acidification (Borensztaijn and Kurylowicz, 1952; Burton and Lochhead, 1951; Jackson et al., 1951; Janicki et al., 1954; McCormack et al., 1953; Merck and Co., Inc., 1956; Pagano and Greenspan, 1954; Rickes and Wood, 1955a, b), treatment of the cells with alcohols (Leviton, 1956b; Meyer and DeVries, 1952; Mulli and Schmid, 1956), autoclaving or heating the cells at temperatures above 80” C. (Chiao and Peterson, 1953; Distillers Co. Ltd. and McCombie, 1953; Farbenfabriken Bayer, 1952; Garibaldi et al., 1951; Lewis et al., 1949; Rickes and Wood, 1955a), breaking the cells with sonic vibrations (Pagano and Greenspan, 1954), and breaking the cells by increasing the osmotic pressure (McCormack et al., 1953; Pagano and Greenspan, 1954). Extraction of sewage samples with hot water has been found to be effective in removing the cobamides present (Kamikubo and Tanaka, 1955a; Kamikubo et al., 1957). Apparently the above treatments irreversibly denature the proteins which combine with vitamin Blz-like materials and make them unavailable to microorganisms used in bioassays (Bird and Hoevet, 1951; Burkholder, 1952; Davis et al., 1952; Ford et al., 1955; Gregory and Holdsworth, 1953; Kristensen, 1956).

111. Microbial Processes for Synthesis of Naturally Occurring Cobamides

A. SELECTION OF MICROORGANISM As mentioned above, fermented feces and manures were found to have higher cobamide content (as measured by stimulation of the growth of chicks fed a ration low in “animal protein factor”) than the unfermented material. A culture identified as F . solare (Petty, 1950;Petty and Matrishin, 1949) isolated from hen feces produced the material used by Stokstad et al. (1948) with patients afflicted with pernicious anemia. Other reports showed that many other organisms from this source were capable of producing cobamides when grown in appropriate media (Ansbacher et al., 1949; Ansbacher and Hill, 1949; Burton and Lochhead, 1951; Garibaldi et al., 1951, 1953; Halbrook et al., 1950; Hall et al. 1950a; McGinnis et al., 1949). In one study Burton and Lochhead (1951) found that 347 of 537 cultures of bacteria isolated from soils, manures, poultry litter, and seeds produced factors stimulating the growth of L. lactis (Dorner), while filtrates from 450 of 576 actinomycetes gave positive results in this microbial assay. Others have shown that many proteolytic bacteria produce cobamides, as do many of the aerobic organisms found in the intestinal

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contents of animals (Hodak, 1956; Kaleja, 1956, 1957). Among the organisms shown to produce cobamides are bacteria from the following genera: Aerobacter (Smith et al., 1952a; Witkus et al., 1954) Agrobacterium (Hoogerheide, 1957) Alcaligenes (Rickes and Wood, 1955a) Azotobucter (Almon, 1958; Burton and Lochhead, 1951) BaciZlus (Fantes and O’Callaghan, 1956; Garibaldi et al., 1953; Lugones and Mundel, 1952) Clostridium (Fantes and O’Callaghan, 1956; Wood and Hendlin, 1952) Corynebacterium (Clarke, 1958; Jannes, 1953; Pawelkiewicz and Zodrow, 1956b) Escherichia (Halbrook et al., 1950) Flavobacterium (Petty and Matrishin, 1949; Petty, 1950) Mycobacterium (Kocher and Sorkin, 1952; Peterson and Pope, 1952; Rickes et al., 1948~) Propimibacterium (Hargrove and Leviton, 1952; Janicki and Pawelkiewicz, 1954; Leviton and Hargrove, 1952; Sudarsky and Fisher, 1957) Proteus (Hanson and Hodge, 1957; Hodge et al., 1952) Pseudommas (Ordanik, 1951; Rickes and Wood, 1955a) Rhizobium (Burton and Lochhead, 1952; Vyas and Prasad, 1956) Serratia (Hill and Branion, 1953) Streptococcus (Halbrook, et al., 1950; Hanson and Hodge, 1957; Hodge et al., 1952; Porter and Dollar, 1958) Xanthommas (Fantes and O’Callaghan, 1957). Most of the streptomycetes studied produced significant quantities of cobamides (as shown by microbial assays) (Borensztaijn and Kurylowics, 1953; Hall et al., 1950a, b; Matsuda, 1952; Pridham et al., 1951; Sakai, 1953b; Saunders et al., 1952; Shull and Routien, 1951; Simek et al., 1955). The following streptomycete species have been reported to produce significant amounts of cobamides when grown on appropriate media: S. albidojlavus (Rickes and Wood, 1955b) S. antibioticus (Rickes and Wood, 195513) S. aureofaciens (Tarr, 1951b) S. aureus (Borensztaijn and Kurylowicz, 1953) S. farinosus (Hall et al., 1954) S. fradiae (Nelson et al., 1950; Rickes and Wood, 1955a) S. griseus (Rickes et al., 1948c, 1955b) S. olivuceus (Hall, 1953; Hall et al., 1953; Sakai, 1953b) S. roseochromogenes (Rickes et al., 1948c; Rickes and Wood, 1955b) S. winaceus (Jones, 1951). Other references and organisms are mentioned in the review by Darken (1953).

MICROBIAL SYNTHESIS OF COBAMIDES

99

Small quantities of cobamide-like activity have also been found in cultures of Ustilago zeae (Haskins et al., 1950), Aspergillus niger (Nicholas, 1952; Sakai, 1953a), Neurospora species (Maki and Ushikoshi, 1956; Sakai, 1953a), Ashbya gossypii (Smiley et al., 1951), Eremothecium species (Rickes and Wood, 1955a), lichens (Sjostrom and Ericson, 1953), and algae (Ericson, 1952; Ericson and Lewis, 1953). Further study suggested that at least some of the cobamides found in the algae were the result of bacterial contamination (Ericson, 1952; Ericson and Lewis, 1953). Isolation of the cobamides formed by the above-named microorganisms has been carried out in many instances, and examinat,ions of the isolated materials have shown that several types of cobamides were often present. Those whose structures have been elucidated are listed in Table I together with the sources. In most instances it was not possible to isolate in crystalline form more than half of the cobamide present, and it is likely that there are many undiscovered cobamides present in certain of the cultures. TABLE I SOMECOBAMIDES FOUND I N MICROBIAL PRODUCTS Cobamide 5,6-Dimethyl-a-benzimidazolylcobamide a-Adenylcobamide

2-Methyl -a-adenylcobamide

5-Hydroxy -a-benzimidazolylcobamide 2-Methylmercapto-aadenylcobamide a-Guanylcobamide Cobinamide

5-Methyl -a-benzimidazolylcobamide a-Benzimidazolylcobamide

Sources

References

Bacilli Propionibacteria Sewage Streptomyces Anaerobes Propionibacteria Rumen samples Sewage Anaerobes Propionibacteria Rumen samples Sewage Sewage

Lewis et al. (1949) Hargrove and Leviton (1955) Bernhauer and Friedrich (1954) Rickes and Wood (1955b) Pfiffner et al. (1951); Barker et al. (1958) Pawelkiewicz and Nowakowska (1955) Ford et al. (1951) Bernhauer and Friedrich (1954) Pfiffner et al. (1954) Pawelkiewicz (1955a), Porter and Dollar (1958) Ford el al. (1953) Bernhauer and Friedrich (1954) Bernhauer and Friedrich (1954)

Sewage

Friedrich and Bernhauer (1957~)

Nocardia Propionibacteria Streptomyces Rumen samples Sewage

Barchielli et al. (1957) Janicki and Pawelkiewicz (1954) Smith (1954) Ford and Porter (1953) Friedrich and Bernhauer (195th)

Sewage

Friedrich and Bernhauer (1958a)

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The accomplishments of the microbial geneticists in increasing the yields of antibiotics and other metabolites of microorganisms have been recognized in the fermentation industries. Efforts directed toward isolation of strains of organisms producing more cobamide than the parent culture have not been successful in many laboratories (Burton and Lochhead, 1951; Saunders et al., 1952; Shull and Routien, 1951). In one instance strains of S. griseus capable of producing more streptomycin than the parent were found to have lost the ability to produce cobamide (Dulaney, 1951). JuHiard (1957) found that mutants from a cobamide-producing B. megaterium culture produced a series of substances that antagonized cobamides as far as growth promotion of cobamide-requiring bacteria was concerned. Another report mentions that no change in cobamide production was observed in “degenerate” strains of S. griseus which had lost their ability to produce streptomycin but still produced cobamides (Perlman et al., 1954). However, some success has also been reported: Pagano and Greenspan (1954) isolated a mutant from an unidentified streptomycete which produced 5.7 mg. of cobamide per liter when grown under certain conditions, or about three times as much as the parent culture.

B. MEDIAUSEDIN THE PRODUCTION OF COBAMIDES A number of media have been used in studying the biosynthesis of cobsmides by microorganisms. The formulations of a few of these have been summarized in Table 11. Many of the media contained materials of natural origin including soybean oil meal, distillers’ solubles, fishery wastes or fish meal, yeast preparations, meat extract, animal stick liquor, cornsteep liquor, casein or casein hydrolyzates, and mash residues from penicillin-producing fermentations. [Darken (1953) has listed some media not included in Table 11, and her review shows some of the variations that have found favor at various times.] Several studies have shown no direct relationships between the amino acid composition of these media and production of cobamides (Ganguly and Roy, 1955; Kurz and Nielson, 1957). Synthetic media containing amino acids, inorganic salts, and glucose have been used to grow streptomycetes and gave yields of cobamides equivalent to those obtained in media containing natural products (Dulaney and Williams, 1953; Perlman et al., 1955). In most experiments the cobamide yields have been correlated with cellular mass. In experiments where the microorganisms produced acids from carbohydrates present in the media, it was necessary to neutralize this acid in order to obtain maximum cobamide yields. Continuous neutralization as well as buffers have been used. In some experiments higher yields of cobamides were obtained when the energy source, e.g,, glucose (N. V. Philips Gleoilampenfabriken, 1957) or lipids (E. R. Squibb and Sons, 1952), were added continuously or intermittently to the growing

101

MICROBIAL SYNTHESIS OF COBAMIDES

TABLE I1 MICROBIAL S Y N T H E S I S O F 5, 6-DIMETHYL-(YBENZIMIDAZOLYL-COBAMIDE CYANIDE^

PROCESSES FOR

Microorganism

Ingredients of Medium

Yield :me./ Liter)

Referenoe

__

Beet molasses; ammonium phosphate; cobalt salt; inorganic salts Flavobacterium solare Yeast extract; malt extract; glucose; penicillin mash residue; inorganic salts Flavobacterium devo- Glucose; soybean meal; ram cornsteep liquor; cobalt salt; buffer salts Propionibaceriu~nj r e u Glucose; casein hydrolyden reichii sate; yeast extract; cobalt salt; lactic acid Propionibacterium freu Glucose; peptone; yeast; den reichii KzHPO, ; cobalt salt Propionibacterium Yeast extract; glucose: co8 hermanii balt salt; alkali Streptom yces griseus Glucose; soybean meal; cobalt salt Glucose; brewer’s yeast; Streptomyces fradiae soybean meal; cobalt salt; inorganic salts Streptomyces olivaceus Glucose; soybean meal : distiller’s solubles; cobalt salt; inorganic salts Glucose; animal stick liqStreptomyces species uor; cobalt salt Soybean meal; glucose; Streptomyces species KzHPOi ; COClz Bacillus megaterium

0.45

Lewis et al. (1949)

0.6 Petty (1950)

0.6

Hall and Tsuchiya (1951)

3.0

Leviton and Hargrove (1952)

2.4

Aso et al. (1954)

3.0

Hinz (1957)

0 . 3 Wood and Hendlin (1952) 0 . 7 Nelson et al. (1950)

3.3

Hall el al. (1953)

2.0

Garey et al. (1951)

5.7

Pagano and Greenspan 1954

-

While the cultures are presumed to produce the cobamide hydroxide, the vitamin is usually isolated in the cyanide form (Wolf, 1950).

streptomycetes. Mixed culture fermentations have been used in producing cobamides; in one system, lactose-containing media were fermented with lactobacilli and after the lactic acid content had reached a maximum the cobamide-producing propionibacteria were added (Leviton, 1956a; Leviton and Hargrove, 1952); in another system, a medium containing soybean oil meal was fermented by a mixture of Pseudomonas sp., Streptococcus bovis, Proteus vulgaris, and Clostridium putriJicum to give higher cobamide yields than were obtained when any one of the cultures was used (Hodge

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et al., 1952). Certain processes have been noticeably inhibited by products resulting from the procedures used in sterilizing the ingredients of the media. In experiments with S. olivaceus, yields obtained when the medium was sterilized in a flash sterilizer were higher than those obtained when the medium was sterilized in a batch-type cooker (Pfeifer et al., 1954). Other experiments with S. olivaceus have been interpreted as showing that aeration is not an important variable in the process since maximum yields were obtained as long as moderate aeration rates were used (Makarevich et al., 1958; Pfeifer and Vojnovich, 1952). Appreciable quantities of aadenylcobamides were found in the fermented media when suboptimal aeration rates were used, while, when adequate aeration rates were used, only 5,6-dimethyl-a-benzimidazolylcobamide was found (Makarevich et al., 1958).

C. PRECURSORS FOR THE BIOSYNTHESIS OF COBAMIDES The observations that cobalt is part of the cobamide molecule led to use of media containing this element (Hendlin and Ruger, 1950;Lewis et al., 1949). Marked increases in production of cobamide activity (as measured by microbial assays) were found when cobalt salts were included in the media. Growth of many of the streptomycetes used in cobamide-producing fermentations was unaffected by concentrations of cobalt of the order of 5 p.p.m., but inhibition was observed when the levels were raised to 50 p.p.m. or more (Kojima and Matsuki, 1956; Principe and Thornberry, 1952; Wood and Hendlin, 1952). When less than 1 p.p.m. of cobalt was present in the media, as much as 75 % of the cobalt added was incorporated into the cobamide formed; this rate of utilization dropped sharply when higher concentrations were used (Perlman and O’Brien, 1954;Smith et al., 1952a). An organic complex formed by growing yeast in a cobalt-containing medium was more efficiently used by this S. griseus strain in the biosynthesis of cobamide activity than was the cobalt of cobalt nitrate (Perlman and O’Brien, 1954). Much of the cobalt present in the yeast was found to be firmly bound to the cell protein, and could only be released by acid treatment. Certain compounds have been found to inhibit cobalt utilization by streptomycetes; sodium fluoride was the most effective inhibitor in one series of experiments (Iwamoto, 1952). While most media used for the microbial production of cobamides have contained cobalt salts as essential ingredients, a few processes have used media where cobalt salts were not added. Petty (1950)reported that when his strain of F. solare was grown in a medium containing such materials as malt extract and mash residue from penicillin-producing fermentations, cobamide yields of the order of 0.6 mg. per liter were obtained. Sudarsky and Fisher (1957) obtained about 3 mg. of cobamide per liter when their

MICROBIAL SYNTHESIS OF COBAMIDES

103

culture of P . freudenrekhii was grown in a medium based on cornsteep liquor and beet molasses. It seems probable that the raw materials provided enough cobalt to meet the requirements for cobamide synthesis. The precursors of the other portions of the molecule have also received attention. Sahashi el aZ. (1950) in experiments with an unidentified streptomycete found that addition of 5,6-dimethylbenzimidazoleto the fermentation resulted in increased cobamide titers. Several years later Weygand el al. (1954a) showed that some of the Clclabeled 5,6-dimethylbenzimidazole added to growing cultures of S. olivarceus was incorporated in the 5,6dimethyla-benzimidazolylcobamide formed by this culture. Similar conclusions were reached by Minogata and Sakai (1953) who used cultures of B. subtilis, B. mgaterium, and B. natto in their experiments. Shemin et al. (1956) and Corcoran and Shemin (1957) have shown that b-aminolevulinic acid acted as the precursor for much of the cobinamide portion of the cobamide molecule, and in experiments using N16-labeled2-threonine, Krasna et al. (1957) found that this amino acid was the precursor of the fragment which yielded ~~-1-amin0-2-propanol on acid hydrolysis of cobamides. Methionine has been known to supply the “odd” methyl groups of the porphyrin-like moiety (Bray and Shemin, 1958). Studies using porphobili110gen-C~~ showed that this moiety was incorporated into the cobamides formed by an unnamed bacterial culture (Schwartr et al., 1958). OF RADIOACTIVE COBAMIDES D. PREPARATION The first form of labeled cobamide to be prepared was Coso-labeled 5,6dimethyl-cu-benzimidazolylcobamidecyanide. This was formed as a result of growing streptomycetes in a medium containing Coaosalts (Chaiet et al., 1950; Rosenblum and Woodbury, 1951; Smith et al., 1952a). The fiveyear half-life of the Co60limited the amounts that could be used in animal experiments, and for this reason cobamide labeled with the shorter-lived isotopes of cobalt (Cobs,Cob’, and C O ~were ) prepared by the biosynthetic process (Bradley et al., 1954; Smith, 1957~).While cobalt isotopes have been incorporated into the cobamide molecules biosynthetically, this exchange has not been accomplished by ordinary chemical means (Fantes et aZ., 1950b). Direct neutron irradiation of the crystalline vitamin gave very low specific activities (Anderson and Delabarre, 1951; Maddock and Coehlo, 1954; Numerof and Kowald, 1953; Woodbury and Rosenblum, 1953). Specific activities obtained with the biosynthetic process have ranged from 0.8 mc. per milligram when Co60was used (Chaiet et al., 1950; Mollin and Smith, 1956; Smith, 1952d, e) to about 12 mc. per milligram when carrier-free CoS6and (made by cyclotron treatment of iron) were used (Mollin and Smith, 1956; Smith, 1957~). The cyanide group of 5,6-dimethyl-cu-benrimidazolylcobamide was la-

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beled with C14 by exchange in alkaline solutions with C14N ions (Smith, 1952c, 1957c) or by reacting the cobamide hydroxide with C14N ions (Boxer et al., 1951).An S3& thiocyanato cobamide was prepared in a similar manner (Smith, 1957~).Carbon-14 has been introduced into the imidaeole ring by fermentation in the presence of C14-labeled5 ,6-dimethylbenzimidazole (Weygand et al., 1954s). As mentioned above, experiments with labeled 6-aminolevulinic acid showed that this material was incorporated into the cobinamide portion of the molecule (Shemin el al., 1956; Smith, 1957~). When a 8.griseus culture was grown in a medium containing radioactive phosphate, some of the phosphate of the cobamide formed was labeled (Smith, 1952a, c, d). A cobamide containing Co60and PS2was also prepared (Smith et al., 1952~).

E. OCCURRENCE

OF

5 , ~-DIMETHYL-Lu-BENZIMIDAZOIAYLCOBAMIDE CYANIDE IN MICROBIAL FERMENTATIONS

The presence of cyanide as in the vitamin BIZ isolated from liver and from microbial sources was first reported by Brink et al. (1950). Somewhat later Wolf (1950) found that the cobamides present in fermented media could be converted into the stable cyanide salts by the addition of soluble cyanides to the concentrates. Processes for this conversion during the fermentation operation have been described in patents (McDaniel and Woodruff, 1953; Pagano and Greenspan, 1954). These include the addition of solutions of potassium cyanide, calcium dicyanide, potassium ferricyanide and potassium ferrocyanide (McDaniel and Woodruff , 1953), acetone cyanhydrin (Perlman et al., 1956), or nitriles (Pugano and Greenspan, 1954) to the growing cultures. Inasmuch as cyanide inhibits growth of many of the cobamide-producing cultures, care must be taken not to exceed the toxic limit.

F. VITAMINBIZ AND COBAMIDES FROM SEWAGE Within a few years after Hoover’s report (Hoover et al., 1951) of the occurrence of vitamin Blz-like substances in sewage, a number of other laboratories confirmed the finding. The results reported show that levels range from 4 to about 10 mg. of cobamide per kilogram of dry sewage sludge solids, depending on the method of operation of the digester and seasonal variations (Hoover et al., 1951, 1952; Kamikubo and Miyawaki, 1955a; Kamikubo and Takata, 1953; Kocher and Corti, 1952; Neujahr, 1955a, b; Takata et al., 1956; Whitmarsh et al., 1955). These potencies are higher than those of some commercial feed grade cobamide supplements which often contain about 3.3 mg. cobamide per pound (Murdock, 1952). The identity of the cobamides present in various sewage sludges has been of considerable interest. Pure crystalline 5 ,6-dimethyl-cu-benzimida-

MICROBIAL SYNTHESIS OF COBAMIDES

105

zolylcobamide cyanide has been isolated from a number of samples (Friedrich et al., 1957; Janicki et al., 1956; Kamikubo and Tanaka, 1955a, c, 1956b; Stevens et al., 1955). Friedrich and associates have also crystallized a-adenylcobamide cyanide, 2-methyl-a-adenylcobamide cyanide, 5-hydroxy-a-benzimidazolylcobamide cyanide, and 2-mercaptomethyl-a-adenylcobamide cyanide from sewage sludge (Friedrich et al., 1957). Sewage sludges from a yeast plant were found to contain a-benzimidazolylcobamide cyanide and 5-methyl-a-benzimidazolylcobamide cyanide (Friedrich and Bernhauer, 195th). They also mention that there are some seventeen other cobamides present in their extracts of sewage sludge, and it is likely that this group includes some of seven mentioned by Neujahr (1956) and the four by Rabek et al. (1956). It is likely that the extracts which have been only partially purified contain many of these cobamides (Kamikubo, 1955; Kamikubo and Tanaka, 1955b, 1956a; Kamikubo et al., 1957; Miner and Wolnak, 1953; Takata, 1955). Addition of cobalt salts to the sewage digesters resulted in only a slight increase in cobamide content of the sewage sludge (Elbowicz, 1957; Kamikubo et al., 1955; Neujahr, 1958). Other studies showed that addition of chlortetracycline, penicillin, or neomycin to the sewage digesters enhanced the biosynthesis of certain of the group of cobamides present (Neujahr, 1957). In the latter study it was apparent that aeration of the sludge definitely resulted in increased 5 ,6-dimethyl-a-benzimidazolylcobamideproduction and reduction in the yield of the 5-hydroxy-a-benzimidazolylcobamide. On the other hand, anaerobiasis was shown to result in increased cobamide synthesis by pure cultures of Methanobacterium omelianski (Pehany, 1958). G. COBAMIDE PEPTIDES FROM MICROORGANISMS The native form of cobamides in microorganisms and liver has been the subject of investigations in several laboratories. Wijmenga et al. (1949; Wijmenga, 1951,1952; Wijmenga et at., 1954a, b) commented on the properties of what apparently was a cobamide-peptide in which the cobamide was linked to a polypeptide or protein rather than to cyanide. The binding of cobamides by proteins had been observed by Ternberg and Eakin (1949) and others (Bird and Hoevet, 1951; Gregory, 1953; Gregory and Holdsworth, 1953) who found that these complexes did not stimulate the growth of cobamide-requiring microorganisms. Hausmann (1949) described a polypeptide entity derived from S. griseus which contained cobamides. This material was ineffective in treatment of pernicious anemia but became active after digestion with enzymes. In later publications (Hausmann, 1953; Hnusmann and Mulli, 1952a, b; Hausmann et al., 1953; Mulli and Schmid, 1956) the properties of a number of cobamide-peptides derived

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from microbial sources (as well as from fish and animal sources) were described in some detail. These red-colored materials were found to stimulate the growth of cobamide-requiring bacteria (as opposed to the inactivity of cobamide-protein complexes) and were active in varying degrees in the treatment of pernicious anemia. The purest preparations (Mulli and Schmid, 1956) contained about 23% cobamide. The value of cobamidepeptides was demonstrated by Heathcote and Mooney (1958) who used a preparation derived from a microbial source to treat pernicious anemia. They found that the preparation was active orally. The preparation used by Heathcote and Mooney had a molecular weight of about 10,000 and stimulated the growth of 0. mlharnensis. Apparently the same factor was isolated from a streptomycete and another (unidentified) microorganism. It seems probable that the cobamide-containing coenzymes studied by Barker et al. (1958; Barker, 1959) account for some of the activity of these polypeptide-containing entities.

IV. Microbial Synthesis of “Unnatural Cobamides”3 All of the cobamides mentioned in the above discussion have been found in fermented media, and are presumed to be synthesized by microorganisms from relatively simple chemical substances, e.g., glucose, amino acids, cobalt salts, etc. As the structural studies of the cobamide group progressed, several degradation products were identified, and it was only natural that these were used in experiments to determine whether the cultures could utilize them as precursors in the biosynthesis of cobamides. Two types of processes were studied: a process in which the organisms used only a base (purine or benzimidazole) and supplied their own cobinamide, and a process in which microbial cells combined externally supplied cobinamide and purine, benzimidazole, or other base. For purposes of classification, the former process (with the culture supplying its own cobinamide) will be called a “total synthesis,” and the latter a “partial synthesis.” The products will be called “unnatural cobamides” even though some of them have been found in certain fermentations.

A. TOTAL SYNTHESIS Dulaney and Williams (1953) found that addition of o-phenylenediamine, o-dinitroaniline, o-xylidine, or 3,4-diaminotoluene to the growing S. griseus cultures resulted in increased cobamide synthesis RB measured by microbial assay. Further study using ClP-labeled o-phenylenediamine ,

8 After this manuscript was completed, copies of the excellent review by Kon and Pawelkiewicz entitled “Biosynthesis of Vitamin B12 Analogues” were distributed in preprint form. These authors have summarized much of the information on the chemistry and biological properties of the cobamide analogs (Kon and Pawelkiewicz, 1958).

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107

showed that only traces of the labeled material could have been incorporated into the 5,6-dimethyl-a-benzimidazolylcobamide synthesized. Fantes and O’Callaghan (1954, 1955) examined this fermentation process and isolated from the fermented medium a new cobamide which they identified as a-benzimidazolylcobamide. In later experiments Fantes and O’Callaghan (1956, 1957) used this process to prepare a number of cobamides including 5,6-dichloro-cr-benzimidazolylcobamide, 2 , 3-naphthimidazolylcobamide, 5amino*-benzimidazolylcobamide, 5-hydroxy-ar-benzimidazolylcobamide, and trifluoromethy1.a-benzimidazolylcobamideby adding different benzimidazoles or other bases to the growing cultures. They also observed that cultures of S. olivaceus, S. fradiae, S. aureofaciens, B . megaterium, and X . juglandis could be substituted for the S. griseus culture. These experiments confirmed and extended Woolley’s hypotheses based on experiments with growing cultures of B . megaterium (Woolley, 1950; Woolley et al., 1951). Investigations in other laboratories confirmed the formation by S. griseus of a-benzimidazolylcobamide when o-phenylenediamine was added to the growing cultures (Ganguly and Roy, 1956). However, a similar synthesis, the incorporation of 5,6-dimethylbenzimidazole into 5,6-dimethyla-benzimidazolylcobamide has not always been successful, and may depend in part on the cultural conditions used in growing the 8. griseus cells (Pawelkiewicz, 1954; Sahashi et al., 1950; Sakai, 1956; Surikova and Popova, 1957). Concurrently with the experiments by Fantes and O’Callaghan, Pawelkiewicz and Janicki were studying the biosynthesis of cobamides by a strain of P. shermanii. They found that under certain conditions this culture produced a material later identified as cobinamide (Janicki and Pawelkiewicz, 1954, 1955, 1956; Pawelkiewicx, 1954). When benximidazoles, phenylenediamines, or purines were added to the growing cultures, significant amounts of the added substances were found in the cobamides. Some of the cobamides formed by this and other processes are listed in Table 111. Pawelkiewicz (1955b) also reported that cultural conditions markedly affected this conversion, and that the addition of small amounts of chlortetracycline to the cultures increased the formation of the new cobamide and decreased formation of cobinamide. Sulfanilamide was also found to have this effect (Janicki and Pedziwilk, 1958). Bernhauer et al. (1958) have examined the mechanism of synthesis of new cobamides by P. shermanii, and have concluded that an exchange may be operative in which the added base (benximidazole, purine, etc.) displaces the adenine normally used by the cells in formation of a-adenylcobamide. Experiments with P. arabinosum have confirmed this hypothesis (Perlman, 1959). They have also observed that when 5-methylbenzimidaxole was added to the culture, both 5-methyl~-benzimidazolylcobamideand 6-

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TABLE I11 SOMEUNNATURAL COBAMIDES SYNTHESIZEI) BY MICROBIAL PROCESSES

Cobamide

References on microbial synthesis Totala

a-Benzimidazolylcobamide

5-Methy1-a-benzimidazolylcobamide 6-Methyl-~-benzimidazolylcobamide 4,6-Dimethyl-a-beneimidaeolylcobamide 5(6) -Trifluoromethyl-a-benzimidazolylcobamide 4(7) -Methyl-a-benzimidazolylcobamide 5,6-Diethyl -a-benzimidaeolylcobamide 5(6) -Propyl-6 (5)-ethyl-a-benzimidazolylcobamide 5,6-Dimethoxy-a-benzimidazolylcobamide 5(6) -Methoxy-a-beneimidazolylcobamide 5(6) -Ethoxy-a-benzimidaeolylcobamide 5,6-Dichloro-~~-benzimidazolylcobamide 5(6)-Chloro-6(5) -methyl-a-beneimidazolylcobamide 5(6) -Bromo-6(5) -methyl-a-benzimidazolylcobamide 5(6) -Bromo-7 (4)-methyl-a-benzimidazolylcobamide 4(7) -Bromo-6(5) -methoxy-a-beneimidazolylcobamide 5(6) -Amino-a-benzimidaeolylcobamide 5(6) -Nitro-a-beneimidazol ylcobamide 5,6-Dinitro-a-beneimida~olylcobamide 5(6)-Nitro-6 (5)-methyl-a-benzimidazolylcobamide 5(6)-Hydroxy-a-benaimidazolylcobamide 4(7)-Hydroxy-a-benzimidazolylcobamide 4-Chloro-a-benzotriazolylcobamide 5,6-Imidazo-a-benzimidazolylcobamide 2-Mercapto-a-beneimidazolylcobamide 2-Amino-a-benzimida~olylcobamide 2,3-Naphthimidaeolylcobamide a-Benzotriazolylcobamide a-Benzothiazolylcobamide 2,3-Dimethylpyrazinoimidazolylcobamide a-Adenylcobamide 2-Methyl-a-adenylcobamide 8-Aza-a-adenylcobamide 2-Amino-a-adenylcobamide 2,s-Dichloro-a-adenylcobamide 2-Methylthio -a-adenylcobamide 6-Methylthio-a-adenylcobamide 2-Hydroxy-a-adenylcobamide 2-Thio-a-adenylcobamide Phenazinylcobamide

Partial b

;2;8; 9; ia la; 3;4;l2; 17;18;20 24 2;13a;16; li la;3;4;6;7; 20 11; 12 2;13a 13 la;3 10;20 6 6;7 6 la; 3; 6; 7 6 6;7 3 3 17;18 9;10 3;11; 12 la 6 6 6 6; 7 20 3; 11; 12; 13 9; 10 17;18;20 11; 12;24 17;18; 19 17;18 9; 10 23 6 6; 7 11; 12 17;18 17;18 24 5 9;10; 18 17;18;20 20 3;11; 24 21;22 la;11;12;24 12 12 11; 12;13 20 11; 12;13 12;13 21;22 15 24 20

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MICROBIAL SYNTHESIS OF COBAMIDES

TABLE 111 (Continued) References on microbial synthesis

Cobamide

Total’ 2-Hydroxyphenazinylcobamide 3,4-Dihydro-~-4-oxo-quinazolinylcobamide 4-Chloro-8-nitroquinazolinylcobamide Quinoxalinylcobamide 5-Carboxylic acidamide-a-benzimidazolylcobamide 3 ,5,6-Trimethyl-~-benzimidazolylcobamide

Partial b

20 20 20 20

la; 4 14

a Cobamides synthesized by adding benzimidazoles, purines, or other bases to growing cultures of Nocardia, Propionibacteria, Streptomyces, etc. Cobamides synthesized by adding benzimidazoles, purines, or other bases together with cobinamide to cells of E . coli or other bacteria.

References for Table 111

1. Barker (1959) la. Bernhauer and Friedrich (1954) 2. Bernhauer et al. (1958) 3. Dellweg et al. (1956a) 4. Dellweg et al. (1956~) 5. Dellweg et al. (1956d) 6. DiMarco et al. (1957a) 7. DiMarco et al. (1957b) 8. Fantes and O’Callaghan (1955) 9. Fantes and O’Callaghan (1956) 10. Fantes and O’Callaghan (1957) 11. Ford et al. (1954) 12. Ford et al. (1955)

13. Ford and Hutner (1955) 13a. Friedrich and Bernhauer (1958b) 14. Heinrich (1958) 15. Kon (1957) 16. Pawelkiewicz (1954) 17. Pawelkiewicz (1955~) 18. Pawelkiewicz and Nowakowska (1955) 19. Pawelkiewicz and Zodrow (19560) 20. Perlman and Barrett (1968) 21. Porter (1956) 22. Porter (1957) 23. Robinson et al. (1955) 24. Southcott and Tarr (1957)

methyl-a-benzimidazolylcobamide were formed (Bernhauer and Friedrich, 1958; Friedrich and Bernhauer, 1958b). Other cultures have been substituted for the P. s h e m n i i , including strains of Corynebacteriumdiphtheriae (Pawelkiewicz and Zodrow, 1956b, c), a strain of Nocurdiu rugosa (DiMarco et al., 1957a), and a strain of Propionibacterium urubinosum (Perlman and Barrett, 1958). Many of the cobamides synthesized using these organisms have not been isolated in pure form, and characterization has depended on measurements of mobilities in a number of paper chromatographic and paper ionophoretic systems. Since these analytical methods are quite sensitive and specific, the products formed have been listed in Table 111. Barker (1959) has isolated from appropriately supplemented Clostridium tetanomorphum fermentations pure

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coenzymes containing a-adenylcobamide, a-benzimidazolylcobamide and 5 ,6-dimethyl-a-benzimidazolylcobamide. In studying the biosynthesis of these new cobamides, Pawelkiewicz and Zodrow (1956a) found that their culture of P. shermunii normally produced significant amounts of porphyrin and coproporphyrin 111, and that synthesis of these was significantly decreased when cobalt salts were added to the media. In similar studies using N . rugosa (Bardi et al., 1958) significant amounts of uroporphyrin and coproporphyrin I11 were found in the cultures, and synthesis of these substances was increased when b-aminolevulinic acid was added to the medium.

B. PARTIAL SYNTHESIS In an investigation of the biological properties of the vitamin Bl2-like factors found in calf feces, Ford el al. (1952b) showed that when cobinamide was added to cells of E. coli (mutant 113-3) two new cobamides were formed. Further study of this system (Ford and Holdsworth, 1954; Ford and Porter, 1953; Ford et al., 1954, 1955) showed that it was possible to produce many new cobamides by adding purines, benzimidazoles, or related substances together with cobinamide to these bacterial cells. The reviews by Ford and Hutner (1955), by Kon (1955; 1957), and by Porter (1957) include discussions of the biological properties of these new cobamides and some of the problems encountered in their synthesis. Bernhauer and associates (Bernhauer, 1956; Bernhauer and Friedrich, 1954; Dellweg et al., 1956a, b, c, d; Friedrich and Bernhauer, 1957c) have used this system to prepare cobamides, many of which are mentioned in Table 111.Less extensive studies on this system were carried out by DiMarco et al. (1957b) and on a related system by Southcott and Tarr (1957). The biosynthetic method was used by Robinson et al. (1955) in proving the structure of 5-hydroxy-c~-benzimidazolylcobamide cyanide. They mixed cobinamide and 5-hydroxybenzimidazole together with the E. coli cells and isolated two red pigmented substances, one of which had the same behavior in filter paper chromatographic systems as 5-hydroxy-a-benzimidazolylcobamide cyanide (Bernhauer’s factor 111). The E . coli methods were also used to prepare the 5,6-dichloro-a-benzimidazolylcobamidecyanide used by Kamper and Hodgkin (1955) in X-ray crystallographic studies leading to the elucidation of the structure of cobamides and cobinamide.

c. NUTRITIONAL VALUE

OF COBAMIDES

While an extended discussion of the nutritional value and methods of determining the nutritional value of cobamides is outside the scope of this review, a brief summary of the findings concerning cobamides synthesized

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by microbial processes may be of interest, since most of the “unnatural” cobamides were prepared in the hope that they would be more useful than the “natural” product. A number of factors apparently affect the reproducibility of the chick test as shown by Coates et al. (1951b, 1953); Coates and Ford (1955), Coates and Kon (1957), by Briggs and Daft (1955), and by Spivey et al. (1954). Pfiffner et al. (1951, 1954) showed that certain of the naturally occurring cobamides, namely a-adenylcobamide cyanide and 2-methyl-a-adenylcobamidecyanide, were inactive in the promotion of the growth of chicks fed a ration low in animal protein factor and in the treatment of pernicious anemia. Other studies mentioned by Kaczka el al. (1951) suggested that 5,6-dimethyl-a-benximidazolylcobamidehydroxide was less active than 5,6-dimethylbenzimidazolylcobamidecyanide in the treatment of pernicious anemia, but later experiments (Coates and Kon, 1957) showed that these observations were erroneous. After Bernhauer et al. (1955) showed that 5-hydroxy-a-benzimidazolylcobamide cyanide was effective in the treatment of pernicious anemia, the hypothesis that 5,6-dimethylabenzimidazolylcobamide cyanide was the unlg active material was abandoned. More recent reports have confirmed this observation and shown that 5-hydroxy-c~-benzimidaxolylcobamidecyanide also stimulates the growth of chicks (Coates et al., 1956). Other cobamides found useful in the treatment of pernicious anemia include :a-benzimidaxolylcobamide cyanide (Blumberger et al., 1957; Fantes and O’Callaghan, 1955); 5,6-dichloro-abenzimidazolylcobahide cyanide (Blumberger et al., 1957; Fantes and O’Callaghan, 1957); 5-methyl-cu-benzimidazolylcobamidecyanide (Blumberger et al., 1957); 5-carboxido-a-benzimidazolylcobamidecyanide (Blumberger et al., 1957); 5,6-diethyl-c~-benzimidazolylcobamide cyanide (Blumberger et al., 1957);and 2,3-naphthimidazolylcobamidecyanide (Blumberger et al., 1957). Experimental programs studying the relationship of structure to activity in the cobamide group have shown that in the chick test a number of the above cobamides are quite potent. These include: a-benzimidazolylcobamide cyanide (Briggs and Fox, 1955; Coates et al., 1956);5,6-dichloro~-benzimidazolylcobamide cyanide (Coates et al., 1956); 5-methyl-a-benzimidazolylcobamidecyanide (Coates et al., 1956); 5-hydroxy-a-benzimidazolylcobamidecyanide (Coates et al., 1956; Fox et al., 1957); and 2,3-naphthimidazolylcobamide cyanide (Coates et aE., 1956). The experiments by Coates and associates showed that the cobamide analogs were probably active per se as they were found unchanged in the livers and other organs of chicks whose growth was stimulated by the addition of these cobamides to the all grain ration. Many of the other cobamides listed in Table I11 have not been tested for activity in either the chick test or in the treatment of pernicious anemia, and it is likely that other cobamides will also be found which have activity in these tests. Barker (1959) has

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found that practically all of the cobamide present in rabbit liver exists in the coenzyme form.

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Factors Affecting the Antimicrobial Activity of Phenols E. 0. BENNETT Department of Biology, University of Houston, Houston, Texas

I. Introduction.. . . . . . . . . . . . . . . . . . ........................ .................................. 11. The Effect of Temperature., , , . 111. The Effect of Oxygen Tension.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. The Effect of p H . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. The Effect of Extraneous Materials. . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Inorganic Salts.. . . . . . . . . . . . . . ........... . . . . . . . . . . . . . . 126

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

1. introduction Almost one hundred years have elapsed since Kuchenmeister (1860) first recommended the use of phenol for controlling the growth of microorganisms. Perhaps this is an appropriate time to pause and consider the status of our knowledge of these compounds after a century of research and use. The research that has been done with phenols can be divided into two broad areas. First, workers have endeavored to find more active inhibitors by making substitutions on the molecule; and second, investigators have studied the factors that may increase or decrease the inhibitory activity of these compounds. Many workers have studied the effect of molecular substitution on the antimicrobial activity of phenols, and excellent reviews have been published on this subject by Suter (1941) and Klarmann (1957). We need not be ashamed of our progress in this area as the work has been both intensive and fruitful. We cannot be proud of our progress in understanding the basic factors that affect the antimicrobial activity of phenols. Even after one hundred years, we still do not appreciate the importance of such factors as pH, 123

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temperature, the presence of inorganic salts, etc., on the inhibitory activity of these compounds. One sometimes has the feeIing that research in this area has been deliberately avoided for fear that some weakness of these compounds might be detected. Unjustified stress on phenol coefficients has also resulted in neglect of the factors affecting the antimicrobial activity of phenols. Many years ago Lockemann and Ulrich (1932) stated that phenol coefficients are not reliable criteria for estimating antibacterial activity and since that time other critical comments have appeared in the literature. In spite of these reports many workers still use phenol coefficients as the basis for grading antimicrobial agents. The factors that can increase or decrease the antimicrobial activity of inhibitors are of basic importance in selecting compounds for use against microorganisms. This review was written with two objectives in mind: first, to draw attention to the factors that can decrease or increase the inhibitory activity of phenols; and second, to point out areas where additional research is needed. It is hoped that the paper will be helpful to workers who are involved in the selection of inhibitors for use in controlling the growth of microorganisms.

II. The Effect of Temperature Bacteria may be more resistant to phenol when they m e cultivated at elevated temperatures prior to contact with the inhibitor. Chick (1908), Smyth (1934), and Grubb and Edwards (1946) observed that Salmonella lyphoscs and Straphylococcus aureus were more resistant to phenol when the cells were grown at increased temperatures. It should be noted that Grubb and Edwards (1946) could not increase the resistance of all strains of staphylococci under the same conditions, so it is possible that the phenomenon is specific for certain strains. There are not enough studies to determine whether increased resistance to phenols after growth at elevated temperatures is characteristic for all organisms and whether it is exhibited with all types of phenolic compounds. It has been reported that bacteria exhibit a relationship between sensitivity to heat and phenol. Davis et al. (1948) carried out a study of this type with heat-sensitive and resistant strains of Bacillus globigii. They observed that the spores from the heat-resistant culture survived longer in 5 % phenol than did the spores of the heat-sensitive strain. A similar observation was made by Szeremi (1951) who observed that strains of Escherichia coli isolated from warm-blooded animals were more resistant to heat and phenol than were strains obtained from cold-blooded animals. Cicconi (1941) may have indirectly demonstrated the relationship when he found that pseudomonads exhibited a relationship between sensitivity to phenol

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and low temperature (3°C.); strains that were sensitive to cold were also sensitive to phenol. These papers may have some importance with regard to the control of microbial growth. For example, quite often the flora of two similar deterioration problems may exhibit significant differences in their sensitivity to a phenolic compound, and the variation may be due to differences in the environmental temperatures. It is also possible that greater concentrations of inhibitor would be necessary to control microbial growth in the summer than in the winter if the environmental temperature fluctuates with the season. Phenols are much more effective against bacteria when the temperature is increased while the organisms are in contact with the inhibitoSfCooper and Haines (1928) observed that increasing the temperature from 20" to 37°C. doubled the effectiveness of phenol against Escherichia coli and pseudomonads. Shimizu and Ueno (1955) noted that pentachlorophenol was also more effective against Bacillus mesentericus and Bacillus mycoides spores a t elevated temperatures. Tilley (1942) found that the inhibitory activity of phenol, cresols, resorcinol, and butylphenols was increased against Staphylococcus aureus and Salmonella typhosa when the temperature was increased. It appears that if the environmental temperature can be increased simultaneously with the addition of the phenol the inhibitor will function more effectively.

111. The Effect of Oxygen Tension There are very few observations in the literature pertaining to the effect of oxygen tension on the inhibitory activity of phenols. In general, there are indications that anaerobic bacteria are more resistant to phenols than aerobic organisms (Scott, 1928, 1937; Gould et al., 1957). Scott (1937) observed that anaerobic bacteria survived for one month in 0.5% phenol while the most resistant aerobes studied did not survive for more than two weeks under the same conditions. Oxygen tension may have a significant effect on the sensitivity of facultative organisms to phenols. Gould et al. (1957) noted that an unidentified diplobacillus that was sensitive to hexachlorophene under aerobic conditions was highly resistant when grown under anaerobic conditions. This observation may be of practical importance in deterioration problems where there may be a fluctuation from aerobic to anaerobic conditions. Organisms such as pseudomonads may be inhibited in the presence of oxygen, but when anaerobic conditions prevail the organisms multiply and possibly even oxidize the phenol to levels that are no longer inhibitory under aerobic conditions.

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There is also an indication in the literature that it may be possible to increase the antibacterial activity of at least one phenol by manipulating the oxygen tension simultaneously with the addition of the inhibitor. Fowler (1951) noted that 2,4-dinitrophenol interferes with the shift by Escherichia coli from aerobiosis to anaerobiosis. It may be possible to enhance the effectiveness of this inhibitor and others by shifting from aerobic to anaerobic conditions at the same time the inhibitor is added. Much additional research is needed before any definite conclusion can be made as to the effect of oxygen tension on the antibacterial activity of phenols. IV. The Effect of pH 3 It is @ell-knownthat phenols are most effective in an acid environment and that their antibacterial activity decreases as the pH becomes more alkaline. It has been observed that phenol, cresols, and chlorophenols exhibit reduced activity in alkaline solutions (Kuroda, 1926; Lundy, 1938), and other workers have reported similar results with n-butylphenol (Rettger et al., 1929b), and dinitrophenol (Barron et al., 1950). Ordal (1941) showed that sixty-eight times as much 2,4,6-trichlorophenol was required to kill StaphylococcusauTeus a t a pH of 9.8 a8 at 5.8, and Wolf and Westveer (1952) found that eight times as much 2 , 3 ,44richlorophenol was required to kill Salmonella typhosa at a pH of 8.2 as at 7.2. These results indicate that some phenols at least cannot be expected to function properly in an alkaline environment and that possibly the use of these inhibitors should be avoided in such situations. The specific effect of an alkaline environment on the antibacterial activity of phenols may depend partly upon the organism used in the study. Lundy (1938) observed that the antibacterial activity of sec-amyltricresol decreased against Straphylococcus aureus as the pH increased; on the other hand, when Eschem'chia coli was used the antibacterial activity increased as the pH became more alkaline. Klarmann et al. (1934) noted that there was no reduction in activity of 2-n-amyl-4-chlorophenol and 2-cyclohexyl4-chlorophenol against Mycobdctem'um tuberculosis in a pH range from 6.6 to 8.2. However, there was a reduction in activity when Staphylococcus auTeus was used as the test organism. It is possible that some phenols may be effective against specific organisms in an alkaline environment. Additional research is needed in this area before an intelligent conclusion can be made, however.

V. The Effect of Extraneous Materials A. INORGANIC SALTS The fact that inorganic salts can influence the inhibitory activity of phenols was noted many years ago when it was discovered that sodium

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chloride increased the absorption and antibacterial activity of phenol (Meyer, 1901; Das Gupta, 1937). Lundy (1938) was the first worker to make an extensive study of this observation using Staphylococcus aureus as the test organism. He found that 2 % concentrations of FeC1, , Fez(SOJa , and CuS04 increased the bactericidal activity of phenol, and CuSOr and ferric alum increased the effects of sec-amyltricresol. Lundy (1938) suggested that oxidation-reduction potentials may be involved in the ability of some salts to increase the killing power of phenols. Salle and Guest (1944) proved that the inhibitory activity of phenols can be increased in the presence of oxidation-reduction systems. These workers observed that there was a 18-fold increase in the efficiency of phenol against Staphylococcusaureus when one-half gram mole (one equivalent) of ferric sulfate was added to one gram mole of phenol. Similar results were obtained with a mixture composed of one gram mole each of ferric chloride and phenol. The addition to the solution of phenol and ferric chloride of another oxidation-reduction system, composed of one gram mole of ferrous chloride to two gram moles of ferric chloride, produced a 15- to 45-fold increase in the efficiency of phenol. Similar results also were obtained with cresol and hexylresorcinol. Copper ions potentiate the antibacterial activity of at least three different phenols against Mycobacterium tuberculosis. Erlenmeyer et al. (1953) discovered that the presence of a 0.0002 M concentration of copper ions reduced the inhibitory concentrations of o-(p-nitrobenzylideneamin0)phenol and o-(4-pyredylmethyleneamino)phenol to about one-half those needed in the absence of the metal ion. Hahn et al. (1953) observed that the presence of copper ions also increased the effectiveness of o-aminophenol against the tubercle bacillus. Other metal ions can increase the efficacy of phenols. Erlenmeyer et al, (1953) found that a 0.001 M concentration of cobalt ions increased the antimicrobial activity of o-(p-nitrobenzy1ideneamino)phenol and 0-(4pyredylrnethy1eneamino)phenol. The cobalt ions were about as effective as copper ions for increasing the activity of the two compounds. It is possible that the specific effect of salts may be dependent upon the test organism used in the study. Lundy (1938) observed that 2% concentrations of FeC13, ferric alum, Fea(S04)3, KaCr&, and KMN04 enhanced the inhibitory activity of sec-amyltricresol against Escherichia coli but had no effect on the effectiveness of the same compound against Staphylococcus aureus. Inorganic salts can also interfere with the inhibitory activity of phenols. Lundy (1938) noted that the antibacterial activity of phenol against Staphylococcus aureus was reduced in the presence of 10 % Na3POc. However, the author believed that this reduction may have been due to in-

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creased alkalinity. Maurice (1957) reported that a 2 X M concentration of phosphate buffer (potassium) at a pH of 6.7 more than doubled the sterilization time of 1.2 % phenol against Escherichia coli. Similar studies showing some interference of activity by salts have been reported by Haydon (1956) and Kravitz and Stedman (1957). B. SOAPSAND SIMILARCOMPOUNDS The inhibitory activity of phenols is believed to be partly dependent upon their tendency to migrate from the aqueous to the lipoidal phase as represented by the bacterial cell. Fogg and Lodge (1945) and Pujimoto (1951) have shown that there is a relationship between the distribution coefficient and inhibitory activity. The phenols that are the best inhibitors also migrate rapidly from the aqueous to the lipoidal phase. Soaps and related compounds can interfere with the activity of phenols by creating an additional lipoidal phase. In this instance, the inhibitor migrates into the lipoidal phase and the concentration available to inhibit the bacteria is reduced in proportion to the concentration of soap, Soaps have been shown to interfere with the antimicrobial activity of phenol (Frobisher, 1927; Hampil, 1928), sec-butylphenol (Hampil, 1928), m-cresol (Hampil, 1928), n-butylresorcinol (Hampil, 1928; Rettger et al., 1929a), and hexylresorcinol (Hampil, 1928 ; Frobisher, 1927). Glycerol has been reported to interfere with the antimicrobial activity of phenol (Cooper, 1948), cresols (Bartos, 1937 ; Cooper, 1948), o-phenylphenol (Tilley and Schaffer, 1931), chlorophenols (Cooper, 1948), and hexylresorcinol (Tilley and Schaffer, 1931). Cooper (1948) observed that glycerol reduced the activity of chlorophenols more than it did phenol or p-cresol. Bile and a lipid fraction from the liver were found to interfere with the inhibitory activity of phenol and dinitrophenol (Barnabie and Brighenti, 1954; Poe and O’Kelly, 1949). Glycols also interfere with the effectiveness of phenols. Cooper (1948, 1949), and Higuchi and Lach (1954) observed that ethylene, polyethylene, and propylene glycols reduced the inhibitory activity of phenols. The Tweens and other nonionics have been shown to reduce the inhibitory activity of bis-phenols (Bolle and Mirimanoff, 1951 ; Lawrence and Erlandson, 1953; Erlandson and Lawrence, 1953; Einola, 1955; Schoog, 1957), the Dowicides (Erlandson and Lawrence, 1953), and o-benzyl-pchlorophenol (Erlandson and Lawrence, 1953). These reports indicate that phenols may not function properly in the presence of large quantities of soaps or related compounds. There are several papers in the literature that verify this point. Barr and Tice (1957) found that phenols were not effective as preservatives for solutions containing nonionic surfactants of the sorbitan-partial-fatty-acid and poly-

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oxyethylene-ester types. Kabelik (1947) observed that phenols, chlorophenols, nitrophenols, and cresols were unable to preserve oils used in the manufacture of leather. Allred (1954) found that he had to compensate for the loss of tetrachlorophenate into the oil phase in water floor operations. He found that from 20 to 25 % of the inhibitor migrated from the water to the oil phase. Bennett (1957) reported that phenolic compounds are of very little value in the control of bacterial growth in emulsion oils. If the type and quantity of soap can be controlled it is possible to increase the antibacterial activity of phenols with these compounds. Schaffer and Tilley (1930) found that castor-oil and linseed-oil soaps mixed in the proportion of one part soap to two parts phenol doubled the germicidal efficiency of phenol, cresol, and hexylresorcinol. Rapps (1933) found that 3-methyl-4chlorophenol was about twice as effective in a castor-oil soap solution as in water. Ordal et al. (1941) observed that the antibacterial activity of phenols against Staphylococcus aureus was increased by the addition of pure soaps such as sodium oleate: Cade (1935) showed that soaps increased the antibacterial activity of phenols in both acid and alkaline environments. Schaffer and Tilley (1930) noted that the enhancement of germicidal activity was generally greater against gram-negative organisms. Conflicting reports have appeared in the literature pertaining to the effect of wetting agents on the antibacterial activity of phenols. Gershenfeld and W i t h (1941) noted that thirty-nine surface-tension-reducing compounds commonly employed as wetting agents did not increase the bactericidal or bacteriostatic efficiencies of sixteen phenolic compounds. The same year Ordal et al. (1941) studied the effect of sodium laurate on the germicidal power of phenols. He observed that the presence of the wetting agent increased the inhibitory activity of phenol, thymol, o-phenylphenol, and 2-chloro-o-phenylphenol against .Staphylococcus aureus. The following year, Fisher (1942) noted that cafionic, anionic, and neutral surface-tension-reducing substances did not increase the bactericidal activity of phenol or cresol. Later, Ordal and Deromedi (1943) found that sodium lauryl sulfonate increased the germicidal action of 2,4-dichlorophenol and 2,4,6-trichlorophenol against Staphylococcus aureus. Shute and North (1953) observed that sodium lauryl sulfonate increased the inhibitory activity of phenol against Pseudomonas aeruginosa. Trim and Alexander (1944) found that the antibacterial activity of hexylresorcinol was increased by the addition of cetyl trimethyl ammonium bromide. Several studies have shown that Aerosol OT, the dioctyl ester of sodium sulfosuccinate, increased the antibacterial activity of phenol, 2,4-dichlorophenol, 2,4,6-trichlorophenol, cresol, and hexylresorcinol against Staphylococcus aureus (Gershenfeld and Perlstein, 1941; Ordal and Deromedi, 1943; Tobie and Orr, 1945). The potentiating action of Aerosol OT is presumably

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due, at least in part, to a reduction of the interfacial tension between the bacterial cells and the surrounding liquid so that the germicide penetrates the cell more readily. Gershenfeld and Perlstein (1941) found that Aerosol OT produced greater potentiation at pH 4 than at 7, and Ordal and Deromedi (1943) noted that it nearly disappeared at pH 7.7; and at 9.7 the solution of phenol and Aerosol OT was no more effective than the same concentration of Aerosol OT alone. It appears that the enhancement of the antibacterial activity of phenols by Aerosol OT was due to a synergistic action between the wetting agent and the undissociated molecule. There are indications that the effect of surface active agents on the activity of phenols may, among other things, be dependent upon the pH, the particular surface active agent used, the particular phenol used, the ratio of surface active agent to phenol, and the organism used in the test. Perhaps the complexity of the problem accounts for the confusion in the literature.

C. OTHER TYPESOF ORGANICMATTER It is well known that organic material can interfere with the antibacterial activity of phenols. Several years ago Klarmann et al. (1929) observed that the presence of small quantities of peptone and gelatin caused a 17% reduction in the effectiveness of phenol against Staphylococcus aureus and Salmonella typhosa. These workers observed that organic matter a180 interfered with the bactericidal activity of the halogenated phenols and that the degree of interference was dependent upon the number of halogen atoms in the molecule. The monosubstituted phenols such as p-chlorophenol showed reductions comparable to that of phenol, whereas the effectiveness of 2,4,6trichlorophenol was reduced about 50 % under the same conditions. Klarmann et al. (1929) also found that the antibacterial activity of the alkyl phenols and the di- and tri-substituted halogen derivatives of resorcinol were reduced in the presence of organic matter. These workers concluded that even exceedingly small quantities of organic material are sufficient to cause a considerable reduction in the germicidal activity of certain phenols. The degree of impairment by organic matter may be dependent upon the test organism and structure of the phenolic compound. Klarmann et al. (1929) observed that organic matter reduced the antibacterial activity of thymol more than 60 % against Staphylococcus aureus. However, there was much less impairment when Salmonella typhoea was used as the test organism. The di- and tri-substituted halogen derivatives of resorcinol also showed greater reduction in effectiveness in the presence of organic matter with Staphylococcus aureus than with Salmonella typhosa. On the other hand, m-cresol was about equally effective against both organisms, and there was no pronounced impairment of germicidal efficacy by organic matter.

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There are scattered observations in the literature pertaining to the interference of other materials with the activity of phenols. It has been reported that alcohol (Das Gupta, 1937; Bartos, 1937), charcoal (Eisler, 1926), serum (Bechhold and Ehrlich, 1906; Rettger et al., 1929a, 1929b), ascorbic acid (Barber and Haslewood, 1946), blood (Barber and Haslewood, 1946), formamide (Cooper and Goddard, 1952), salivary mucus (Klarmann et al., 1951), and dust (Klarmann et al., 1951) interfere with the antimicrobial activity of phenols. Dagley et al. (1949, 1950) observed that phenols inhibited growth of Aerobacter aerogenes by inducing a lag period. These workers suggested that phenol produced this lag phase by retarding production of essential metabolites necessary to initiate cell division. It is possible that the antibacterial activity of phenol could be neutralized by the addition of filtrates of cultures that had grown under normal conditions provided that all the essential metabolites have not been consumed during growth. Dagley et al. (1950) found that filtrates from fully grown cultures of Aerobacter aerogenes could largely abolish lag due to phenol. Several compounds that could be present in culture filtrates have been studied for their ability to reduce effects of phenol on bacterial growth. Dagley and Freeman (1949) observed that acetate and butyrate ions in subbacteriostatic concentrations partially reversed the inhibition of Aerobackr aerogenes produced by a 600 p.p.m. concentration of phenol. Dagley et al. (1950) found that leucine, methionine, glutamate, a-ketoglutarate and succinate all decreased the lag phase produced by phenol. They also observed that amino acids could partially abolish the lag phase produced by p-cresol, p-n-propylphenol, and p-n-butylphenol. These reports indicate that the inhibitory activity of some phenols can be reduced by metabolic products of bacterial growth. The effectiveness of phenols may be reduced if they are used in situations where quantities of these compounds are present. This is one of the reasons why phenols may function more effectively if they are added before growth of bacteria and deterioration has occurred.

VI. The Effect of the Size of the Bacterial Population The effectiveness of phenols a t any given concentration is partly dependent upon the quantity of bacteria in the environment. Smyth (1934) found that doubling or halving the concentration of Staphylococcus aureua cells made a difference of about 17.0 % in the results of phenol death-rate determinations. Garrod (1935) demonstrated an even greater effect with the same organism. He showed that a concentration of 5000 p.p.m. of phenol was required to inhibit a large inoculum, whereas a concentration of about 80 p.p.m. inhibited a small one. Tezuka (1940) also noted that there was a

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reduction in the effectiveness of phenol as the bacterial population increased. The quantity of dead cells is also an important factor in determining the effectiveness of phenols. Tezuka (1940) demonstrated that phenol is absorbed by both living and dead cells and that dead cells take up more phenol than living cells. These reports show that a larger quantity of inhibitor must be used as the number of bacteria increases. This fact is very important in regard to the amount of a particular phenol that may be necessary to destroy the bacterial population in a particular environment. The initial concentration of inhibitor may have to be two or three times greater than that necessary to control growth after the initial population is eliminated. The indiscriminate use of phenols may stimulate deterioration under certain conditions. Even though a large population of different bacteria may be present, quite often deterioration is caused by the growth of only one one or two species. Phenols may kill the extraneous flora but a t the same time stimulate the organisms involved in deterioration. A possible explanation accounting for this observation may be found in the reports pertaining to the leakage of intracellular components after treatment with phenols. Gale and Taylor (1947), Pulvertaft and Lumb (1948), Yanagita and Suzuki (1948), Stedman et al. (1957), and Beckett el al. (1958) observed that the treatment of gram-positive and gramnegative bacteria with phenols caused a leakage of intracellular components. Bean and Walters (1955) suggested that the release of these constituents may influence the viability of the last survivors and alter the course of the bactericidal reaction. It is also conceivable that phenols can cause a leakage of materials from the extraneous flora that can be utilized for growth by the phenol-resistant organisms involved in deterioration.

VII. The Revival of Bacteria After Treatment with Phenol There is very little information available regarding whether or not bacteria can be revived after contact with supposedly lethal concentrations of phenols. One report in the literature indicates that Escherichia coli can resume growth if the concentration of inhibitor is reduced by dilution; however, this may not be true revival (Roberts and Rahn, 1946). Charcoal and ferric chloride have been studied for their ability to neutralize the lethal effects of phenols on bacteria and divergent results have been published. The controversy began when Flett et al. (1945) observed that Staphylococcus uurezls and Salmonella typhosa could be revived after treatment with phenol in concentrations that normally were lethal for the organisms. This revival was accomplished by adding charcoal (0.1 %) or ferric chloride (0.03%) to the subculture medium. Tilley (1948) repeated

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the experiment and reported that the presence of ferric chloride (0.03 to 0.1 %) did not reverse the inhibition of Staphylococcus aureus or Salmonella typhosa. A few years ago, Jacobs and Harris (1954) made an attempt to resolve this descrepancy. They found that activated charcoal (0.1 %) and ferric chloride (0.03%) did not revive Escherichia coli. It is impossible at this time to make a definite conclusion as to whether bacteria can be revived after treatment with lethal concentrations of phenols. If this fact can be shown in the future, the information will have considerable practical value.

VIII. The Development of Resistance to Phenols There are conflicting reports in the literature regarding whether or not bacteria can acquire resistance to phenols. Davies et al. (1944) observed that the resistance of Aerobacter aerogenes could not be increased even after 100 passages in the presence of a partially inhibitory concentration of phenol. Several other workers have also reported that they were unable to increase the resistance of microorganisms to these compounds (Fogg and Lodge, 1945; Yanagita et al., 1950; Dean and Hinshelwood, 1952; Wild and Hinshelwood, 1954). Gould et al. (1957) found that they could not increase the resistance of Bacillus subtilis, Pseudomonas aeruginosa, and Staphylococcus aureus to hexachlorophene. On the other hand, there are reports that some bacteria can develop resistance to these inhibitors. Escherichia coli developed resistance to phenol, hexylresorcinol (Meader and Feirer, 1926), and hexachlorophene (Gould et al., 1957), while the resistances of Salmonella typhosa and Aerobacter aerogenes were increased to hexylresorcinol and phenol (Meader and Feirer, 1926). Harm (1951) and Berger and Wyss (1953) increased the resistance of StaphyZococcus aurem to phenol. Berger and Wyss (1953) trained a strain sensitive to 1600 p.p.m. phenol to grow in the presence of 3000 p.p.m. of the compound, and resistance was retained through 40 transfers in the absence of the inhibitor. The resistant strain could not compete in a mixed culture with the sensitive wild strain when grown in a phenol-free medium. This observation may account for some of the difficulty encountered by other workers in producing resistant strains. Phillips and Hinshelwood (1953) approximately doubled the resistance of Aerobacter aerogenes to various alkyl phenols. These workers also noted some cross resistance; for example, resistance to o-isopropylphenol resulted in partial resistance to both phenol and thymol. Growth after 10 passages through a drug-free medium showed that fairly rapid though incomplete loss of resistance took place. However, the loss of resistance occurred less readily the longer the original training had been continued.

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It is probable that microorganisms can develop resistance to phenols under proper conditions, and this is one of the reasons that these compounds sometimes do not function properly. Workers who use phenols to control the growth of bacteria should always be on guard for the development of resistant strains. IX. The Oxidation of Phenols by Bacteria Many workers have reported the isolation of microorganisms that can oxidize phenols. One of the first papers on this topic was written by Wagner (1914) who found that phenol could serve as a source of carbon and energy for soil bacteria. Later, Sen Gupta (1921) showed that phenol disappeared from normal soil but not from sterilized soil, and that second and third applications of the compound disappeared much faster than the first application. Several studies have been reported pertaining to the quantities of phenol that can be utilized by bacteria. Most of these investigations have shown that approximately 1000 p.p.m. of phenol was optimum for oxidation (Gray and Thornton, 1928; Kalabina and Rogowskaya, 1934; Ergorova, 1946). Kalabina and Rogowskaya (1934) observed that bacteria could oxidize an average of 57.0 mg. of phenol per day. Phenol is oxidized faster under aerobic conditions; however, some attack of the compound can take place under anaerobic conditions as well (Meissner, 1953; Hamdy et al., 1954). Ettinger et al. (1951) observed that up to 50 p.p.m. of phenol may be dissimilated in approximately 2 weeks by sewage bacteria under anaerobic conditions and low temperature. Different workers have noted the effect of pH on the oxidation of phenol. Investigators have found that a pH of 7.0 to 7.5 is optimum for oxidation of phenol by bacteria (Meissner, 1953; Ergorova, 1946; Hamdy et al., 1956). The effect of temperature on the oxidation of phenol has also been studied. Ergorova (1946) observed that a temperature of 55" to 6OOC. was optimum for oxidation by Bacillus thermophenolicus. Hamdy et al. (1954) found that a temperature of 37" to 55OC. was optimum for oxidation of phenol by sewage bacteria, and Ettinger et al. (1951) found that phenol could be oxidized at 4°C. by sewage bacteria. Cresol was also one of the first compounds to be studied for its susceptibility to bacterial attack, and many years ago Wagner (1914) found that it could be oxidized by microorganisms. Since that time other workers have reported that soil bacteria can oxidize p-cresol, o-cresol, m-cresol, and 4,6-dinitro-o-cresol (Kramer and Doetsch, 1950; Dagley and Patel, 1957; Gundersen and Jensen, 1956). Gundersen and Jensen (1956) observed that

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a pH of 8.0 was optimum for oxidation of 4,6-dinitro-o-cresol. Bacteria oxidized o-cresol under both aerobic and anaerobic conditions and a t temperatures as low as 4°C. (Ettinger et al., 1951; Meissner, 1953). Organic material may stimulate the oxidation of phenols by bacteria. Gundersen and Jensen (1956) found that the oxidation of 4,6-dinitro-ocresol by a Corynebacterium was stimulated by small quantities (0.01 to 0.08 %) of yeast extract, alanine, asparagine, lysine, aspartic acid, cystine, cysteine, tyrosine, valine, glycine, glutamic acid, leucine, methionine, or histidine. Ammonia alone, or ammonia together with acetate or propionate could not substitute for the yeast extract or amino acids. Inorganic salts may be necessary for the oxidation of phenols by bacteria. Gundersen and Jensen (1956) reported that the presence of tap water was essential for the decomposition of 4 ,6-dinitro-o-cresol by Corynebacterium simplex. The compound was not oxidized when they used distilled water filtered through an Amberlite cation column. The addition of 0.1 % calcium carbonate to the medium allowed some growth and oxidation of the compound. These workers concluded that one or more trace elements seemed to be needed for the oxidation of the inhibitor. Bacteria have been studied for their ability to oxidize nitrophenols. Kramer and Doetsch (1950), and Gundersen and Jensen (1956) reported that different cultures of soil bacteria could not oxidize o-nitrophenol, m-nitrophenol, 2,4-dinitrophenol, 2,5-dinitrophenol, 2,6-dinitrophenoll and 2,4 ,6-trinitrophenol. Jensen and Gundersen (1955) and Gundersen and Jensen (1956) isolated a strain of Corynebacterium simplex that oxidized p-nitrophenol, 2 ,4-dinitrophenol, and 2 ,4,6-trinitrophenol. The culture oxidized p-nitrophenol in concentrations up to 500 p.p.m., survived in 1000p.p.m. concentrations, and was killed by a concentration of 2000 p.p.m. The organisms oxidized 2,4 ,6-trinitrophenol and 2,4-dinitrophenol in concentrations as high as 200 p.p.m. Simpson and Evans (1953) isolated two species of pseudomonads that oxidized up to 100 p.p.m. of o-nitrophenol and p-nitrophenol. Each strain utilized the appropriate nitrophenol but did not oxidize the other isomer, and neither strain oxidized the compounds under anaerobic conditions. Gundersen and Jensen (1956) studied the effect of pH on the oxidation of 2 ,4 ,6-trinitrophenol. The compound was oxidized over a fairly wide range with maximum oxidation between pH 6 and 7. These workers found that maximum oxidation of 2 ,4-dinitrophenol, and p-nitrophenol occurred at a pH near 8 and none of the compounds were oxidized at pH 7. Simpson and Evans (1953) observed that optimum pH for the oxidation of o-nitrophenol was 7 to 7.5 and 7.5 to 8.0 for p-nitrophenol. There also is a report in the literature pertaining to the oxidation of

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chlorophenols by microorganisms. Walker (1954) observed that soil bacteria were able to oxidize o-chlorophenol, p-chlorophenol, and 2,4-dichlorophenol. It is now evident that bacteria belonging to several diverse groups can oxidize phenols under a variety of conditions, and it is particularly important to use these inhibitors with care against such organisms. The author isolated pseudomonads that could oxidize phenols from metal-cutting fluids that had been treated with these inhibitors. In such instances the addition of phenols may stimulate growth of the bacteria and deterioration of the product.

X. The Use of Phenols with Other Inhibitors A. COMBINATIONS THATINCREASE INHIBITORY ACTIVITY

It has been reported that the combination of phenol and merthiolate produces a synergistic effect against bacteria. Rosenstein and Levin (1935a, b) found that a mixture of 50 p.p.m. of merthiolate and 2500 p.p.m. of phenol killed diphtheroids, staphylococci, and pseudomonads in 48 hours a t 5°C. When the compounds were used individually, some of the organisms were not killed by 5000 p.p.m. of phenol or by 100 p.p.m. of merthiolate. Falk and Aplington (1936) used the same ratio and observed that a mixture of merthiolate (100 p.p.m.) and phenol (5000 p.p.m.) killed Pseudomonus aeruginosa in serum after 24 hours exposure a t 20°C., whereas 295 p.p.m. merthiolate and 8000 p.p.m. of phenol were required when used singly. The mixture did not lose its potency for a t least 10 months in biological products (Rosenstein and Levin, 1935b). Sulfanilamide and sulfathiazole exhibit increased activity against Escherichia coli and Salmonella typhimurium when combined with phenol or resorcinol (Kayser et al., 1948, 1951; Kayser and Hadot, 1951). Kayser et al. (1948) observed that individually sulfanilamide (100 p.p.m.) and phenol (400 p.p.m.) had little effect on growth of Escherichia coli, but when they were combined they inhibited growth for 24 hours. Hayakaya (1955) found that the effectiveness of streptomycin and paminosalicylic acid against tubercle bacilli could be increased by combination with o-aminophenol. It was noted that the combinations were even effective against streptomycin and p-aminosalicylic acid-resistant strains of tubercle bacilli. There are two other reports pertaining to antimicrobial properties of mixtures containing phenols and antibiotics. The combinations of phenol with citrinin and 2 ,2'-thiobis(4 ,6-dichlorophenol) with tyrothricin have shown increased inhibitory activity against bacteria (Terui and Shibazaki, 1949; Florestano et al., 1956).

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Several other compounds are able to increase the effectiveness of phenols. Kojima (1931), and Barber and Haslewood (1946) found that the presence of oxidizing agents such as hydrogen peroxide increased the antibacterial activity of phenol and aminophenols. Bartos (1937) observed that the inhibitory activity of m-cresol increased when combined with ether or carbon tetrachloride. Cooper (1945, 1947) showed that acetone, acetylmethylcarbonol, and diacetone alcohol in less than germicidal amounts increased the bactericidal activity of phenol and p-cresol. This worker noted that acetone increased the killing power of phenol more than it did that of p-cresol or chlorophenols. Cooper and Coodard (1952) found that the activity of phenol was increased slightly in the presence of 20 % formamide. It is likely that in the future many other compounds will be shown to produce synergistic effects with phenols. It is also possible that the combination of two or more phenols will result in increased antimicrobial activity.

B. COMBINATIONS THATDECREASE INHIBITORY ACTIVITY There are reports in the literature that phenols can interfere with the antimicrobial activity of other compounds. Neumann (1946) found that phenols interfered with the effectiveness of p-phenylsulfamide against aspergilli. Bonicke (1952) found that m-aminophenol, phenol, and resorcinol interfered with the inhibitory activity of isonicotinic acid hydrazide against Mycobacterium phlei. Plaxco and Husa (1956) observed that bacitracin and phenol were incompatible, and Tinker and Husa (1957) showed that erythromycin lost its antibacterial activity in the presence of phenol. DiMarco and BiE (1951) and Cooper (1954) reported that small quantities of 2 ,4-dinitrophenol and phenol inhibited lysis of Staphylococcusaweus by Penicillin. DiMarco and Biffi (1951) suggested that 2,4-dinitrophenol slowed the growth of the organisms and thereby reduced their sensitivity to penicillin. Smith .(1952) demonstrated that the inhibition of Escherichia coli by chloramphenicol could be reversed by 2 ,4-dinitrophenol, 2,4-dichlorophenol, and 2-amino-4-nitrophenol. He suggested that 2 ,4-dinitrophenol may have increased the metabolic activity of the cells which, in turn, stimulated the oxidation of the antibiotic. It is likely that additional compounds will be observed to be incompatible with phenols in the future.

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Ordal, E. J., Wilson, J. L., and Borg, A. F. (1941). J. Bacteriot. 43, 117-126. Phillips, L. S., and Hinshelwood, C. (1953). J. Chem. SOC.3679-3683. Plaxco, J. M., and Husa, W. J. (1956). J. Am. Pharm. Assoc., Sci. Ed. 46, 141-145. Poe, c. F., and O'Kelly, R. J. (1949). J. Bacleriol. 67, 385-386. Pulvertaft, R. J. V., and Lnmb, G. D. (1948). J. Hyg. 46, 62-64. Rapps, N. F. (1933). J . Soc. Chem. Ind. (London) 62, 175-176, Rettger, L. F., Valley, G., and Plastridge, W. N. (1929~).Zentr. Bakteriol. Parasitenk. Abt. I . Orig. 110, 80-92. Rettger, L. F., Plastridge, W. N., and Valley, G. (192913). Zentr. Bakleriol. Parasitenk. Abt. I. Orig. 111, 287-296. Roberts, M. H., and Rahn, 0. (1946). J . Bactepiol. 62, 639-644. Rosenstein, C., and Levin, I. (1935a). J . Bacteriol. aS, 6. Rosenstein, C., and Levin, I. (1935b). Am. J. Hyg. 21, 260-279. Salle, A. J., and Guest, H. L. (1944). PTOC. SOC.Ezptl. B i d . Med. 66, 26-28. Schaffer, J. M., and Tilley, F. W. (1930). J . Agr. Research 41,737-747. Schoog, M. (1957). Artneimittel-Forsch. 7, 400-402. Scott, J. P. (1928). J . Infectious Diseases 43, 90-92. Scott, J. P. (1937). J. Znfectious Diseases 61, 103-109. Sen Gupta, N. N. (1921). J . Agr. Sci. 11, 136-158. Shimizu, W., and Ueno, S. (1955). Bull. Japan. Soe. Sci. Fisheries 20, 927-929. Shute, C. C. D., and North, R. A. (1953). J . Roy. Znst. Public Health and Hyg. 16,7481. Simpson, J. R., and Evans, W. C. (1953). Biochem. J. 66, xxiv. Smith, G. N. (1952). Arch. Biochem. Biophys. 40, 314-322. Smyth, H. F. (1934). J . Bacteriol. 28,333-341. Stedman, R. L., Kravitz, E., and King, J. D. (1957). J . Bacteriol. 75,655460. Suter, C . M. (1941). Chern. Revs. 28, 269-299. Szeremi, K. (1951). Ann. hug. publ., ind. et sociale 28, 21-34. Terui, G., and Shibaxaki, I. (1949). HakkB Kdgaku Zasshi 27, 150-156. Tezuka, E. (1940). Japan. J . Ezptl. Med. 18.387433. Tilley, F. W. (1942). J. Bacteriol. 43, 521-526. Tilley, F. W. (1948). J . Bacteriol. 66,479-488. Tilley, F . W., and Schaffer, J. M. (1931). J. Agr. Research 43,611517. Tinker, R. B., and Husa, W. J. (1957). J . Am. Pharm. ASSOC., Sci. Ed. 46,243-246. Tobie, W . C., and Orr, M. L. (1945). 3. Lab. Clin. Med. 30,741-744. Trim, A. R., and Alexander, A. E. (1944). Nature 164, 177-178. Wagner, R. (1914). 2,Glirungsphysiol.4,289-319. Walker, N . (1954). Plant and Soil 6, 194-204. Wild, D. G., and Hinshelwood, N. (1954). Proc. Roy. SOC.B142, 427-436. Wolf, P. A,, and Westveer, W. M. (1952). Arch. Biochem. Biophys. 40,306-309. Yanagita, T., and Suzuki, Y. (1948). J. Antibiotics (Japan) 2, 197-204. Yanagita, T., Tsutomu, S., Yuji, M., Takashi, U., and Shigeo, F. (1950). J. Antibiotics (Japan) 3, 3842.

Germfree Animal Techniques and Their Applications' ARTHUR W . PHILLIPS AND JAMES E. SMITH

Biological and Food Research Laboratories, Department of Bacteriology and Botany, Syracuse University, Syracuse, New York

V. Additional Research Needs. . . . . .

,...,.....

B. Animals Free from Antigenic Stimulation.. .

F. Nomenclature and Symbolism.

1. Introduction The development of the germfree (gf) vertebrate animal as a generally useful experimental system has only recently attained a suitable stage of fruition. More than sixty years of international effort by remarkably few investigators has resulted in the establishment of breeding colonies of gf rats and mice and short-term rearing of other species. These achievements have been accompanied by an increased interest in the applicability of the gf animal for routine experimental work since it provides the only method of assuring direct and complete control of the microbial inhabitants of a host animal. It is well established now that no 1 Studies conducted in our laboratory were aided by grants from the National Institutes of Health, United States Public Health Service, and the Syracuse University Research Institute.

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viable microorganisms are present in any part of the gf animal; however, the question of their being virus-free remains to be fully answered. Obviously, a gf animal has to be confined to an isolated environment which, in turn, must be free from microbial contamination; this requires appropriate isolation equipment which, for many applications, need not be either complex or expensive. Any desired microorganism, or combination of organisms, may be introduced to the gf animal, thereby permitting the determination of precise interactions of the host and its specified flora. This represents an important advance in the eventual realization of a research animal that is defined with respect to genetics, nutrition, environment, and exposure to foreign antigens. This essay treats the chief currents of progress and thought concerning the gf vertebrate. In addition, some of its potential applications are discussed.

II. Methods and Equipment Isolation equipment for rearing and handling gf vertebrates has undergone much refinement during the last sixty years. The past decade, however, has shown the greatest improvement in apparatus by investigators in the United States, Sweden, and Japan.

A. APPARATUB AND METHODS 1. Historical

The first detailed illustrated description of gf apparatus was provided by Nuttal and Thierfelder (1895) for use with guinea pigs. The animals were maintained in a glass bell jar system equipped with air filters and a sterile food supply. Cohendy (1912) described a similar apparatus for chickens. Kuster (1915) described apparatus which is substantially the basis for present day equipment. Gustafsson (1948) has discussed the design and operation of all the equipment mentioned above. 2. Modern Apparatus

Isolation equipment may be classified as high- and low-pressure types. The former will withstand internal pressures necessary for steam sterilization; the latter must be sterilized a t much lower pressures or within an autoclave. a. High-pressure Type Isolators. Reyniers (1943) and Reyniers and Trexler (1943) have described their equipment. Essentially the apparatus is a modified autoclave (gf rearing unit) with a smaller autoclave attached (food clave). An inner door serves to separate the two autoclaves which can function independently, thus permitting the transfer of objects without

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contamination. The main autoclave, or gf isolator, is equipped with glass viewing ports as well as rubber gloves sealed into the wall of the unit for handling objects within. In addition, the unit is equipped with air filters for providing sterile air. These consist of metal tubes packed with glass wool. The entire assembly is sterilized as a unit by steam under pressure followed by vacuum drying, Protective plates bolted over the glove ports prevent glove expansion and breakage during sterilization. An alternative to the food clave for transferring materials in the above system is the germicidal trap containing 2 % mercuric chloride solution which seals the main gf unit from the exterior preventing the passage of contaminated air into the gf unit. Objects such as fertile eggs, for instance, are put into the trap where they remain until their surfaces are sterilized, after which they are brought into the gf unit where chicks are hatched and reared in the gf state. Sterile objects can be passed from one gf isolator to another without exposure or contamination by connecting the two units through a sterilized food clave as described by Reyniers (1943). This is a great advantage in the maintenance of equipment and redistribution of animals, particularly in long term experimentation. Further modifications of the above gf unit have been described (Reyniers et al., 1946; Reyniers, 1956) for special purposes such as performing Caesarean sections, examinations, and storage. The basic isolator unit consists of a steel (stainless preferred) cylindrical unit about 32 inches in diameter by 42 inches in length. These units will contain 5 adult white Leghorn chickens or 25 adult rats. Miyakawa et al. (1958b) also described autoclave type gf apparatus similar in size and design to that described above but with important modifications. Recognizing the inherent weakness of rubber gloves, Miyakawa and his co-workers avoided the need for gloves by the use of remotecontrolled mechanical manipulators operated from outside the gf chambers. Manipulations such as parenteral injections into animals were performed with ease in this apparatus. Sterile air to the rearing units is provided by passing air through glass wool filters and then through an electric heating unit which elevates air temperature to 500°C.; the air is cooled before entering the isolator. Exhaust air from the isolators is passed through glass wool filters. Provision is also made for temperature and humidity control of air in the rearing units. The capacity of each unit is 30 guinea pigs. The application of remote controlled manipulators to gf isolators by Miyakawa represents an important advance in gf methodology particularly for long term maintenance of gf animals. No doubt further advances will be made along these lines. Because of capacity limitations of the units described above, a much

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larger rearing unit was built with a capacity of 2,000 rats of all ages (Reyniers, 1956). This is a stainless steel cylindrical autoclave-type unit 8 ft. in diameter by 15 ft. in length and sterilized by steam under pressure. A large germicidal tank attached to the colony unit served as a trap and permitted the entry of an operator sealed in a plastic suit. A food clave (lock) at one end of the large unit allowed the transfer of gf animals, sterilized food, etc. to or from a smaller gf unit attached to the gf colony unit. b. Low-Pressure Isolators. Since this equipment may be constructed of relatively thin metal, plastic, rubber, or glass, the cost of this equipment is substantially less than high-pressure apparatus. Low-pressure isolators are sterilized in an autoclave or with chemical germicides. A good example of steam sterilized equipment is that presented by Gustafsson (1948). It is a rectangular plan, approximately 3 ft. in length, 2 ft. high, 18 inches wide, and made of inch stainless steel. A large removable, rectangular plate glass window in the top provides complete visibility of the interior. This is a great advantage over the much smaller circular viewing ports of the high pressure isolator. Rubber gloves are attached to the wall as in previous units. A germicidal trap attached to the unit permits transfer of objects to the interior. The exhaust air from the isolator is bubbled out through the germicidal trap. The entire unit is leak-tested by filling the unit with ammonia gas and applying moistened red litmus paper over exposed joints, Air is sterilized by passing through a combination filtration and heating unit (500°C.) ; layers of asbestos, glass wool, and carborundum are used in the air sterilization unit. Sterilization of the assembled gf isolator is achieved by wheeling the unit into a steam autoclave. This is equipped with a germicide reservoir from which the trap of the isolator as well as the window seal are properly filled with germicide before the sterile unit is removed from the autoclave. Apparatus made of transparent plastics have been used with gf animals. For instance, Trexler and Reynolds (1957) described a flexible film isolator. “Plexiglas” (Rohm and Haas Co.) isolators have been used in our laboratory during the past several years. Plastic isolators may be sterilized by means of chemical germicides such as peracetic acid or ethylene oxide; however, those made of heat-resistant plastics may be sterilized by steam in an autoclave. In addition, glass jar isolators for rearing gf chickens up to about 3 weeks of age have been described (Reyniers et al., 1949b and Luckey, 1956a, b). c . Shipping Isolators. Reyniers and Sacksteder (1958b) described apparatus for shipping gf animals. Buccessful transportation of animals up to distances of 2,000 miles was reported.

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3. Procedures

a. Sterilization of Air. Various types of filters, alone or in combination with heat or electrostatic precipitation, have been used. More commonly, only filters (glass wool, asbestos) are used except in the apparatus of Gustafsson, and also that of Miyakawa. Prefilters should be used to reduce clogging of sterilizing filters. Exhaust air may be discharged through a filter or by bubbling through a germicidal liquid as used by Gustafsson (1948). b. Sterilization of Diets. Steam sterilization is generally used although radiation is currently being investigated. Diets are usually supplemented to compensate for losses in vitamins during steam sterilization. Reyniers et al. (1950) and Luckey et al. (1955~)have reported on the adequacy of diets sterilized by steam and electron beams. Detailed procedures for sterilization of diets have been reported by Reyniers et al. (1946), Gustafsson (1948), and Miyakawa et al. (1958a). c. Obtaining Gf Mammals. Mammals are obtained by Caesarean section with delivery of the young directly into the sterile isolator as described by Reyniers and associates (1943, 1946), Gustafsson (1947, 1948) and Miyakawa et al. (1958a). Animals must be obtained close to term and properly prepared with a germicidal seal to avoid contamination during delivery. d . Gf Birds. Fertile eggs are immersed in a germicidal solution for sterilization and then introduced directly into a sterile isolator which is maintained under proper temperature control (Reyniers et al., 1949b). Chickens have been so reared through the first generation. I n addition, turkeys have been raised in the gf state. B. REARINGOF GERMFREE ANIMALS 1. Birds

a. Chickens. Since the first attempt by Nuttal and Thierfelder (1895) to rear gf chickens, a number of efforts have been described by Cohendy (1912), Schottelius (1913),Balzam (1937), and Reyniers and Trexler (1943). Reyniers et al. (1949b) have described in detail their methods of rearing gf chickens. They have presented the results of extensive studies on egg surface sterilization and reviewed the work of others. Bacterial contamination of egg contents was occasionally encountered. Their data indicated that only eggs containing live embryos should be admitted to a gf isolator for hatching. In the procedure of Reyniers and co-workers, eggs are dipped in a detergent solution, brushed, and immersed in 1% HgC12 solution a t 38OC. for 5 to 10 minutes and put into the gf isolator where the germicide is allowed

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to dry on the egg surfaces during incubation. Results showed that only 95% of fertile eggs were gf. More recently, Luckey (1952) scrubbed eggs with a detergent solution, rinsed, dipped in 2% HgCla solution for 4 minutes, and incubated. Eggs were candled at 20 days, the viable ones placed in the germicidal trap attached to a gf isolator for 4 minutes, and then transferred into the sterile chamber for hatching. The trap on this unit also contained 2 % HgCll solution. Reyniers et al. (194913) described both practical and semi-synthetic diets for rearing chickens. These diets had to be supplemented with vitamins to compensate for losses during steam sterilization (Reyniers et al., 1950). b. Turkeys. The successful rearing of gf turkeys has been described by Luckey (1952, 195613). It was stated that gf turkeys were very similar to conventional birds in appearance and growth at least up to 4 weeks of age (Luckey, 1956b). 2. Mammals

The parent generation of gf mammals must be obtained by Caesarean delivery into a sterile isolator as described above. Only gf rats and mice have been bred and the gf young reared. a. Rats. Reyniers et al. (1946) and Gustafsson (1947, 1948) have given complete descriptions of the hand-rearing of gf rats as well as recommended diets. Reyniers’ group started feeding 3 hours after birth on an hourly schedule; pipettes with rubber teats were used for feeding. As much milk was given as the rat would take. Care must be used in the manner of feeding to avoid getting milk into the respiratory tract and air into the stomach and to prevent injury of the delicate young by handling. Deserving special attention is the technique of stimulating urination and defecation after each feeding by gentle stroking of the perianal region and urethral papilla with cotton. Weaning was started at 18 days and usually completed at 25 to 30 days. The milk diet was based on cow cream supplemented with casein, lactalbumin, salts, yeast, liver extract; vitamin C improved the growth of young rats. Gustafsson (1948) found the rubber teat feeding unsatisfactory and used a stomach tube which he reported to be a more efficient procedure than mouth feeding. Rats were fed every 4 hours; the amount of milk given increased from 0.8 to 8 gm. per day during the hand-feeding period. The milk diet used was cow cream supplemented with casein hydrolyaate, minerals, vitamins and liver extract. The reports of Gustafsson and of Reyniers and co-workers should be consulted for further details regarding the handrearing of rats. b. Guinea Pigs. Because guinea pigs are comparatively well developed at birth, they were employed by the earliest workers. Nuttal and Thier-

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felder (1895, 1896, 1897) reared gf guinea pigs on sterile cow milk for 2 weeks. Enlarged ceca and poor digestion complicated their work. Somewhat better results were obtained with yeast-supplemented diets by Cohendy and Wollman (1914,1922). Small numbers of gf guinea pigs up to 60 days of age were reared by Glimstedt (1936) who reported satisfactory growth of the animals, although the distended cecum problem was encountered. This syndrome has plagued all who have worked with gf guinea pigs. Reyniers and Trexler (1943) briefly described the rearing of this species. Phillips et al. (1955) reared gf guinea pigs for about 40 days on a weaning diet of Purina laboratory chow, oats, Cerophyl, Alphacel, dextrose, and salts. Miyakawa et al. (1958a) have described the most extensive studies on gf guinea pigs. They fed a milk diet supplemented with vitamins, yeast and liver for the first 5 days, then the milk diet was mixed with the solid diet until 30 days when the latter only was given. The solid diet consisted of roasted soy bean flour, sucrose, gum arabic, salts, vitamins, yeast, and liver. Growth weight curves for the gf and conventional controls on the same diet were comparable, although somewhat less than conventional animals. These workers have handled some 200 guinea pigs of which one gf animal was reared for 150 days during attempts to breed the animals. Nevertheless, mortality was highest during the first 20 days of life. c. Monkeys. Reyniers (1942, 1943) briefly reported the f i s t and only attempts to rear monkeys (Macacus rhesus) under gf conditions. Animals were given a supplemented diet that simulated the composition of monkey milk; additional vitamins were added. Also a precooked cereal and vegetable supplements were given. Weaned animals were fed sterilized carrots, apples, turnips, peas, spinach, tomatoes, corn, potatoes, bananas, pears. The growth rate and general appearance of the animals were comparable to controls, and monkeys were among the easiest animals to hand-rear under gf conditions. d . Goats. Kuster (1912) obtained good growth of gf goats reared on sterile goat milk which may have been largely responsible for the ease in rearing such animals. However, contamination occurred before the animals could be weaned. e. Rabbits. No reports are available on the rearing of this species. However, during 1939-1941 one of us (A.W.P.) attempted to rear gf rabbits. Although larger at birth than rats, rabbits are similarly underdeveloped and almost as delicate. While a variety of milk diets were used, including sterile rabbit milk, mortality was high and very few animals were weaned. The problem did not appear to be nutritional. Generally, the young animals had a distended bladder and defecation was infrequent. Rabbits that were weaned were given practical diets based on commercial rabbit chow. No gf

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rabbits were reared longer than 2 months, and distended ceca were of common occurrence. It appears possible that part of the problem of rearing gf rabbits might have been avvided by employing the stimulation technique subsequently found successful for hand-rearing gf rats. It should now be possible to rear gf rabbits with the same techniques used for rats. Indeed, Reyniers (1957) mentions that 10 gf rabbits were reared in 1956-1957. f. Other Species. Gf mice, dogs, and cats have also been reared, but no details are presently available (Reyniers, 1957; Gustafsson, 1948; and Wagner, 1958).

111. Characteristics of Germfree Animals

A. GENERAL CHARACTERISTICS Most of the observations discussed here were made on gf chickens and rats which have been reared through several generations. Other species have been studied in less detail, via. guinea pig, rabbit, mouse, dog, goat. monkey, and turkey. The general health and appearance of the gf animals are comparable to conventional animals (Gordon, 1955). Indeed growth rate, and red cell and hemoglobin concentrations may be slightly higher in the gf animal. While maturity and reproduction may begin a little earlier, the important question of their longevity has not been answered. However, they live a t least as long as normal animals. There are important characteristics peculiar to the gf animal which are presumably due to the absence of microorganisms. For instance, some organs and tissues that are ordinarily exposed to microorganisms, such as the gastrointestinal tract and the adjacent and associated lymphoid tissues, are smaller in weight in the gf animal than in the conventional one. In addition, the total leucocyte count is lower in the gf state. The serological picture also reflects the absence of microorganisms except for nonviable organisms found in sterilized diets (Wagner, 1955). The gastrointestinal tract, notably the small intestine and the ileocecal tonsil in the chicken, is more uniform in weight in the gf animal and rather variable in the conventional state (Gordon, 1955). The latter appears to have more connective tissue in this organ than its gf counterpart. These and other data seem to reflect certain rather profound effects on the host of intestinal microorganisms. 1 . Lymphocytes

Certain lymphoid tissues such as the ileocecal tonsil of the gf chicken may have one-tenth the lymphocyte content and less than one-half the weight of this organ in the conventional bird (Gordon, 1955). While no

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differences were seen in the spleen and its lymphocyte content, the blood of the gf animal contains markedly fewer lymphocytes than conventional blood. However, the difference between lymphocyte content and production or release of lymphocytes is exemplified by the reduced lymphocyte produp tion of the gf spleen. On the other hand, there is a marked increase in the lymphocytes released by the spleen of the gf animal following bacterial contamination. During the first two weeks of bacterial stimulation this induced production of lymphocytes increased greatly and after the third week subsided somewhat although still higher than the initial level. This stimulation was followed by increased antibody production.

2. Serological Characteristics Heterohemagglutinins against rabbit erythrocytes were found in gf chickens more than 30 days of age as well as conventional birds in about the same titer (Wagner, 1955). On the other hand, some of the antibacterial agglutinins characteristic of conventional chickens were not observed in gf birds, although agglutinins against small numbers of nonviable bacteria present in the diet eventually became manifest in gf birds over 2 months old. Parenteral injections of Salmonella pullorurn vaccine or beef serum into gf and conventional chickens produced the same antibody titers.

B. THEGERMFREE CHICKEN Much of our knowledge of gf life has been obtained on the chicken. Satisfactory growth and development of gf chicks was reported by Cohendy (1912), Balzam (1937), Gordon et al. (1958) using crude diets, and by Reyniers et al. (1949b) and Luckey et al. (1955a, 1956) using purified diets. 1. Growth and Development

The successful rearing of more than one generation of apparently healthy gf chickens was achieved by Reyniers et al. (1949a, b, c). Growth and morphology of the gf birds compared well with conventional birds on commercial broiler rations. White Leghorns and Rose Comb White Wyandotte bantam chicks grew equally well in the gf state. The purified diets supported good growth and development. Perosis was occasionally seen in some groups of gf birds. Egg production and hatchability were poor in gf birds. Morphological and histological analyses of body tissues such as skin, muscle, bones, and circulatory, respiratory, and other organs indicated no abnormalities in gf birds, with the exception of the following differences in the gastrointestinal tract and lymphoid tissues. The proventriculus, small intestine, and ceca were smaller in the gf animal which also had less peribronchial and intestinal lymphoid tissue than did the conventional bird. The gf animal occasionally showed a heavier thymus gland with a higher concen-

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tration of thymocytes; adrenals were slightly smaller. The increased weight of the gf thymus was observed on another occasion (Gordon, 1952) but more recently was not encountered in a larger group of gf birds (Gordon et al., 1958). A histological study of gf chicken lymphoid tissue by Thorbccke el al. (1957) indicated the absence of plasma cells and secondary nodules in the lining of intestinal mucosa which are common in normal birds. Lymphoid tissue, secondary nodules, and plasma cells are sparse in gf animals but occasionally are found in spleen and the ileocecal-colic junction especially in older gf animals. An important observation that requires further study is the low liver weight in older gf birds (Gordon, 1952). Young birds, 30 days old, did not show this difference. Gf chicks may have a slightly higher growth rate than controls (Reyniers et al., 1950; Gordon, 1955), although this is not consistent (Gordon et al., 1958). 2. Chemical Composition

Comparative analyses (Reyniers et al., 1949c) of liver, muscle, bone, ceca, brain, and serum of gf and conventional birds were reported for their fat, ash, dry weight, phosphorus, potassium, riboflavin, pantothenate, niacin, biotin, and folic acid. The gf birds had a high brain potassium level and high levels of biotin and niacin in the ceca. Riboflavin and vitamin C of the ceca were normal, but somewhat lower values of pantothenic acid, folic acid, and vitamin BI2were observed in gf chicks (Reynierset al., 1950). Further studies by Reyniers et al. (1950) on the vitamin content of the liver and ceca of gf and control chickens at 4 weeks of age indicated comparable levels of liver vitamin A, thiamin, riboflavin, pantothenic acid, and folic acid. But ascorbic acid in the gf liver and adrenals was lower than in control birds. A recent study of Gordon et al. (1958) failed to show a significant difference in adrenal ascorbic acid of gf and conventional birds. Gf chicks had higher levels of bile riboflavin and biotin, liver vitamin BIZ,and blood niacin (Luckey, 1956a). 8. Serum Antibodies

The serological picture of the gf chicken showed important differences from the conventional controls (Reyniers et al., 1949~).Although both gf and control sera contained natural hemagglutinins against rabbit, rat, and horse erythrocytes and not against sheep and human cells, the gf sera had a markedly lower titer than conventional sera for rabbit erythrocytes. Natural antibacterial agglutinins against Paracolobactrum uerogenoides, Escherichia coli, and Lactobacillus sp. were absent in gf sera in contrast to

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conventional sera. However, agglutinins were found in gf sera against Staphylococcus aureus and another Lactobacillus sp., although in lower titer than the controls. This was attributed to a mild oral vaccination of the gf animals by small numbers of dead bacteria present in the sterile diets; indeed, prior to sterilization these diets were shown to contain viable staphylococci and lactobacilli. The y-globulin content in serum of gf chicks remains at a constant lower level during growth from 4 to 17 weeks, whereas in conventional chicks it is somewhat higher a t 4 weeks, increasing to markedly higher levels at 17 weeks (Thorbecke et al., 1957). These workers found the total serum proteins to be the same in gf and conventional birds. A monocontamination in gf birds at 3 weeks of age failed to provoke an increase in y-globulin within one week; similarly, no y-globulin increase was observed in gf chicks after 4 weeks of B. subtilis contamination of 8-week-old gf birds. However, immunization of the monocontaminated animals having low y-globulin with a killed bacterial vaccine resulted in a rise of serum y-globulin to levels comparable to those found in immunized and nonimmunized conventional control birds.

4. Blood Picture Hematological observations (Reyniers et al., 1950) on 4-week-old gf birds showed no consistent differences in hemoglobin, hematocrit, or RBC. The total white-cell count was markedly lower in the gf birds which also had lower average values for the percentages of lymphocytes and eosinophiles and somewhat higher values for basophiles and heterophiles than did control birds. The most marked difference in the above data occurred in the relatively low eosinophile levels of the gf birds. There is also a more recent suggestion (Gordon, 1955) that gf animals have a slightly higher red-cell and hemoglobin level. RAT C. THEGERMFREE In comparison with the work done on chickens, there are fewer published details on the characteristics of the gf rat. Much of the work on this species has been conducted at Notre Dame University where a colony of gf rats exists in the eighth generation (Gordon, 1955) since the first report of this group (Reyniers et al., 1946). In addition, important studies have been reported by Gustafsson at the University of Lund, Sweden. 1 . Growth and Development

As might be expected, there is a great deal of difference between the growth rate and appearance of hand-reared gf rats obtained by Caesarean

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section and dam-reared gf rats, although these differences generally diminish some time after weaning. Comparative data on growth rates up to weaning have been reported by Reyniers et al. (1946) and Gustafsson (1948) who observed substantially lower growth rates in all hand-reared rats, whether gf or contaminated; mortality may be high in both groups, especially during the first 5 or 6 days. Bloating is rather common in the younger rat. Handreared gf rats sacrificed at 28 days showed a greatly distended cecum containing green-yellowfluid having a sweet odor (of course, conventional animals have an obnoxious odor). Reyniers et al. (1946) stated that when the hsnd-reared gf rat is weaned on a solid diet its growth improves and mortality is 15% or less. It is interesting that these workers also observed that mortality was higher in the hand-reared, contaminated young rats than among the hand-reared gf rats. They also stated that dietary ascorbic acid reduced bloating and mortality in hand-reared gf rats. Reyniers et al. (1946) also reported the successful rearing and breeding of gf rats. Several animals were hand-reared under gf conditions and fully weaned in about a month. Twenty gf young were born from 3 gf mothers, and 14 of the young were weaned. Morphological observations indicated less lymphoid tissue and distended ceca in all of the gf animals. Gustafsson (1948) observed a somewhat smaller thymus and spleen in 30day-old hand-reared gf rats, and abnormally small lymph glands in both gf and Contaminated hand-reared rats. I n the same age group, gf rats also showed a greatly distended intestinal tract, especially the cecum. A brownblack fluid was often seen in the gf tract. In the above report, Gustafsson also presented a clear picture of the histological differences in the cecal wall of the gf and conventional 30-day rats; in the gf rat a marked distension of the crypts of Lieberkuhn was seen as well as an absence of free cells in the epithelium and subepithelial connective tissue. Gustafsson concluded that older (more than 50 days) rats must be included in studies of lymphoid development. Orland et al. (1954) reported that while distended ceca and lens cataracts were occasionally seen in hand-reared gf rats these abnormalities were not found in gf rats reared by their gf mothers. They also showed a favorable osseous development of the cranium and femur of the gf animal. Orlsnd et al. (1954) presented data showing a sex difference in the body weights of 160-day gf and conventional rats; conventional males were about 100 grams heavier than gf males, whereas the females of both groups had comparable weights. Luckey et al. (1955b) found that with equivalent food consumption, conventional dam-reared rats grew faster and attained a higher body weight in 120 days than did gf rats on the same diet. Results did not indicate whether unknown dietary factors or preweaning status were involved.

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2. Chemical Composition of Tissues Luckey et al. (1955b) found that gf rats up to 9 months old were similar to conventional rats in the moisture, ash, fat, and nitrogen content of muscle, bone, spleen, liver, kidney, and brain. 3. Blood Picture

Only a brief description is available. Luckey et al. (1955b) showed that 146-day gf rats had the expected low total white cell count, a differential count similar to conventional rats, and somewhat lower hemoglobin values. It has been reported (Reyniers, 1956) that there was less blood supply to the intestinal tract of the gf rat, as well as other gf animals, and this organ lacked tonus, connective tissue, and lymphocytic cells. Intestinal villi were not as well developed in gf animals as in a control group.

4. Lymphoid

Systems

In practically all essential details investigated, the gf rat shows the characteristics described above for chickens (Gordon, 1955). This investigator commented that rats and chickens showed the same pattern of response to bacterial stimulation of lymphocytic elements. Gf animals appeared to have normal endocrine function, although except for the data on adrenal function, few hormone data are available. Adrenal cholesterol and ascorbic acid values indicated a normal pattern in gf animals. In the above report, Gordon also summarized data on lymphocyte removal from blood a t various levels of the rat intestinal tract. His results indicated a slight lymphocytic activity in the lower gut of the gf rat, although this activity was much less than in the conventional animal. The change in splenic lymphocyte production in gf rats exposed to mixed bacterial contamination has been briefly described by Gordon (1955). A very marked increase in lymphocyte production occurred during the first 2 weeks following contamination.

IV. Applications of Germfree Animals The application of gf animals to specific problems will be exemplified by the following descriptions of selected investigations. Only applications involving the living vertebrate animal will be discussed here. In addition, several potential applications of gf animals are suggested.

A. ESTABLISHED APPLICATIONS 1. Historical

Some of the earliest studies in this field indicated the application of gf animals to experimental biology and medicine. For instance, Nuttal and

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Thierfelder (1897) reported the absence of phenols in the urine of gf guinea pigs, whereas such compounds are normally found in conventional animals. Cohendy and Wollman (1922) used gf guinea pigs in their studies on Vibrio comma in which they showed that infection was possible in the absence of other organisms. Glimstedt (1936) used the same gf species in an investigation of lymphoid development, and Schottelius (1913) and Balzam (1937) employed gf chickens in studies of vitamin B complex deficiency. He showed that gf and conventional control chicks responded similarly to a diet deficient in B-vitamins. Cohendy (1912) showed that growth rates of gf and control chicks were comparable. Kuster (1912) reported that urinary ammonia excretion is less in gf goats than in controls and that urea excretion was higher in gf animals. Most of the early workers in the field were handicapped with very small numbers of gf animals; nevertheless, their observations generally have been confirmed by more recent investigations. Although the work summarized above was performed with relatively crude and limited facilities, the results show that much information can be harvested from short-term experiments with gf animals. 2. Modern Investigations

a. Etiology of dental caries. It has been observed that gf rats do not develop dental caries on a diet that produces a high incidence of caries in control animals (Orland et al., 1954). Carious lesions were seen only after deliberate contamination of gf rats with a Lactobacillus sp. (Orland et al., 1955). These results suggest the need for future studies on details of the carious process. b. Amoebic Dysentery (amoebiasis). The gf guinea pig enabled Phillips and co-workers (1955, 1958) to establish the necessity of synergism between bacteria and Entamoeba histolytica in the etiology of amoebiasis. The intracecal inoculation of amoebae into control animals resulted in ulcerative amoebiasis, whereas in gf guinea pigs the amoebae failed to survive longer than 5 days. However, after deliberate contamination of gf guinea pigs with either Escherichia coli or Aerobacter aerogenes, marked acute ulcerative amoebiasis resulted following inoculation with amoebae. These results together with further details regarding biochemical patterns of this interesting and important symbiosis should serve to encourage the investigation of other diseases having a possible symbiotic relationship in their etiology. The present work should be extended to studies of symbiosis of E. histolytica with other organisms, for there appears to be no data confining the synergism to the bacterial species mentioned. c. Radiation Injury. Bacterial infection following acute radiation injury due to whole-body exposure to high levels of ionizing radiations serves to seriously complicate the treatment and interpretation of radiation effects

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per se. The gastrointestinal tract and hemopoietic system, being acutely sensitive to radiation, are responsible in the main for permitting the invasion of host tissues by intestinal bacteria. The gf animal offers a direct approach to the study of radiation injury apart from microbial influence. A few brief reports are available on gf rats and chicks as applied to this problem. Gf rats exposed to a single dose of total body X-rays were reported to have a survival time twice that of conventional control rats with doses greater than 400 r. (Reyniers et al., 1956). As these workers pointed out, interpretation of the results must allow for the underdeveloped lymphoid tissues in the gf animal. This difficulty might be at least partially avoided with the aid of gf animals antigenically stimulated to render the gastrointestinal tract and lymphoid system more comparable to control animals. McLaughlin et al. (1958) also obtained increased survival in 10-day-old gf chicks exposed to X-ray doses under 800 r. Above this dose, all animals eventually succumbed, although the gf birds lived longer than the conventional irradiated animals. d. Liver Necrosis. Because of evidence that intestinal microorganisms may have a role in dietary liver necrosis, Luckey et aE. (1954) investigated the effects of a necrogenic diet on gf rats. Although necrosis was not seen in gf rats with twice the food consumption of controls, necrosis was encountered in the gf animals having the same daily food consumption as the control rats. Nevertheless, oral antibiotics were found to prolong the lives of conventional rats on a necrogenic diet (Forbes et al., 1953). They reported that while changes in intestinal flora occurred, these could not be clearly related to necrosis since a synthetic surface active agent gave similar results without altering the flora. It was suggested that antibiotic alterations of metabolic patterns of the flora may be involved. Evidence to be discussed in another section offers support to this possibility. e. Growth Promotion Effect of Antibiotics. The growth stimulation effect of antibiotic-supplemented diets has been well established for many species of animals (Jukes and Williams, 1953; Stokstad, 1954; Porter, 1957). Intestinal microorganisms appear to be involved, but the precise mechanism is not known. There is a possibility that antibiotic inhibition of bacterial decarboxylases may result in preventing the formation of growth-depressing amines (Melnykowycz and Johansson, 1955). Gf chickens and turkeys have been used in studying the growth-promotion effect of antibiotics. Although some inconsistencies have been encountered (Gordon, 1952; Luckey, 1952; Luckey et al., 1956), a more recent report indicates that no growth-promotion effects occur in gf chicks (Gordon et al., 1958). Indeed, these investigators observed that gf birds and antibiotic-fed conventional birds had smaller weights of the small intestine and

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ileocecal tonsil and had a higher lymphocyte concentration in the thymus than untreated conventional birds. More work is needed before the antibiotic growth effect can be interpreted in terms of metabolic changes of intestinal bacteria. Gf animals offer a direct approach to such investigations since the flora can be controlled and specific effects attributed to known organisms. f. Transmethylation and Intestinal Flora. The question of bacterial participation in the synthesis of biologically labile methyl groups was attacked by du Vigneaud et at. (1950, 1951) by feeding deuterium oxide to gf rats. Synthesis of the labile methyl group in the gf animals was comparable to that in controls. g. Vitamin Metabolism. Reyniers el al. (1950) reported no additional vitamin requirements of the gf chick over those of control birds. Gf birds had large amounts of vitamins in their ceca. Luckey et al. (1955a) reported that gf and conventional chicks showed similar qualitative requirements for B-group vitamins. However, the gf birds showed more acute deficiencies of thiamin, riboflavin, niacin, and folic acid when fed diets deficient in these vitamins. Vitamin-deficient birds (gf) excreted amounts of vitamins which if absorbed would have been nutritionally adequate. Peculiarly, gf birds recovered spontaneously from vitamin K deficiency. Further studies are needed on vitamin metabolism in gf animals with a controlled intestinal flora. Luckey et al. (1955b) showed that the gf rat, unlike the conventional rat, requires biotin presumably for folic acid synthesis. These results suggest IL role of intestinal flora in providing the host with these vitamins. Balance studies on gf rats showed unaccountable losses of niacin, riboflavin, and biotin but not pantothenic acid and vitamin Biz. Folic acid was synthesized upon the addition of biotin to the diet. h. Biogenesis of Histamine. Since evidence exists indicating bacterial formation of histamine in the intestinal tract, Gustafsson and co-workers (195713) conducted a comparative study of the histamine content of gf and conventional rats. Histamine distribution and content was the same in both groups of animals; daily excretions of histamine were equivalent. Differences between the two groups of animals were largely in the amounts of conjugated histamine excreted; this appeared to be correlated with sex of the animals. Thus, conventional male rats and gf females excreted virtually all of their histamine in a conjugated form, whereas the conventional females and gf males excreted more free histamine. No explanation was given for these somewhat unexpected results. The above workers also noted in their preliminary examinations that both gf and conventional rats contained the same relative numbers of mast cells in the mesentery. Perhaps con-

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trolled flora studies employing organisms with high histidine decarboxylase activity might yield somewhat different results. i. Bile Acids. Lindstedt and Norman (1956) showed that chemotherapeutic agents greatly reduced the rate of bile acid excretion in rats. This effect was explored by Gustafsson et al. (1957a) using gf rats fed cholic acid24-C1*.The half-life of cholic acid in the gf animals was five times greater than in control rats. However, this difference disappeared when the gf rats became grossly contaminated; this occurred after the animals were removed from isolation. This effect of mixed contamination on cholic acid turnover could not be achieved with monocontamination by either Aspergillus niger or Clostridium perfringens.Taurocholic acid was the only metabolite found in feces of gf rats fed labeled cholic acid. Similar results were obtained in conventional rats given chemotherapeutics (Norman, 1955). On the other hand, untreated conventional rats convert cholic acid to a number of compounds apparently through intestinal bacterial action. Some of the organisms splitting the peptide bond of taurocholic acid have been isolated (Norman and Grubb, 1955); other organisms attack the steroid nucleus. j. Hemorrhagic Shock. Many studies have suggested a role of bacterial endotoxins in hemorrhagic shock. However, the issue has been controversial, possibly due in some degree to the presence of an intestinal flora in conventional experimental animals. This difficulty was avoided by Zweifach el al. (1958) in their study of irreversible shock in gf rats. Results did not indicate that bacteria or bacterial products were involved in the shock syndrome. Gf and conventional rats were similar in their response to bleeding, duration of hypotensive episode, and other pathological changes associated with shock. These experiments probably should be extended to other gf animals such as the dog, using controlled intestinal floras. k. Lymphoid Cells and Tissues. The gf animal is particularly suited to the investigation of various aspects of lymphoid systems. The underdevelopment of lymphoid tissue in gf animals due to reduced antigenic stimulation has been adequately documented by a number of workers as discussed elsewhere in this essay. In their studies of the lymphoid system of gf and monocontaminated chicks, Thorbecke et al. (1957) observed significant increases in spleen plasma cells and secondary nodules as well as serum y-globulin in response to immunization by injection. Monocontamination without immunization failed to produce these results. Gross contamination, however, would seem to affect y-globulin levels since Wostman and Gordon (1958) reported lower globulin values in antibiotic fed chickens than in untreated birds. This difference was apparent in 4-month- but not 2-month-old birds.

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The reaction of the gf guinea pig to monocontamination and injections of egg albumin, ribonucleic acid (RNA) and deoxyribonucleic acid (DNA) prcparations, cortisone, and ACTH have been recently described by Miyakawa et ul. (1957). Monocontamination resulted in the appearance of many clear-center nodules and pyronin-staining cells in the medullary cord. Albumin injection resulted in no clear centered nodules; however, large pyronin-staining cells with enlarged nucleoli appeared in the cortex of the medullary cord. Injections of DNA and RNA also failed to produce clear centered nodules in the lymph nodes. It is of interest that cortisone and ACTH did not produce a response in gf guinea pigs in contrast to pronounced lymphocyte depression in conventional animals. 1. Indole Production. This is but one aspect of the important problem of microbial metabolism in the gut and its significance for the host. Wagner (1958) compared the fecal indole and urinary indican of gf rats, mice, and chickens with their controls. Neither of these compounds was seen in gf animals, but both were present in the conventional groups; no indican was observed in feces of conventional chicks. Monocontamination with an indole-producing organism resulted in the excretion of indole and indican by rats and mice and of indole by the chickens. m. Starch Digestion. The possible influence of intestinal flora on the intestinal carbohydrases-oligo-1 ,6-glucosidase, maltase, and invertasewas studied by Larner and Gillespie (1957) with gf rats. No marked differences were reported between gf and control rats. These results may indicate that the intestinal flora of the control animals failed to produce effectively the types of amines that these workers had previously found to be inhibitory toward the above enzymes in vitro. It would be of interest to determine the extent of carbohydrase inhibition using animals having a flora known to produce an arylamine inhibitor of the enzymes. n. Wo2m.d Healing. Miyakawa et al. (1958b) reported that bacterial contamination of guinea pigs was important as a stimulant to the production of new capillaries in the mesenchymal tissue of a cut wound. Capillary formation was rather scant in the gf animals. On the other hand, completion of the epidermal bridge was more rapid in the gf animal. These workers also suggested that microbial contamination offered a nonspecific stimulation to host tissues which influenced exudative and proliferative processes. There is a hint here that normal microbial contamination may influence collagen synthesis. Blood clotting mechanisms also deserve consideration in gf animals. 0 . Virology. A great amount of groundwork must be accomplished before the value of the gf technique for virological studies can be assessed properly. The published literature on the subject is lacking in such rudimentary in-

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formation as the species of virus which can be carried through serial passages in g f animals. Reyniers (1949) and Reyniers and Sacksteder (1958a) reported the possible occurrence of transmissible viruses under gf conditions. Pooled human serum from cases of infectious hepatitis was filtered through UF glass filters and administered to gf chickens. Animals which were 30 to 60 days old showed a marked distention of the crop (which filled with a “foamy liquid”) followed by extreme weakness, paralysis, and death. The disease could be carried through at least three passages in White Leghorns. Since no liver damage was observed, further identification of the infectious agent was not made. Another disease of g f birds was characterized by tremors (“jitters”) which developed in newly hatched chicks and increased in severity until the animals died at about 7 days. Lesions of the dura were observed. Interestingly, this disease was confined to g f birds and was arrested by deliberate contamination with bacteria. Transmission of the etiological agent from bird to bird could be accomplished only with bacteria-free macerates of brain tissue. Reyniers and Sacksteder also revealed that six generations of gf C3H mice failed to develop tumors. The parents of these mice had been taken by Caesarotomy from a conventional dam, and suckled by gf Swiss strain foster-mothers (low-tumor incidence strain). The conventional controls exhibited 98% mammary tumors in the females and 40 to 50% hepatomas in the males. Tentatively, it appears that the Bitther factor can be eliminated by this technique. p . Immunology and Serology. Of the uses for which g f animals are considered, the studies of antigen-induced synthesis of antibodies seem to be the least contrived reason to justify the nuisance and expense. The few published papers describing the serological reactions and the structure of lymph tissues in gf animals have already yielded some interesting observations. The differences in antibody responses to various bacterial species (Wagner, 1955) merit further investigation. Indeed, the mechanism of antibody stimulation by dead bacteria in the gut can be attacked directly with the aid of gf animals. Immunological aspects of the gf animal are treated further under the section on characteristics of these animals. Thorbecke et al. (1957) investigated lymphoid tissue and serum proteins of gf and conventional chickens. The lymphoid tissue of the g f chicken was underdeveloped and contained fewer plasma cells and secondary nodules. And, as mentioned above, they found interesting effects in their serum protein studies. The albumin content of gf chickens and rats tends to be higher than their conventional controls according to Wostman and Gordon (1958). They also showed that the 7-globulin content of conventional birds on a peni-

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cillin-containing diet was higher than in the gf animal but lower than in conventional birds not fed penicillin. These important effects were not seen in 2-month birds but were evident in birds 4 months of age. The identity of the substance responsible for inciting the globulin synthesis should be determined.

B. POTENTIAL APPLICATIONS 1. The Growth-Depressing Action of Intestinal Flora

Evidence exists that the intestinal flora inhibits the growth of young animals. This is supported chiefly by the extensive literature on the growth promoting effects of diets supplemented with antibiotics, and the general lack of this response in gf animals. This inhibition deserves greater attention from the standpoint of mechanism studies, since virtually nothing is known of the factors responsible for this phenomenon. Melnykowycz and Johansson (1955) summarized evidence for a bacterial role in the inhibition effect. More detailed studies are needed regarding the nature and amounts of potentially harmful microbial products formed in the gut and their effects upon the host. Although, as previously mentioned, some data are available on the production of physiologically active bacterial products such as amines, ammonia, and phenols, our knowledge is meager with respect to their net effects on the healthy animal. Similarly, intestinal synthesis of toxic bacterial products-such as the toxins of Clostridia sp. and the lipopolysaccharides of Escherichia coli and other gram-negative bacteriashould be investigated. The gf animal should be most helpful in identifying the specific nature and source of inhibitory compounds since a given organism may be established as a pure culture in the intestinal tract of these animals. Mixed cultures of known composition may likewise be established in the gut of the gf animal to explore microbial interrelationships and their effects on the host. Presumably adult animals are also susceptible to a normal inhibition by the gut flora, although this has not been as obvious as in the growing animal. Some hint in this direction is available from the work of Wostman and Gordon (1958) who found comparatively lower levels of serum y-globulin in conventional chickens fed antibiotics than in a control group of $-monthold birds not receiving antibiotics. In this case, it appears that intestinal microbial action results in the production of a substance stimulating y-globulin formation in the host. Verification of the inhibitory action of intestinal flora may have an important bearing on studies of disease and aging. The gf animal will be useful in the solution of these problems.

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2. Virology The course of a generalized virus infection from the time of inoculation to the expression of symptoms has been described by Fenner (1948, 1949) in work with mouse ectromelia and by Burnet (1955) in a discussion of virus disease pathogenesis. Fenner concluded that ectromelia virus first multiplies in a primary, localized lesion and then spreads to the regional lymph node where further multiplication occurs. A primary viremia occurs as virus passes from the regional lymph node into the blood stream by means of which it then infects the spleen and liver. The large populations of virus particles produced in the viscera result in a secondary viremia when they in turn appear in the blood stream. Widespread infection of epidermal tissue then ensues. Similar patterns of dispersion have been shown for polio virus (Wenner and Kamitsuka, 1957) and for canine distemper virus (Liu and Coffin, 1957; Coffin and Liu, 1957). However, the latter virus did not multiply or cause a lesion in the nasal epithelium; by means of Coons’ fluorescent antibody technique, the primary site of virus multiplication was demonstrated to be in the regional lymph node. There are numerous unsolved problems associated with each of the various steps of the virus dissemination pathway. The gf animal with its underdeveloped lymphatic tissues and low phagocyte response (Reyniers, 1949) may help resolve some of the issues. For example, there are interesting observations to be made concerning the multiplication of the viruses at the site of inoculation. Does the absence of a microflora and of antigenic materials in the food and dust of the cage decrease the variation in virus susceptibility among inbred animals inoculated with the same dose? Are the local lesions in gf animals and conventional controls produced simultaneously at different dose levels? Does the gf state significantly alter the yield of active virus from the local lesion or change its hyperplastic and necrotic response? Do uninfective strains of virus produce lesions or yield infective virus when the host tissues are treated with methylcholanthrene and cortisone? I n experiments such as the latter, Duran-Reynals (1952, 1957a, b) found that latent or subinfectious virus of vaccinia and fowl pox produced lesions in mice, chickens, and pigeons. Newly isolated viruses are frequently cultured in newborn animals, embryonated eggs, brain tissue, and tissue cultures. It is also the common experience of everyone who attempts to infect mature laboratory animals with virus isolated from nature that a number of passages in the new host may be necessary before typical lesions are produced regularly. These procedures are predicated in part on the thesis that the virus can multiply most readily in tissues which have had limited exposure to antigens and

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that large virus populations must be assembled before the expression of disease symptoms can occur. In the case of ectromelia (Burnet, 1955) the significant level for the expression of symptoms is about lo6infective particles per gram of tissues. It remains to be seen whether the reduced amount of lymphatic tissue in the gf animal can produce enough virus particles to overcome the “normal” antibodies and phagocytes and establish a viremia; this would be particularly interesting with Japanese B encephalitis (Hammon et al., 1946) and Murray Valley encephalitis (McLean, 1953) which do not cause a lesion at the site of inoculation and probably multiply in the vascular endothelium. One of the crucial issues which must be examined is whether or not the gf animal is also virus-free. Certainly the systems in current use are capable of physically excluding the occasional virus contaminant which might be encountered. But the very real possibility of transplacental passage of viruses remains as a most unsettling thought. Egg transmission of mouse leukemia (Gross, 1957) and of chicken visceral lymphomatosis (Burmester, 1957) are well established observations. The natural existence of subinfectious populations in perfectly healthy hosts has become evident with the isolation of herpes, Coxsackie, ECHO, and adenoviruses from literally thousands of specimens of feces and pharyngeal scrapings (Huebner, 1957,1958).Working with lymphocytic choriomeningitis in mice, Hotchin (1958) found that mice inoculated with the LCM virus shortly before or after birth carried the virus indefinitely. Neither inflammation nor neutralizing antibodies are observed in these animals, and the disease symptoms could not be induced by using cerebral trauma, stress, X-irradiation, or viral superinfection. Measles virus, attenuated hog cholera virus, and swine encephalitis virus almost certainly can pass the placental barrier. The examples cited are characterized by a viremia which is detected readily. The use of the gf system must eventually require some assurance that the experiments can be performed without being compromised by the existence of viruses which are (a) in a latent phase analogous to the prophage, (b) are in low concentrations after an immunological tolerance has been established in the host, or (c) are maintained as cytoplasmic particles which are distributed during mitosis. 3. Immunology and Serology In view of Wagner’s observation (1955) that gf animals respond well to antigenic stimulus, it is probable that autoantibodies can be demonstrated in them also. Burnet (1956) suggests that during embryogenesis a warning “self-marker” mechanism is established in the cell which prevents the formation of antibodies against the animal’s own tissue proteins. Other authors reviewed by Boyd (1956) have proposed that an animal’s antibody-

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forming sites are so saturated with its own antigens that they cannot respond, or that exposure to an antigen in the fetal state causes an “actively acquired tolerance.” Support for the latter view is suggested by the observation of Hotchin (1958) that mice injected with lymphocytic choriomeningitis virus shortly after birth fail to develop neutralizing antibodies even though a high population of viruses can be demonstrated throughout their lives. Thyroid extracts have been shown to be antigenic for homologous species (Witebsky and Rose, 1956; Witebsky et al., 1956, 1957; Rose and Witebsky, 1956); thyroglobulin is probably the most active antigen in the extracts. It is possible that the differences of opinions concerning the lack of antigenicity for tissue components may be resolved by quantitative studies employing purified thyroglobulin as an antigen, gf animals (or tissues from them), and techniques similar to those used by the virologists for studying the synthesis of viral proteins. The same techniques might be applied to the study of several serious diseases which have been associated with the autoantibody phenomenon (Boyd, 1956); these are acquired hemolytic anemia, thrombocytic purpura (abnormal bleeding attributed to a low platelet count), rheumatic fever, rheumatoid arthritis, multiple sclerosis, and certain types of dermatitis. Concerning these diseases, Boyd comments : “All or nearly all the diseases . . . have certain things in common: they often follow an infectious disease, they are often familial, suggesting that the patient has inherited a tendency to produce antibodies readily and perhaps unnecessarily, they often produce ‘biologically false-positive’ tests for syphilis, and they are often relieved or improved by ACTH or cortisone.’’ One concept of the problem is that products resulting from an infection combine with a normal tissue constituent and alter it so that it then serves as a nonmetabolizable antigen. The evidence for the autoimmunization hypothesis is largely suggestive, but by means of a controlled antibody response in the gf animal and control over such factors as collagen synthesis in the mouse with cortisone and estradiol (Duran-Reynals, 1957b)) an experimental approach can be devised. Of particular interest might be young chicks which when inoculated with Fuginami or Rous sarcoma viruses show an acute hemorrhagic disease (Duran-Reynals, 1940, 1950) that is somewhat analogous to the hemolytic anemia which occurs subsequent to virus pneumonia. In this latter disease antibodies against the host’s erythrocytes are formed. A completely satisfactory explanation is yet to be found for the persistanee of antibodies resulting from some virus diseases. Yellow fever, smallpox, chicken pox, and German measles confer lifetime immunity while herpes simplex and influenza virus provide no immunity at all. Burnet (1956) considers that strong immunity results (u) from a viremia which

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distributed virus to all the antibody-producing sites b y means of the vascular system and (b) from the cells of the lymphocytic series which not only are stimulated to produce antibody but will pass that ability to progeny. It was observed by Felton (1949) and confirmed by Stark (1955) that antibody-producing sites can be saturated with large doses of antigen which cause the cessation of antibody against the antigen for a long period of time. If the antibody-producing sites are located principally in the lymphopoietic tissues, it would seem that an “immunologically paralyzing’’ dose for the gf animal would correspond to the reduced mass of its lymphatic tissue. In gf birds the lymph tissue is about one-fifth the weight of the conventional bird’s lymph tissues. Such an approach may also bring about a better understanding of how animals can sometimes tolerate huge populations of virus particles circulating in the vascular system; for example, Beard (1958 a, b) mentions that chicks may not succumb to a total administered dose of 145 billion particles of erythromyeloblastosis virus. The study of “maturation resistance” to virus infection-increasing resistance acquired with age-is of great importance if we are to understand the pathogenesis of poliomyelitis, mumps, a clinical variety of nonspecific encephalomyelitis and encephalitis, and mononucleosis. Two types of maturation resistance were recognized by Lennette (1957) : ( a ) a resistance resulting from structural “barriers” which arise along the neural pathways at different ages and prevent the virus from passing into the central nervous system (CNS) and (6) resistance in which the multiplication of the virus in the CNS ceases or is held to a low level. Bodian (1957) describes the virtual disappearance of polio virus from the spinal cords of rhesus monkeys 2 to 4 days after the onset of paralysis. This phenomenon was observed to occur at that time regardless of the number of neurons destroyed or the titer of the virus. Both types of maturation resistance are amenable to study in the gf system since type ( a ) can be demonstrated with mice and type ( b ) with rats.

4. Aging Studies The gf animal may be used in determining the role of specific microorganisms and their products in the aging process of an animal host. The postulated growth-inhibitory effects noted earlier seem worthy of investigation from the viewpoint of aging problems. However, more comparative data are needed on aging in gf and conventional animals, particularly in regard to connective tissue metabolism. 5. Intestinal Flora in Host Nutrition

Specific organisms can be studied in vivo with respect to their contribution to the nutrition of the host. Problems such as competition between host

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and flora for nutrients, minerals, vitamins, and cofactors, or synthesis of compounds useful to the host can be investigated under conditions of a controlled intestinal flora. 6. Microbial Metabolism of Sterols in the Gut

Although beginnings have been made in this field (Gustafsson et al., 1957a), more work is indicated on the degradation of sterols by intestinal microorganisms and the influence of the products on the host. The use of the gf animal would aid in specifying the causal relationships involved in the reduction of cholesterol, 0-sitosterol, and 7-dehydrocholesterol by fecal microorganisms (Coleman and Baumann, 1957).

7. Conjugation Mechanisms in Detoxication It seems likely that gf animals should be of use in studying enzymatic mechanisms of detoxication, especially in regard to the origin and metabolic control of requisite enzyme systems. Certainly the gf animal has less demand for such systems, although no data of this sort is yet available. 8. Intestinal Enzymes and Digestion

Marked changes occur in some of the digestive enzymes of the growing animal. The young pig has been studied by Lewis et al. (1957), who found that the low initial pepsin activity of the stomach tissues at birth was greatly increased after 3 weeks of age. Since enzyme production by the normal intestinal flora may complicate studies of digestive enzymes, use of the gf animal should avoid this difficulty. 9. ~ n t e s ~ i Absorption n~l and P e r m e a ~ l i t ~

Important changes occur in the permeability of the intestinal tract of young animals wherein globulins are absorbed from the gut lumen without degradation. This has been shown for the rat (Bangham and Terry, 1957) and for the herbivore (Deutsch and Smith, 1957). Globulin absorption declines in the rat up to the twentieth day after birth when permeability of the gut wall to globulins is abruptly terminated (Halliday and Kekwick, 1957). The events responsible for changes in intestinal permeability have not been defined. However, there are marked changes in the flora of animals, especially upon weaning, which may have a bearing on intestinal permeability through bacterial stimulation of the gut wall. It should be of interest to learn whether gf rats demonstrate similar permeability changes; if not, it would provide evidence of microbial participation and suggest the use of gf animals in the elucidation of the phenomenon. More information is desirable concerning the permeability and absorption characteristics of the

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gf gut. Future studies should also include the transport of viruses and bacteria through the intestinal tissues, relating this traffic to the absorption of particulate structures from the lumen or the shedding of such structures into it. 10. Gastrointestinal Dysfunctions

Evidence for the role of intestinal flora in a number of dysfunctions has been reviewed recently by Gardner (1957). He has discussed factors which modify the distribution of the gastrointestinal flora, such as anatomical changes in the gut, and the consequent effects on absorption and vitamin metabolism. The gf animal should be helpful in unraveling some of the microbial factors involved in gastrointestinal dysfunctions. It is essential to identify specific toxic substances, their sources, and their specific effects on intestinal absorption, liver and kidney function, and erythropoiesis. 11. Microbial Ecology in the Host

The gf animal offers a direct means of investigating metabolic interrelationships between known mixtures of microorganisms in their natural habitat, e.g., the mouth, respiratory tract, and intestinal lumen. Microbial ecology in relation to host and parasite has been discussed recently in a Symposium of the Society for General Microbiology (1957). Among other things, the requirement for controlled studies of mixed cultures of microorganisms in their natural habitats was stressed.

IS. Host Metabolism in Relation to Resistance to Infection As Dubos (1954) has emphasized, physiological status of the host in relation to resistance and infection has been much neglected in spite of its relevance. The gf animal seems to offer a satisfactory experimental basis for establishing a much needed biochemical concept of the ecology of pathogenesis. Thus, Elberg (1956) stated in his review of resistance and infection that the gf animal permits the study of determinant activities of multiflora populations. 13. Allergy

This topic suggests a reassessment of the concept of a gf animal in terms of an animal free of exposure to foreign antigens, a concept which is discussed in greater detail elsewhere in this essay. Although no attempts have been reported to obtain such animals, they eventually should prove to be of greater value than the gf animal. One reason for this is the increasing attention being given to allergic reaction in disease. Baird (1957) has reviewed some of the vast literature on allergy to bacterial products in relation to diseases including rheumatic fever, arthritis, hypertension, glau-

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coma, multiple sclerosis, glomerulonephritis, ulcerative colitis, chronic cholecytitis, and juvenile diabetes. However, as he commented, considerable controversy exists regarding the role of allergy in these diseases. A more decisive experimental method, such as using an animal free of foreign antigens, is required to assure the necessary control of the variables involved. The gf animal is an important step towards such a goal. 14. Oncology

The use of the gf technique to study neoplastic growth is one of its most logical applications, especially if transplacental virus infections can be controlled and if normal lymphatic development can be stimulated artificially. Certainly the common methods for inducing tumors in laboratory animals will be tried under gf conditions. It also seems equally logical to use the gf animal to obtain more uniform responses when studying the effects of hormonal factors, tissue culture filtrates, fractions of tissue homogenates, or carcinogens on tumor formation. The use of the gf system is indicated in studies of the relationships between subinfectious (or indigenous) viruses, irritants which cause the localized increase of lymphatic tissue, and neoplasms. An intriguing possibility is the use of the gf technique for the reexamination of compounds which have shown antitumor properties against implanted tumors in animals; it would be of interest to see whether weak agents had their activity enhanced or whether stronger ones remained effective. Useful observations of tumor tissue transplants and cytotoxic effects might be made since homografts have been shown by Miyakawa et al. (1958~)to take more effectively in the gf guinea pig. Particulates isolated from cells or serum of leukemic animals should be tested in this system in an attempt to carry out Koch’s postulates and to establish the identity of the etiologic agent.

V. Additional Research Needs Although a feasible gf methodology has been developed, consideration must be given to improvements which will enhance its usefulness. Following are some of the areas that merit attention.

A. MANUALOF GERMFREE VERTEBRATES The greatest difficulty encountered by those interested in the details of gf animals, and the apparatus and methods in current use, is the lack of a convenient source of information. There is an obvious and rather urgent need for a handbook or manual on the subject. This manual should contain all descriptive details of apparatus, procedures, and characteristics of various species of gf animals. It should contain usable data on all types of gf

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apparatus in current use. The manual should be sufficient to enable an investigator to assess the applications to a specific problem as well as to implement the technique in his laboratory. Periodically, the handbook should be revised or supplemented to include improvements in apparatus and procedures as well as new data on the characteristics of gf animals. Revision is particularly important in view of the recent interest in this field and prominent increase in gf life facilities in the United States as well as other countries. The entire field promises to undergo an accelerated development during the next few years.

B. ANIMALSFREEFROM ANTIGENIC STIMULATION An ideal experimental animal is one that is known genetically, microbially, nutritionally, and antigenically. Recent efforts have brought this concept much closer to realization with the development of diets of known chemical composition (Schultze, 1957; Greenstein et al., 1957). The latter workers suggested that their dietary solutions should be adequate €OF gf animals; as yet no such results have been published. Purified diets for ruminants have been used by Matrone et al. (1957). It remains to be shown that gf animals are free of viruses as measured by sensitive techniques. Since no thorough study of the problem has been conducted, this remains perhaps the largest gap to close in the attainment of a truly gf animal; this is a prerequisite for an animal free of exposure to foreign antigens. These considerations have an interesting relationship to the speculative possibility of the de novo origin of viruses.

C. GERMFREE APPARATUS There is a necessity for readily available equipment at a cost which is within reach of the ordinary laboratory budget. Although some efforts are being made in the form of low-pressure isolators (vide infra) sterilized by chemical agents, further consideration should be given to steam-sterilized, low-pressure isolators made of plastics, rubber, and light metals. Films that are readily punctured or subject to cracking, corrosion, and early deterioration should be avoided, While chemical germicides can be effective, more work needs to be done on them; steam sterilization of isolators is generally preferred at the present time. Further development is needed on gloves and remote-controlled manipulators, particularly for long-term use as in the maintenance of gf colonies. Gloves less susceptible to punctures and breaking are needed. The development of simplified and inexpensive remote-controlled manipulators would, of course, eliminate the need for gloves which are one of the weak points in most gf isolators today. Air sterilization devices can be improved with the addition of an incin-

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erator, and these ought to be available to those constructing their own apparatus. As mentioned above, the Swedish and Japanese apparatus employ this method of air sterilization; the device does not seem to be in supply in this country, however. D. STERILIZED DIETS FOR GERMFREE ANIMALS Although steam-sterilized diets are generally employed, other methods may have some advantages. For instance, preservation of the physical and chemical characteristics of a diet during sterilization can be achieved best perhaps by ionizing radiations. It is of interest that radiation sterilization is currently being intensively studied under the auspices of the Quartermaster Command and the Surgeon General of the Department of the Army, Results of this research will be applicable to the sterilization of diets for gf animals. In our laboratory, diets sterilized by electron beams have been routinely employed with gf chicks.

E. CHARACTERISTICS OF GERMFREE ANIMALS Important information is needed regarding biochemical details of metabolism, histology, and cytology of the gf animals. 1 . Metabolic Studies

Much data is needed about the enzyme levels and metabolic patterns in the liver, kidney, lymphoid system, the gastrointestinal tract, and endocrine organs of gf animals. The marked underdevelopment of the lymphoid tissues of the gf animal, as observed by gross and histological inspection, should be reflected in biochemical patterns of protein and nucleic acid metabolism and associated systems. Are there related deviations in hormone metabolism? Suitable answers to these questions should help to establish a much needed biochemical concept of microbial stimulation of the lymphoid system. The necessity for examining hormone metabolism rests partly upon evidence of hormonal involvement in antibody formation. Hormone dysfunction may be associated also with growth inhibition of a host by its intestinal flora. Some of the intestinal anomalies of the gf animal, e.g., lack of tonus, may be a result of serotonin deficiency. Additional qualitative and quantitative work is required on substances excreted by gf animals. Compounds of microbial origin known to possess significant pharmacological activity should be further examined for their distribution and effects on host tissues. For example, how efficient are detoxication processes under multiple challenge by various intestinal microorganisms? Is there any evidence for increased uncoupling of phosphorylation in the tissues of conventional animals in comparison with the gf animal? Are acet-

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ylation and glucuronide-forming systems, for instance, significantly different in gf and conventional animals? Does adaptive enzyme formation occur in the host in response to microbial products? Simply on the basis of a greater detoxication demand in the conventional animal, one would expect some affirmative answers to these questions. 2. Histology

Studies on lymphoid cells and tissues should be expanded together with the addition of cytochemical and enzymatic analyses. Further comparative investigations of leukocytic functions are desirable, especially those of the lymphocyte and plasma cell. Similarly, a clearer picture of histological differences in the intestine, liver, lungs, heart, kidneys, and marrow of gf and conventional animals would be most helpful. It is desirable to have histological and biochemical observations of connective tissue and ground substance, red cells and erythropoiesis, and mast cells.

F. NOMENCLATURE AND SYMBOLISM There is a growing need for a system of nomenclature and symbolism concerning gf animals contaminated with a specific multiple microbial flora or specific foreign antigens. One is thus confronted with the problem of two conceptual levels of definition: the foreign antigen-free animal and the gf animal. Obviously, the animal that is defined environmentally, nutritionally, genetically, and antigenically constitutes a more generic concept than that of the gf animal. The generic concept appears to be a feasible one now. Thus, the term “gnotobiotics” (Reyniers et al., 1949d), from the Greek equivalent of “known life,” possibly should be reserved for the more generically defined animal since it encompasses a greater knowledge of the animal than does the gf concept. A symbolic notation is required for the defined biological systems given above and their applications. This is a problem of great importance to the theoretical and experimental development of such systems.

G. SOURCES OF GERMFREE ANIMALS The effort required to rear gf animals to maturity may have turned many workers away from important applications of this material. A most urgent need in this field today is for readily available gf animals. Possibly the best way of effectively fulfilling this need is the establishment of several gf animal colonies which are appropriately located for efficient distribution of animals to investigators for use in their own laboratories. Colonies of gf animals of specified genetic strains should be established for the common laboratory species. Further development of devices for shipping gf animals will be needed in connection with the above facilities.

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H. NUTRITION The literature is incomplete with respect to the nutritional requirements of the gf rat and guinea pig and almost no dietary information has been reported on gf mice, rabbits, monkeys, and dogs. Nutritional effects, if any, of the relatively large amounts of inactivated vitamins in steam sterilized diets deserve attention. Attempts ought to be made to rear gf animals on chemically defined diets as suggested by Greenstein et al. (1957).

VI. Summary The methodology of gf birds and mammals, their characteristics, and some of their applications were described. Consideration was also given to various means of rendering this technique more accessible to all interested investigators. Facilities for using gf animals can be relatively simple and inexpensive. However, long-term rearing and the development of breeding colonies of gf mammals and birds require more extensive operations. More such colonies are needed if the full advantages of this technique are to be realized.

ACKNOWLEDGMENT One of us (A.W.P.) expresses his gratitude to the University of Notre Dame and James A. Reyniers for many years of adventures in germfree animal research.

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Iluran-Reynals, F. (1952).Ann. N . Y . Acad. Sci. 64,977-991. Duran-Reynals, F. (1957a). Ann. N . Y . Acad. Sci. 68, 430-440. Duran-Reynals, F. (1957b).Texas Repts. Biol. and Med. 16, 754-781. du Vigneaud, V.,Ressler, C., and Rachele, J. R. (1950). Science 112, 267-271. du Vigneaud, V., Ressler, C., Rachele, J. R., Reyniers, J. A,, and Luckey, T. D. (1951).J . Nutrition 46, 361-376. Elberg, S. S. (1956). Ann. Rev. Microbiol. 10, 1-20. Felton, L. D.(1949).J . Immunol. 61, 107-117. Fenner, F. (1948).Lancet ii, 915-920. Fenner, F. (1949).J . Immunol. 63, 341-373. Forbes, M.,Schockman, G. D., Chu, E., and Gyorgy, P. (1953). Antibiotics & Chemotherapy 3, 1104-1110. Gardner, F.H. (1957).A m . J . Digest. Diseases 2 , 175-194. Glimstedt, G. (1936).Acta Pathol. Microbiol. Scand. Suppl. 30, 1-295. Gordon, H.A. (1952).In “Colloquium on Growth Effects of Antibiotics in Germfree Animals.” Lobund Institute, Univ. Notre Dame, Notre Dame, Indiana. Gordon, H. A. (1955).Bull. N . Y . Acad. Med. 31,239-242. Gordon, H. A., Wagner, M., and Wostman, B. S. (1958).Antibiotics Ann. 1967-1968,

248-255. Greenstein, J. P.,Birnbaum, S. M., Winitz, M. and Otey, M.C. (1957). Arch. Biochem. Biophys. 73, 396-416. Gross, L. (1957). Texas Repts. Biol. and Med. 16, 603-626. Gustafsson, B. (1947).Acta Anat. 2. 376-391. Gustafsson, B. (1948). Acta Pathol. Microbiol. Scand. Suppl. 73, 130 pp. Gustafsson, B., Bergstrom, S., Lindstedt, S., and Norman, A. (1957a).Proc. SOC. Exptl. Biol. Med. 94, 467-471. Gustafsson, B., Kahlson, G., and Rosengren, E. (1957b). Acta Physiol. Scand. 41,

217-228. Halliday, R.,and Kekwick, R. A. (1957).PTOC. Roy. SOC.B146,431-437. Hammon, W. McD., Reeves, W. C., and Burroughs, R. (1946). Proc. Soc.Ezpt2. Biol. Med. 61, 304-308. Hotchin, J. E. (1958).Zn “Symposium on Latency and Masking in Viral and Rickettsial Infections” (D. L. Walker, R. P. Hanson, and A. S. Evans, eds.), pp. 59-65. Burgess Publ. Co., Minneapolis, Minnesota. Huebner, R. J. (1957).Ann. N . Y . Acad. Sci. 67,430-438. Huebner, R. J. (1958).I n “Symposium on Latency and Masking in Viral and Rickettsial Infections” (D. L. Walker, R. P. Hanson, and A. S. Evans, eds.), pp. 51-58. Burgess Publ. Co., Minneapolis, Minnesota. Jukes, T. H., and Williams, W. L. (1953).Pharmacol. Revs. 6.381420. Kuster, E. (1912). Zentr. Bakteriol., Parasitenk. Abt. I . Ref. 64, 55-58. Kuster, E. (1915).In “Handbuch der Biochemischen Arbeitsmethoden” (E. Abderhalden, ed.), Vol. 8, pp. 311-323. Springer, Berlin. Larner, J., and Gillespie, R. E. (1957).J . B i d . Chem. 226, 279-285. Lennette, E.H.(1957).Texas Repts. Biol. and Med. 16, 603-626. Lewis, C. J., Hartman, P. A., Liu, C. H., Baker, R. O., and Catron, D. V. (1957). J . Agr. Food Chem. 6, 087-690. Lindstedt, S., and Norman, A. (1956). Acta Physiol. Scand. 38, 129-134. Liu, c.,and Coffin, D. L. (1957). virology 3, 115-131. Luckey, T. D.(1952).Zn “Colloquium on Growth Effects of Antibiotics in Germfree Animals.” Lobund Institute, Univ. Notre Dame, Notre Dame, Indiana. Luckey, T. D. (1956a). Texas Repts. Biol. and Med. 14,482-505.

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Luckey, T. D. (1956b). Natl. Acad. Sci. Natl. Research. Council. Publ. 397. Luckey, T. D., Reyniers, J . A., Gyorgy, P., and Forbes, M . (1954). Ann. N . Y . Acad. Sci. 67, 932-935. Luckey, T. D., Pleasants, J. R., and Reyniers, J . A. (1955a). J . Nutrition 66,105-118 Luckey, T. D., Pleasants, J. R., Wagner, M., Gordon, H. A., and Reyniers, J. A. (1955b). J . Nutrition 67, 169-182. Luckey, T. D., Wagner, M., Reyniers, J. A., and Foster, F. L., J r . (1955~).Food Research 20, 180-185. Luckey, T. D., Gordon, H. A., Wagner, M., and Reyniers, J . A. (1956). Antibiotics & Chemotherapy 6, 36-40. McLaughlin, M. M., Dacquisto, M. P., Jacobus, I).P., Forbes, M., and Parks, P. E. (1958). Radiation Research 9, 147. McLean, D. M. (1953). Australian J . Ezptl. Riol. Med. Sci. 31, 491-503. Matrone, G., Ramsey, H. A., and Wise, G. H. (1957). Proc. SOC.Ezptl. Biol. Med. 96, 731-734. Melnykowycz, J . , and Johansson, K. R. (1955). J . Ezptl. Med. 101,507-517. Miyakawa, M., Iijima, S., Kobayashi, R., and Tajima, M. (1957). Acta Pathol. Japon. 7, 183-210. Miyakawa, M., Iijima, S., Kishimoto, €Kobayashi, I., R., Tajima, M., Isomura, N., Asano, M., and Hong, S. C. (1958a). Acta Pathol. Japon. 8 , 55-78. Miyakawa, M., Isomura, N., Shirasawa, H., and Yokoi, K. (195813). Acta Pathol. Japon. 8, 79-97. Miyakawa, M., Kishimoto, H., Itaya, J., Uei, Y., and Kashio, T. (1958~).Acta Pathol. Japon. 8, 177-187. Norman, A. (1955). Acta Physiol. Scand., Suppl. 33, 99-107, 117-121. Norman, A., and Grubb, R. (1955). Acta Pathol. Microbiol. Scand. 36, 537-547. Nuttal, G. H. F., and Thierfelder, H. (1895). 2.Physiol. Chem., Hoppe-Seyler’s 21, 109-121. Nuttal, G. H. F., and Thierfelder, H. (1896). 2.Physiol. Chem., Hoppe-Seyler’s 22, 62-73. Nuttal, G. H. F., and Thierfelder, H. (1897). 2.Physiol. Chem., Hoppe-Seyler’s 23, 231-235. Orland, F. J., Blayney, J . R., Harrison, R. W., Reyniers, J. A., Trexler, P. C., Wagner, M., Gordon, H. A., and Luckey, T. D. (1954). J . Dental Research 33, 147-174. Orland, F. J., Blayney, J. R., Harrison, R. W., Reyniers, J. A,, Trexler, P. C., Ervin, R. F., Gordon, H. A., and Wagner, M. (1955). J . Am. Dental Assoc. 60, 259-272. Phillips, B. P., Wolfe, P. A., Rees, C. W., Gordon, H. A., Wright, W. H., and Reyniers, J . A. (1955). Am. J . Trop. Med. Hyg. 4, 675-692. Phillips, B. P., Wolfe, P. A., and Bartigia, I. L. (1958). A m . J . Trop. Med. Hyg. 7, 392-399. Porter, J . W . G. (1957). Symposia Soc. Erptl. BioE. 11,255-263. Reyniers, J . A. (1942). J . Bacteriol. 43, 778. Reyniers, J . A. (1943). I n “Micrurgical and Germfree Techniques” (J. A. Reyniers, ed.), pp. 95-113. C. C Thomas, Springfield, Illinois. Reyniers, J. A. (1949). Proc. N . Y . State Assoc. Public Health Labs. 28, 60-69. Reyniers, J . A. (1952a). I n “Colloquium on Growth Effects of Antibiotics in Germfree Animals.” Lobund Institute, Univ. Notre Dame, Notre Dame, Indiana. Reyniers, J . A. (1952b).Mich. State Co22. Vet. 13, 178. Reyniers, J. A. (1956). Proc. Intern. Congr. Riochem, 3rd Congr., Brussels, 1966, 458466. Reyniers, J . A. (1957). Am. J . Vet. Research 18,678-687,

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Reyniers, J. A., and Sacksteder, M. R. (1958a). Ann. N . Y.Acad. Sci. 73, 344-356. Reyniers, J. A., and Sacksteder, M. (1958b).Appl. Microbiol. 6, 146-152. Reyniers, J.A.,and Trexler, P. C. (1943).I n “Micrurgical and Germfree Techniques” (J. A. Reyniers, ed.), pp. 114-143.C. C Thomas, Springfield, Illinois. Reyniers, J. A., Trexler, P. C., and Ervin, R. F. (1946).Lobund Repts. 1, 1-84. Reyniers, J. A.,Trexler, P. C., Ervin, R. F., Wagner, M., Luckey, T. D., and Gordon, H.A. (1949a). Nature 163, 67-68. Reyniers, J. A.,Trexler, P. C., Ervin, R. F., Wagner, M., Luckey, T. D., and Gordon, H. A. (1949b).Lobund Repts. 2, 1-116. Reyniers, J. A.,Trexler, 1’. C., Ervin, R. F., Wagner, M., Luckey, T. D., and Gordon, H. A. (194%). Lobund Repts. 2, 120-148. Reyniers, J. A.,Trexler, P. C., Ervin, R. F., Wagner, M., Luckey, T. D., and Gordon, H. A. (1949d).Lobund Repts. 2, 151-162. Reyniers, J. A., Trexler, P. C., Ervin, R. F., Wagner, M., Gordon, H. A . , Luckey, T. D., Brown, R. A., Mannering, G. J., and Campbell, C. J. (1950). J . Nutrition 41, 31-50. Reyniers, J. A . , Trexler, P. C., Scruggs, W., Wagner, M., and Gordon, H. A. (1956). Radiation Research 6 . 591. Rose, N.R., and Witebsky, E. (1956).J . Zmmunol. 76, 417-427. Schottclius, M. (1913).Arch. Hyg. 79, 289-300. Schultze, M.0.(1957).J . Nutrition 61,589-596. Stark, D.K. (1955).J . Immunol. 74, 130-141. Stokstad, E.L.R. (1954).Physiol. Revs. 34, 25-51. Symposium SOC.Gen. Microbiol. (1957).Volume 7. Cambridge Univ. Press, London and New York. Thorbecke, G. J., Gordon, H. A . , Wostman, B . , Wagner, M., and Reyniers, J. A. (1957).J . Infectious Diseases 101. 237-251. Trexler, P. C., and Reynolds, L. I. (1957).Appl. Microbiol. 6,406-412. Wagner, M.(1955).Bull. N . Y.Acad. Med. 31,236-239. Wagner, M. (1958).Bacteriol. Proc. (Soc. Am. Bacteriologists) 11, 88. Wenner, H.A , , and Kamitsuka, P. (1957).Virology 3, 429-443. Wit,ebsky, E.,and Rose, N. R. (1956).J . Immunol. 76, 408-416. Witebsky, E., Rose, N. R., and Shulman, S. (1956).Cancer Research 16,831-841. Witebsky, E., Rose, N. R., Paine, J. R., and Egan, R. W. (1957).Ann. N . Y.Acad. Sci. 69, 669477. Wostman, B. S., and Gordon, H. A. (1958).Proc. SOC.Ezptl. Biol. Med. 97, 832-835. Zweifach, B.W., Gordon, H. A., Wagner, M., and Reyniers, J. A. (1958).J . Ezptl. Med. 107. 437-450.

Insect Microbiology S. R. DUTKY Insect Pathology Laboratory, United States Department of Agriculture, Agricultural Research Service, Beltsville, Maryland

I. Introduction.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Bacterial Diseases. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. Fungous Diseases. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Protozoan Diseases. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Rickettsia1 Diseases. . . . . . . . . . . . . . . . . . . . . . . . . . . . VI. Virus Diseases. . . . . . . ...................... VII. Nematode Diseases. . ...................... VIII. Summary and Conclu ...................... References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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'

Insects are subject to attack by microorganisms, and widespread epizootics frequently terminate a dangerous outbreak that threatens extensive destruction. It has long been hoped that these microorganisms might be employed for control. As they are usually quite specific for insects and are not injurious to plants, man, or animals, this procedure would not involve the hazards presented by the use of insecticides. This aspect is becoming increasingly important. Also, it has become apparent that insects of certain species surviving insecticide applications have given rise to progeny quite resistant to these insecticides, requiring higher dosages to give control as well as development of new insecticides to replace those no longer effective. Our chemical industry has been able to meet the challenge of developing new effective insecticides, and entomologists quickly have established new recommendations for their use, so that we have continued to enjoy effective control of the ever greater variety of insects attacking our crops and persons in spite of this phenomenon. As many of the insecticides now in use are toxic to a wide range of organisms, many feel that serious consequences will develop in the long run due to the unwanted destruction of birds, predators, and parasites that play their part in natural control. Partly in response to the problem of residues and insecticide resistance, partly as a result of recent important and impressive successes in microbial control, and partly as the result of the natural exponential growth of science in general, workers in many parts of the world have renewed the search for new disease organisms and for methods for putting them and ones already known to work in insect control. 175

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Insect pathology as a specialized subject of study is rapidly coming of age. The degree to which this has been accomplished is indicated by the growing number of laboratories throughout the world that have been established for the purpose of basic and applied research in the field. A further indication that this subject is reaching maturity as a science is that it is soon to have its own journal. The microorganisms that attack insects include a large number of forms: bacteria, fungi, protozoa, rickettsia, viruses, and nematodes. Many of them operate against a single host species and in certain instances act in concert. Each of these diverse forms requires specialized methods of study. Often, methods devised for their study in relation to free living forms or as parasites of man, plants, or vertebrates, must be considerably modified to adapt them for greatest usefulness in insect work. The great diversity in the ecology and biology of the hosts also complicates their study. Applied insect pathology is only one phase of insect microbiology. It is a highly important one, and, moreover, a realistic point is that research efforts at a high level in more basic aspects are dependent on a continuing and growing interest in this phase. Because of the extreme diversity of hosts and pathogens, it is expected that by good managment or good luck or both, an adequate number of striking examples of successful application will keep this interest growing until enough basic information will be accumulated so that the workers can assess with accuracy the probability of success of a combination of host and parasite, and select the most suitable pathogen and the best method for its utilization.

II.

Bacterial Diseases

Quite a large number of bacteria pathogenic for insects have been described. These include non-sporeformers and sporeformers. Many nonsporeformers are known to be extremely virulent and frequently cause high losses in insectaries. Attempts to utilize these bacteria for insect control have not thus far given consistently successful results. A good part of the poor progress to date can probably be attributed to their lack of hardiness and our inadequate knowledge of manipulating them to the best advantage. Also, many of these forms are not able to invade readily by feeding and may require certain stresses to make invasion possible. In certain instances, invasion of the hemocele is accomplished by another organism acting as a mechanical vector. An example of this will be described in detail in a discussion of the DD-136 nematode-bacterial disease complex. The bacterial sporeformers causing insect diseases include some of the most successful agents used in microbial control of insects. The only example so far that has reached the goal of commercial production and application

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in this country is Bacillus popilliae Dutky (Dutky, 1940), the causative agent of type A milky disease of the Japanese beetle. Some of these bacterial sporeformers have very wide host ranges. This is especially true where injection is used as the route of infection. When tested by this route, bacteria isolated from one insect species will frequently produce mortality in widely separated groups of insects. By using peroral inoculation, however, many fail to produce consistent infection. Others have very narrow host ranges. Bacillus larvae White, the causative agent of American foulbrood of the honeybee, is without pathogenicity to most other insect species tested. Bacillus popilliae is pathogenic only to closely related species of beetles belonging to the family Scarabaeidae. The narrowness of host range is at the same time an advantage and disadvantage in its use for the microbial control of the insect. It is an advantage because application of the organism for control will affect only the noxious insect intended and have no direct effect on other insect species that are beneficial. But it is a disadvantage where two equally injurious insects attack the same crop and only one is subject to microbial control. Then the immune insect survives to ruin the crop. In some cases a single application of a microbial agent suffices to give lasting control; in others, repeated applications are necessary. This is primarily determined by whether the agent reproduces itself and remains in the field of action. For example, in the case of milky disease for control of Japanese beetle larvae, the spores produced in infected insects become residual in the soil. The larval stages of the Japanese beetles are soil-inhabiting forms and ingest large quantities of soil in their development. It follows, therefore, that the accumulation of spores in the soil will produce a reasonably permanent control. This would also be true in treatment of stored grains and other products. In treatment of crops, on the other hand, most of the infected insects would fall from the plants. Even where the disease agent was deposited on the plant, new growth of the plant and the cleansing effect of wind and rain would soon give an environment relatively free from the agent. The accumulation of a resistant agent on the soil below the plants would not have much effect on the infectiousness of the environment. In the case where an agent accumulates in the field of action, introduction of the agent at levels high enough to produce initial infection of only a small proportion of the population would be adequate to give eventual control. In the case, however, of a nonaccumulative agent, the level of initial application must be high enough to give the degree of control required. While persistence of an agent at continually infective levels over a period of years would be highly desirable, lack of persistence should not rule out microbial control as a practical method. If the microorganism can be pro-

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duced cheaply enough and on a sufficiently large scale, application of a transient microbial insecticide would be satisfactory and competitive with chemical insecticides. It is of some interest that the disease first to find widespread use in the practical control of an injurious insect, type A milky disease of Japanese beetle larvae, should be one with so many features apparently distinctly inimical to application. The milky disease organism does not grow on usual bacteriological media, and, in spite of much effort, even today there is not yet available a technique for mass producing the spores outside the insect body. The disease itself is very unspectacular, requires a long time to kill, and does not produce especially marked manifestation during the life of the doomed insect. Methods of study to produce sufficient material for field application were developed that sidestepped the difficulty of culture. Following the field studies that established the practicability of its use in control (Dutky, 1937; White and Dutky, 1940), methods were worked out for large-scale application and production; and a program of distribution throughout the beetle infested area in 14 eastern states and the District of Columbia was begun in 1939 and largely completed by 1952. The progress of the program has been outlined rather thoroughly (White and Dutky, 1942; White and McCabe, 1951). Since the start of the Federal-State program, 184,000 pounds of spore dust were produced and applied to 140,973 sites totaling 109,119 acres in the 220 counties treated (Report of the Chief of the Bureau of Entomology and Plant Quarantine 1953). Three patents (Dutky, 1941,1942; Dutky and Fest, 1942), were issued covering the major features of the techniques, and several licenses have been issued for the commercial production of the material. Another organism that is today demanding increasing attention as a microbial insecticide and will soon reach the goal of widespread applied use is one that has been recommended for this purpose since its first discovery in 191 1. This organism, Bacillus thuringiensis Berliner, 1915, is an insect pathogenic strain of Bacillus cereus Frankland and Frankland, and a very admirable pathogen it is. Is is easily cultivated on various media and by mass techniques. This organism and several closely related strains attack quite an array of injurious insects of several orders but most particularly Lepidoptera. Not all insects are equally susceptible, and in some cases older larvae of even susceptible species appear to become increasingly resistant as they maturate. The kill of susceptible species is rapid, and the effects obtained are comparable with that of insecticides. There is a growing number of studies attesting to its successful use, and more and more interest is presently being shown it both by entomologists and industry. Selected references to this organism and closely related sporeformers should include Hutz (1928), Hannay and Fitz-James (1955), Steinhaus (1957), LeMoigne

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et al. (1956), Heimpel (1955), Angus (1954), and Le Corroller (1958). These references cover fairly well the subjects of host range, field tests, the crystalline parasporal inclusions, the t,oxicity and taxonomic significance of these inclusions, the transformation of avirulent noncrystallophorous strains to virulent crystallophorous strains, and the correlation between pathogenicity and lecithinase production.

111. Fungous Diseases A large number of species of fungi have been described in association with insects. Representatives are known from the classes Phycomycetes, Ascomycetes, Deuteromycetes, and Basidiomycetes. Not all of these are parasitic, and of those that are, only a comparatively small number produce disease. An interesting group of fungi known to be almost exclusively benign cuticular parasites of insects are the Laboulbeniales. According to Richards and Smith (1956), approximately 1500 species of these curious fungi have been described. Of the fungi that produce definite disease in insects, the best known are the Entomophthorales and certain of the Fungi Imperfecti or Deuteromycetes. These are frequently responsible for large-scale epizootics that quickly reduce dangerous outbreaks of injurious insects in a most dramatic manner. There are many species of Entomophthorales known that attack quite a number of insects of different orders. Some show rather narrow host ranges, while others attack a variety of hosts. In most cases, they attack by direct penetration of the cuticle, and species of the most important family of the order Entomophthoraceae produce spores that are discharged from the host. The spores, falling on the cuticle of a new host when conditions are right, quickly penetrate it. The spores can also germinate and produce secondary conidia, and these secondary conidia are also discharged. Under proper conditions of temperature and humidity, the organism can be disseminated very widely and very rapidly by this process and produce the dramatic epizootic so commonly noted in populations of aphids, grasshoppers, and certain other insects. As a group, these fungi are difficult to culture and are generally short-lived. Resting stages of greater longevity are produced by some species, but conditions required for their production and germination are not completely known. There have been many attempts to use these fungi for microbial control, but until recently at least, they have not given consistently successful results. They hold so much promise that undoubtedly sooner or later a good system for their employment will be found. There are a number of highly infectious and very lethal pathogenic fungi among the Fungi Imperfecti. Perhaps the best example is the green muscardine, Metarrhizium anisopliae Metch. (Sor), named for its host, Anisoplia

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austriaca, the wheat chafer. This fungus has been found in a very large number of insect species and can produce fatal infections in practically every species of insect that has been tested. Experimentally, it is only necessary to dust a small number of spores on the cuticle of the insects, hold the inoculated insects at a high humidity to permit germination of the spores, and the insects become infected. Lesions are produced in the cuticle where the germinating spore thrusts its germ-tube, usually within 24 hours at 30" C., and the insects die several days later. A single spore penetrating the cuticle usually will cause death. The spores can be produced on fairly simple media without difficulty and are reasonably long lived. Dried spores lose their ability to germinate rather rapidly at 30" C. (about 50% in 1 month) but much more slowly at lower temperatures and can be stored for over a year at - 23" C. without measurable loss. Moist spores remain alive for longer periods. The fungus can grow between 10" C. and 31" C. It does not grow at 32" C. or above. It can infect insects through a slightly smaller range of temperature. High humidities are required for spore germination (in excess of go%), but the spores can infect insects in environments of lower humidities. This is especially true of insect species that do not have a high water retentivity (Japanese beetle larvae, for example). The spores survive in moist soil for at least several years and are capable of infecting a high proportion of the soil population under favorable conditions of temperature and soil moisture. In soil, where the humidity is generally high enough to permit germination, infection is governed by another factor. This is the contact between spores adsorbed on soil particles and the insect cuticle. This can only occur at low and at high soil moisture5 (Dutky, 1937). The fungus readily attacks all stages of most insects except the egg stage, and even this stage is sometimes attacked. The newly hatched larvae are readily attacked, however. M . anisopliae has a very long history of suggested and attempted use as a microbial insecticide that begins with its initial describer (Metchnikoff, 1880). Krassilstschik (1888) first produced fungus spores in quantity and made large-scale field tests against Cleonus punctiventris, the sugar beet curculio. Studies to this end are still continuing today. There are many reports of success, partial success, and some of no success with its use. For an adequate summary of these trials, see Steinhaus (1949). I am convinced that sooner or later this magnificent pathogen will find commercial application. Another member of this group of fungi that is highly virulent and attacks a wide variety of insects is the white muscardine, Beauveria bassiana Bals. This fungus has been known as a pathogen of silkworms since 1834 and was perhaps the earliest microorganism to be definitely incriminated as the cause of a disease. Bassi, for whom the species was named, also showed that in-

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sects other than the silkworm could be infected by it. Early attempts were made to use this fungus and species related to it for control of a number of insects, and in some cases a fair degree of success was obtained. The trials were not uniformly successful, however, and use of the fungus for control has not yet been established. Many investigators still feel that species of Beauveria hold promise and are actively working to bring this promise to realization. A great deal of work is being done at the European Corn Borer Laboratory at Ankeny, Iowa, in the study of this fungus and in methods for its use against this insect. Beauveria bassiana and its relatives have rather wide host ranges, are easily cultivated, and are highly lethal to susceptible species of insects. They attack by direct penetration of the insect cuticle. The spores are reasonably long lived (perhaps slightly more so than Metarrhizium) and could be produced economically in quantity. Our tests with both organisms indicate that for many species susceptible to both, Metarrhizium is usually the more virulent. Beauveria occurs more frequently in epizootics among above ground insects probably because of the type of conidia produced. In the case of Beauveria, the conidia are easily separable and air borne, whereas Metarrhizium produces conidia that are massed together (coremia) and are separated with difficulty and are unlikely to become air borne. In my opinion, both fungi deserve continued study. A concerted effort to exploit their use as biological insecticides should be made.

IV. Protozoan Diseases Quite a large number of protozoan species in association with insects have been described. Steinhaus (1947a) has covered some of the more general aspects of these associations rather well, and he has also done so with protozoan infections in his second textbook (Steinhaus, 1949). Some of these species are well-known parasites of man, animals, and plants. Insects and other arthropods serve as mechanical vectors, intermediate hosts, or even as primary hosts for some of these parasites. In the latter cases, the sexual stages of the parasite develop only in the arthropod host. Most species, however, are without effect on other life forms, attacking only their insect hosts. The effect of the association on the insect host ranges from a beneficial one to acute and fatal disease. Examples of all degrees of effect between these two limits have been recorded. Examples of beneficial associations are protozoa and termites, and protozoa and certain wood-eating roaches in which the protozoa appear necessary for digestion of the wood fiber and the insects are dependent on the protozoa for proper nutrition. Protozoa that cause diseases of insects belong to several classes. Mastigophora, Sarcodina, Ciliata, and Sporozoa all contain species described as

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insect parasites. The most noteworthy species are those belonging to the Sporozoa. This class includes many diverse forms with rather complex life cycles, and their study requires a great deal of patient observation as well as a thorough knowledge of the group. Those most commonly affecting insects include species of cephaline gregarines, schizogregarines, coccidia, and microsporidia. The cephaline gregarines have been observed in many insects of nearly all orders and rarely produce fatal infections. They are gut parasites; invasion is limited to the gut epithelial cells, and unless they are very numerous, infections are usually benign. Frequently, nearly the entire population of a species may be parasitized. This is especially true where a population has been in an environment for some time and the parasites can accumulate in the environment as is the case of soil insects and stored-product insects. A number of species of parasitic schizogregarines are known that produce disease in insects. Their pathogenicity is much more pronounced and more general than that of the cephaline gregarines. Some cause rapidly fatal infections, and these species should be considered as candidates for use in microbial control of the susceptible insect species, particularly in cases where the chance of accumulation of the agent would offset the practical difficulties of mass propagation of the parasite. Species of the Coccidia are also definitely pathogenic. Many produce slow but frequently fatal infections. These also should be considered for use in control in much the same way as the schizogregarines. We have observed mixed infections in which all three of these telosporidians were present simultaneously in a single sample of insects and in some cases in the same individual. These samples were from diseased cultures of the dermestid Trogoderma inclusum. Mixed infections are more likely to occur in cultures than among natural populations because of the high population density in cultures. Another contributing factor is the handling of a large number of cultures without adequate safeguards against accidental contamination. Eventually a parasite present in one culture introduced with specimens used as stock, or contaminating the medium used for food, will be transmitted to others in which a second parasite is present, and so on until all cultures will be infected with all parasites to which the insect species are susceptible. In samples of insects taken from quite a large number of wheat storage units under control of the Commodity Credit Corporation, a high incidence of parasitism by a schizogregarine was observed in Trogoderma species in the samples, although other dermestids were relatively free from infection. Marzke and Dicke (1958) are making a preliminary study of the transmissibility of these parasites to see whether further investigation of the possibility of their use in control is indicated. Weiser (1955a, b) and Steinhaus (194713) have made studies on representa-

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tives of this interesting group of protozoa. Kudo (1954) is a good reference to this group of protozoa as well as the other classes. The most important group of the Sporozoa are the Microsporidia. Many species have been reported and many are highly pathogenic and cause a great deal of mortality in insects reared in culture and also in the field. It was one of these, Nosema bombycis, the cause of pebrine of silkworms, that started Pasteur on his way to make his outstanding contributions to microbiology and to human health. Somewhat less dramatic but also important is Nosema apis, a parasite of the honeybee (White, 1919). This parasite rarely destroys a colony directly, but the debilitating effects of the disease cause a considerable loss to the beekeeper even though the exact degree by which yields are reduced through its action cannot always be assessed. These two species have until recently received the most study and are best known. Much of the study on other species is patterned on information available from the literature on these two. This group is also important because of its effect on other beneficial insects. Mass rearing of parasitic and predator insects frequently suffers through their deleterious effects on these insects or on the hosts used for propagation. Allen (1954) reports the effect of parasitism on host and insect parasite and the methods devised to reduce the losses by controlling the disease. Alarge number of species of Microsporidia have been reported in injurious insects. Kudo (1924) has prepared an extensive monograph on Microsporidia and is the American quthority on the group. Species parasitic in insects of many orders have been described, and there are numerous reports of undescribed Microsporidia. Most of the early reports were from Diptera, Lepidoptera, Ephemeroptera, and Hymenoptera. Other orders had only one or two cases of observed parasitism. Dutky and White (1940) noted the occurrence of microsporidian infection among native scarabaeid larvae, apparently the first observed in this group. Taxonomic description of the parasite was not possible because only mature spores were present, and classification of the group is based on their sporogeny. We later observed a large number of Japanese bettle larvae infected with another microsporidian. In this instance mature spores and all stages of sporogeny could be observed. As the sporonts developed into a large number of sporoblasts, the most common number found was 64; the microsporidian was determined to be a member of the genus Plistophora Gurley. Krieg (1955a) has recently described a similar disease in Melolontha spp. and named the parasite Plistiphora melolonthae. I presume his generic spelling is in error and the correct name of the parasite is Plistophora melolonthae Krieg. New species of microsporidia that have been described recently from insects are Thelohania hyphantriae Weiser, Thelohania erigastri Weiser, Nosema lymantriae Weiser, Nosema muscularis Weiser, Nosema aporiae Veber, Plistophora

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aporivora Veber (Weiser 1957 a,b,c). New species described by other workers include Nosema infesta Hall (Hall, 1957a), and Perezia fumiferanae Thomson (Thomson, 1955). Some caution should be used in the description of new species. Microsporidia tend to be rather host specific, but there are many cases on record of interspecies infection, intergeneric infection, and even some claims that a single species may attack insects of entirely different orders. Hall (195713) used the buckeye caterpillar as host to study the sporogeny of Perezia ppausiae Paillot, an important parasite of the quite unrelated European corn borer. Tanada (1953) found in Hawaii extensive field infections in Pieris rapae by a microsporidian first described from the related species Pieris brassicae in France. Many of the species of Microsporidia produce at times extensive mortality. Others that are less spectacular and do not produce acute fatal infections seem to be of considerable importance nonetheless. They may weaken the insect, reduce its potential to survive unfavorable conditions, or lower its fecundity. Thomson (1958) found some evidence of this in his work with spruce budworn. These actions may effect a greater control on the survival of the species than a rapidly fatal disease that has less potential of spread. In many cases infection is transmitted from one brood to another transovarially (Weiser, 1958). With borers or insects that are usually solitary, transovarial transmission may be the only effective means of inoculating an appreciable portion of the population. With insects that are not solitary, the problem of transmission is much simpler. In some diseases, depending mainly on the site of the infection, spores may be discharged in the excrement or from the silk glands while the host still is alive. This would be the case when gut epithelia, Malpighian tubes, or silk glands were heavily infected. The spores thus discharged might be ingested by uninfected insects which would then become infected. In the case of aggressive or cannibalistic insects living together, infected insects might be bitten or eaten by normal ones that would then be inoculated. In species where the infection is in the fat body or other internal structures, spores are not usually released until the death of the infected host. The spores then released might be ingested by normal insects that would then become infected. Microsporidian spores are not particularly resistant nor are they of great longevity. White (1919) showed that Nosema apis spores suspended in water were destroyed in 10 minutes a t 58' C. The dry spores at room temperature survived 2 months, at incubator temperature about 3 weeks, and in the refrigerator about 755 months. Direct exposure to the sun's rays destroyed dry spores in 15 to 32 hours. Allen (1954) showed that tuberworm eggs could be freed of contamination by Nosema destructor spores by heating them at 47' C. for 20 minutes. Weiser (1956) showed that spores of Thelo-

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hania hyphantriae had greatest longevity at 0" C., surviving at this temperature 13 months. At lower temperatures, longevity fell off rapidly, spores surviving only 1 day a t -40" C. and only six days a t -20" C. At temperatures higher than 0' C., longevity decreased rapidly, and spores survived only 30 minutes at 40"C. This lack of hardiness and relatively short longevity even under optimal conditions is a serious drawback to their practical use as a microbial control agent, especially if one had an equal choice of agents. Compare the extreme hardiness and great longevity of bacterial spores and the great longevity of viruses in inclusions that may remain viable for scores of years. Nevertheless, microsporidian spores have an adequate longevity and hardiness to permit their use in tests and to make easily possible maintenance of stocks of infective material. Weiser and Veber (1957) obtained up to 100% infection of Hyphantria cunea by spraying foliage with spores of Thelohania hyphantriae. About 25 % of the infected larvae died in 14 days, and the remainder were dead in about a month. No permanent reduction was obtained in the tests, but the results should be considered most encouraging. T . hyphantriae is an example of the group of microsporidia where the spores are not excreted by the living host and the chances of spread are considerably less than in a disease where this occurs. I would agree with Weiser that the entire group of protozoa should be carefully studied and tested for their possibilities in microbial control.

V. Rickettsia1 Diseases Rickettsia are small, nearly submicroscopic, organisms that are frequently associated with insects or other arthropods. Some are notorious as dangerous pathogens of man and higher animals. These are the most studied and best known. I n some cases they are apparently without effect on their arthropod host, and in others produce fatal infections in this host. Other rickettsia less well-known and not pathogenic to man or animals are reported as benign parasites of insects and other arthropods. Some are inferred to be beneficial or even essential symbiotes. Rickettsia that produce fatal infections of insects but are not infectious to higher animals have been described recently. Blue disease of Japanese beetle and other scarabaeids (Dutky and Gooden, 1950) and "Lorsch" disease of Melolontha vulgaris, also a scarabaeid (Wille and Martignoni, 1952), are the first examples to be recorded. The causal organisms of the two diseases were described as Coxiella popilliae Dutky and Gooden (Dutky and Gooden, 1952), and Rickettsia melolonthae Krieg (Krieg, 1955b). The organisms are very similar. Both are small kidney-shaped rods, 600 millimicrons long and 200 millimicrons wide. Both are filterable. They also are serologically similar (Krieg, 1955~).The histopathology of the diseases is also

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similar. Both have crystalline inclusions, and the affected fat body takes on a bluish coloration. Infected cells become packed with rickettsia and burst, liberating hundreds of thousands or even millions of rickettsia. Nuclei of cells infected soon lose their chromatin and can hardly be distinguished from the cytoplasm. Shortly after, the cell ruptures, releasing the myriads of contained rickettsia and also some of its crystals. Wille and Martignoni insist that in the case of the “Lorsch” disease the crystalline inclusions are present only in the cytoplasm of infected cells and never in the nuclei of fat body cells, oenocytes, or hemocytes. This is in contrast to our description of the sequence in blue disease, where we indicate the nucleus to contain both rickettsia and inclusions. This difference may be true for the two diseases, or perhaps the histopathology is the same, and one group of observers is in error. Undoubtedly further study will clarify this point. Philip (1956) revised the classification of the Rickettsiales and among other changes placed these insect pathogenic rickettsia in a new genus Rickettsiella Philip of a new tribe Wolbachieae of the family Rickettsiaceae. The new name of the blue disease organism was Rickettsiella popilliae (Dutky and Gooden) Philip, the genotype species. Rickettsia melolonthae Krieg, the causal organism of Lorsch disease, was also considered assignable to the new genus. He raised the question of its synonomy, however. Philip (1957) lists a single species only, Rickettsiella popilliae Dutky and Gooden (Dutky and Gooden, 1952; Philip, 1956). Coxiella popilliae and Rickettsia melolonthae are considered as synonyms. Recently two new species have been described, Rickettsiella stethorae Hall and Badgley (Hall and Badgley, 1957), developing in the cytoplasm of the gut epithelial cells of infected Stethorus larvae without formation of typical crystalline inclusions, and Rickettsiella tipulae Muller-Kogler (Muller-Kogler, 1958) that develops in the fat body cells of infected Tipula paludosa larvae. In this latter case, crystalline inclusions are present in the infected cells. Experimental infection was obtained using peroral inoculation in about 25% of the larvae tested. As in the case of the scarabeid diseases, the disease of Tipula is slowly fatal. Infected larvae died 5?4 to 11 weeks after inoculation. Muller-Kogler considers that the taxonomic status of the species described by Hall and Badgley is in doubt because of the lack of crystalline inclusions. It is likely that further study will uncover more diseases of insects caused by this interesting group of organisms. The long period from infection to death and the relative lack of symptoms until just prior to death make this group of diseases easy to overlook. Information on these diseases is still quite meager, but our studies in the United States and those of Niklas (1956, 1958) in Germany indicate that they play a considerable role in natural control and may have value in ap-

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plied microbial control. The long incubation period of these diseases is a serious handicap to their study and makes progress slow and time consuming. It also demands superior quality of larvae used for testing and ideal rearing techniques. Otherwise losses due to incidental factors will seriously affect the tests. With more rapidly fatal diseases, larvae of poorer quality may be used without significantly affecting results. Most of the European studies on infection and transmission of disease have been made using peroral inoculation, or by exposing larvae to inoculated soil. Information on this mode of inoculation is certainly desirable but does not permit very much manipulation of the data obtained. The period of time from exposure to death is extremely long and much more variable than that obtained by injection. Also, the European workers in their report do not give a clear indication of the dosages employed. A summary of the salient points concerning Riciiettsiella popilliae that we have been able to establish in our research with blue disease since publication of our paper in 1952 and not heretofore published except as summaries is given below. 1. The LDao by parenteral injection is less than six rickettsia per insect. 2. The thermal death point is 60" C. for 10 minutes (rickettsia suspended in water). 3. Bacteria-free suspensions of the rickettsia survive without loss of virulence for more than a year at ' 4 C. After 3 years at this temperature, suspensions were still infectious but the titer was only ~ o o , o o oof that of duplicate suspensions stored for a like period frozen in dry ice (-79°C.). There was no decrease in dark-field microscopic counts in either suspensions. 4. They are only partially resistant to drying. There is a very large drop in titer initially but some virulence persists for a long time. 5. The organism is sensitive to dihydrostreptomycin and to sulfadiazine sodium but not to penicillin or aureomycin. Injection of dihydrostreptomycin (20 fig. per larva) or sulfadiazine sodium (200 pg. per larva) protected larvae against an injected challenging dose of 7200 rickettsia. Tnjection of penicillin or aureomycin did not prevent infection. 6. The longevity of infected larvae is prolonged by increasing the amount of food available to the larvae, but this does not prevent infection or their ultimate death. 7. The time required for development of external symptoms is a linear function of the log of the dosage. This would indicate exponential development of the rickettsia during the incubation period. From the data published (Dutky and Gooden, 1952), an apparent doubling time of 6.6 hours can be extracted. This exponential relationship has been established through a number of dosage-time of infection tests, and the apparent doubling

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times extracted varied from about 6.0 to 10.8 hours. This agreement is not too bad since the amount of food, the size of larvae used, and the temperature of incubation varied in the tests. 8. The blue coloration of infected cells observed in the dark field is not due to fluorescence but is the result of Tyndall scattering-probably by the rickettsia they contain. 9. The crystalline inclusions freshly extruded from ruptured infected cells appear by dark-field examination to be enclosed in an enveloping membrane. They appear to lose this membrane after extrusion. X-ray diffraction studies of the crystals recovered from filtrates and similar to the ones observed free in the hemolymph show diffraction patterns resembling those of calcium oxalate. 10. It is possible to produce large amounts of blue disease rickettsia by injecting healthy larvae with suspensions of the organism and then incubating the larvae under satisfactory conditions of temperature, food, and moisture for about 35 days. The rickettsia were harvested by grinding up the living diseased larvae, and filtering the diluted suspension centrifugally through a filter cake of Celite. The filtrate contains few rickettsia and most of the soluble impurities, and can be discarded. The rickettsia entrapped or adsorbed on the Celite were eluted by successive washings with water and the slurry of Celite each time was filtered through paper. The filtrates containing the clean rickettsia can be frozen for storage until use. A yield of about 330 X 109 rickettsia per larva was obtained. As indicated above, tests on frozen storage show that rickettsia retain their virulence for at least 3 years.

VI. Virus Diseases The virus diseases of insects may be divided into three groups: polyhedroses, granuloses, and noninclusion virus diseases. In polyhedroses, crystalloid inclusions are found in the affected tissues and, as the disease progresses, throughout the body of the insect. These inclusions are readily visible by means of the light microscope, using bright-field techniques. In granuloses or capsule virus diseases, the inclusions are very much smaller and are frequently close to the limits of resolution of the light microscope. They are easily observed by dark-field techniques. These inclusions contain a single virus particle surrounded by a protein layer or capsule. In advanced cases of disease, they occur in enormous numbers in the body fluids of the affected insects-several hundred billion per insect. The noninclusion virus diseases in which no regular inclusions are found are the least well known. They are most difficult torecognize, and reported instances of this group of diseases undoubtedly include physiological upsets not of viral or other microbial origin. Furthermore, as inclusions of the first two

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groups are readily soluble in weak alkalies including ammonia, it is not unlikely that insects killed by these diseases will frequently be found denuded of the inclusions. We have observed this several times a t the Insect Pathology Laboratory a t Beltsville. Transmission tests using as inocula suspensions of insect remains showing no inclusions, produced typical cases of polyhedrosis and granulosis. There are several well authenticated types of noninclusion virus diseases that have been adequately studied. Examples of these are sacbrood of honey bees (White, 1913) and a noninclusion virus disease of the armyworm (Wasser, 1952). The polyhedrosis virus diseases might further be subdivided into cytoplasmic and nuclear polyhedroses on the basis of the area of the infected cell in which the virus and inclusions are formed. They might also be subdivided on the basis of the tissue attacked. For example, the nuclear polyhedroses of sawflies may be referred to as gut polyhedroses since attack of the virus is restricted largely to the nuclei of the midgut epithelial cells of these insects. Virus diseases have been described as occurring in a number of orders of insects, but primarily in Lepidoptera and Hymenoptera. The diseases of these orders have been most carefully studied. Hughes (1957) lists 197 species of insects in which virus diseases have been reported. Of this number, 169 are Lepidoptera, 18 Hymenoptera, 6 Diptera, 3 Coleoptera, and 1 Orthoptera. This list is not complete and some of the reports are probably not valid. His annotated list and bibliography of insects reported to have virus diseases is excellent and will give the reader 255 references to the subject of insect viruses published through 1956. The large preponderance of lepidopterous insects reported may mean that these diseases are characteristic of this group or that a great deal of observation has been made on members of this order. It should be remembered that although many more species of Coleoptera are known than Lepidoptera, many of the Coleoptera are more obscure in habitat than are the Lepidoptera. As Sabrosky (1952) indicated that the number of described insect species in 1948 totaled 685,900, it is evident that the 197 reported to have virus diseases is a very small proportion, and that further investigation might quickly wipe out the apparent preponderance of lepidopterous species affected. The presence of these diseases among insects was recognized quite early although the causative agents were not recognized until much later. The frequent occurrence of widespread epizootics that left dead insects hanging from every infested plant quickly captured the imagination of these early investigators. They tried to use these diseases as agents in artificial control. As a generalization, it could be stated that virus diseases of insects, as in the case of virus diseases of man, animals, and plants, are the most viru-

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lent and most readily transmitted of all diseases. This quickly led to an oversimplification of the job at hand and resulted in many disappointing failures. In recent years, however, great strides have been made in our knowledge of these diseases and in their utilization in insect control. As might be expected, there is a great range in the virulence of these microorganisms and in the behavior patterns of the insects they attack. In some cases, the insects are highly susceptible to infection throughout the& life history, whereas in others, susceptibility falls off rapidly as the insect matures. Newly hatched larvae are frequently readily infected, but after a moult or two they may be nearly entirely resistant to infection. This requires, then, a thorough knowledge of the disease and careful planning for proper utilization as a control agent. If applied too late, no apparent effect will be obtained. Also, even though the host might be infected and killed by the application, larger larvae with their increasing feeding capacity might seriously damage the crop before death or disease put an end to their ravages. The problem is further complicated by the fact that much of the transmission from one generation to the next is transovarial, so that early kill of young larvae would have less effect on succeeding generations than survival of older larvae that had apparently escaped infection. These latter might develop to adults and then lay infected eggs that would widely disseminate the disease. The mode of reproduction and development of virus has been studied by examination of ultrathin sections of diseased tissue by electron micrography and by other means. Most of the investigators agree with Bergold (1950) that the virus fist appears as small spherical bodies that elongate to form rods. The rods may escape from their developmental membranes and again form minute spherical bodies. At the end of the reproductive cycle, the rods and some remaining spheres are occluded by protein material to form polyhedra, or’granules, depending on the type of disease. In some polyhedroses, rods are occluded individually, and in others the rods first form into bundles that are then occluded to form polyhedra. In the case of granuloses, usually single rods occur in a granule or occasionally a pair of rods may be occluded. Three recent references to the subject of the development and histopathology of insect viruses are Day et al. (1956) and Bird (1957, 1958). Additional references to important contributions on the subject by himself and others are included in Hughes’ bibliography already referred to (Hughes, 1957). Virus diseases as a group appear to be quite host specific, and infectivity is generally limited to a single species or to closely related species mostly belonging to a common genus. Examples of intergeneric susceptibility (Bergold, 1943; Smith and Xeros, 1953; Tanada, 1954; Bird, 1955), are not uncommonly reported, and some of the reports are well substantiated.

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Phylogenetic relationships of host insects in regard to disease susceptibility may not always follow the morphological relationships employed in their classification. There are numerous examples of viruses attacking a number of species in the same genus, and cross infection has been obtained readily. I n some instances the homologous virus is somewhat more virulent (Dutky et al., 1955b), in others no differences in virulence were noted. It is a frequent practice to propagate the virus from a species on another of the same genus for use in field applications against the original host where for one reason or another the original host could not be used for propagation. Clark and Thompson (1954) used this procedure in tests on control of the Great Basin tent caterpillar, Malacosoma fragilis (Stretch). In these tests, the California tent caterpillar, Malacosoma californicum (Pack), was used as the host for propagation purposes. The virus employed by Chamberlin and Dutky (1958) in tests against the tobacco budworm, Heliothis virescens, was propagated a t the Beltsville Insect Pathology Laboratory by Dr. C. G . Thompson using the corn earworm Heliothis zea, as the propagation host. Factors affecting the susceptibility of insects to virus diseases or the pathogenicity of the agents include the stage of development of the host, temperature, and sometimes humidity and kind of food. The dependence on such factors varies considerably for different diseases and different hosts. Some show little dependence while others appear to be seriously affected. In addition, some diseases are transmissible with such irregularity that workers have intimated that perhaps all individuals of a species are infected with latent virus that develops symptomatically only under “stress conditions.” It would be oversimplification to ignore these often repeated claims, but quite realistic to challenge them and try to find more tangible explanations for the observed effects attributed to various stresses. Spontaneous development of disease in disease-free stocks has been offered in partial support of this theory, but the entomological procedures in common use do not usually afford sufficient security from accidental contamination by an agent whose longevity may be a score of years. Insect stocks have been maintained free from disease for long periods of time even in laboratories that handle disease material in quantity and observe a strict bacteriological discipline. Balch and Bird (1944) had to resort to these techniques in order to obtain disease-free stocks of European spruce sawflies for their studies. With many diseases, susceptibility falls off rapidly as the insect larvae mature. This phenomenon deserves a great deal of attention, and determination of the mechanisms responsible will add much to our knowledge of these diseases. In the case of others, larvae in all feeding stages of development are quite susceptible to infection.

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With some diseases, insects can be infected throughout the range of temperature in which they are active, whereas in others infection can occur only through part of the range. Developing larvae may escape infection a t low temperatures and at high temperatures. Bird (1955) indicates that in larvae of the European spruce sawfly, Diprion hercyniae, continuous rearing a t 85" F. (29.4"C.) inhibits virus multiplication. Dr. C. G. Thompson, Director of the U. S. D. A. Insect Pathology Laboratory a t Beltsville, has been making extensive studies on the effect of temperature on virus infection of a number of insects and should soon publish his interesting results. In all cases, temperature has a marked effect, and the time of development of symptoms and death increases rapidly as the temperature falls. Krieg (1955d) indicates that in the case of the gut polyhedrosis of the European pine sawfly, Neodiprion sertifer, the length of time for 50 % mortality doubles for a 10" C. drop in temperature between 29.6"C. and 11.5" C. Our own work with the virus of Neodiprion pratti-pratti indicates a similar relationship in regard to temperature. In this case, the time to death in days equals 100/(t - 10) for temperatures between 30; C. and 10"C. when larvae were exposed to needles dipped in a suspension containing lo6polyhedra per milliliter. With this virus disease, and apparently with many others, the time to death is more nearly a hyperbolic function of temperature than an exponential one. As a generalization, one could state that every virus disease has a lower and an upper temperature threshhold for infection and development and an optimum. These critical temperatures for the disease may or may not coincide with those of the host insect. This generalization does not restrict itself to virus diseases of insects but applies to all symbiotes. There are many reports that humidity influences the incidence of virus diseases among certain insect populations, especially under field conditions. A recent example is Wallis (1957), who correlated the observed incidence of polyhedrosis in gypsy moth larvae with the relative humidity prevailing at the time of observation. This fails to take into account the period of incubation required for infected larvae to succumb to the disease or to develop frank symptoms. Many investigators working with virus diseases of other Lepidoptera report that humidity has no influence on infection under either field or laboratory conditions (Balch and Bird, 1944; Thompson and Steinhaus, 1950); Krieg, 1955d). It seems reasonable to assume that humidity has generally little direct effect on infection, but that there is an indirect effect through its influence and that of rainfall on the dissemination and persistence of the infectious agent in the zone of action of the host insect. The effect of type of food, feeding of various chemicals, and other stress factors has been summarized by Bergold (1958). Many notable examples of successful use of virus diseases for the control

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of injurious insects have been noted in recent years. Foremost among these are the control of the European spruce sawfly, Diprion hercyniae (Htg.), by means of a gut polyhedrosis (Balch and Bird, 1944), and the control of the alfalfa caterpillar, Colius philodice eurytheme Bdv., by a nuclear polyhedrosis of this species (Thompson and Steinhaus, 1950). The highly successful results obtained by these investigators with these insects restimulated interest in the potentialities of virus diseases as a means of control for both native and introduced insect species. As the result of the rekindled interest, a growing number of insect species have been found where virus diseases, under certain conditions at least, can give economic control for crop protection. This method may be competitive with insecticidal treatment or supplement such treatment to advantage. Recent examples that are especially noteworthy include virus control of the European pine sawfly, Neodiprion sertifer (Geoff.) (Bird, 1950, 1953; Dowden and Girth, 1953; Franz, 1955; Schuder, 1957); virus control of the cabbage looper, Trichoplusia ni (Hbn.) (Hall, 1 9 5 7 ~ ;McEwen and Hervey, 1958); and virus control of cabbageworms, Pieris brassicae L. (Biliotti et al., 1956), and Pieris rapae (L.) (Tanada, 1956). References to many more will be found in Hughes’ bibliography (Hughes, 1957). Large-scale commercial use of viruses should follow when adequate knowledge is available to permit specific recommendations for their use that will ensure adequate results, and when methods of propagation are developed that will provide a large enough supply of the agent economically.

VII. Nematode Diseases Over a thousand species of nematodes have been described in association with insects. These include those forms that have become highly specialized and are fully parasitic and forms that are not highly specialized and are only partially parasitic. The fully parasitic forms tend to be quite host specific, and representative species are found only in association with a very restricted group of insects as hosts, whereas a single species of the semiparasitic forms may attack an unbelievable variety of insects. This latter group, because of its lower degree of specialization, lends itself most readily to manipulation and therefore probably holds greater promise for use in application to insect control. Nematodes that are fully parasitic and develop in the body cavity or tissues of insects of several orders belong for the most part to the families Tetradonematidae, Mermithidae, and Allantonematidae. Some kill their hosts on emergence, others produce sterility. Many are important in the natural control of insects and could be used in application to insect control. This possibility has been pointed out by many investigators (Cobb, 1927;

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Jenkins and West, 1954). A principal difficulty seems to be in propagation of the nematodes in amounts adequate for this use. A great deal of additional information on the biology and life history of these forms must be gathered to permit their practical use. In some forms, the parasites emerge in the pre-adult stage and make final moult to the adult only after periods as long as 1 to 2 years. In others, the final moult may be made sooner after emergence, but oviposition may be delayed for long periods. This behavior is part of the specialization that adapts the species to conform to the ecology of the host and serves to make it a powerful agent of natural control. It presents, however, a considerable difficulty to their satisfactory artificial manipulation and propagation. Among the nematodes that are semiparasitic in insects are a number that show great promise for use in insect control. Some recent examples have been reported by Weiser (195513) and Hoy (1954). The studies on Neoaplectana glaseri Steiner by Glaser and his associates (1940) have done much to indicate that this promise is capable of fulfillment. Glaser and Fox (1930) give the history of the initial discovery of parasitized dead Japanese beetle larvae in 1929 and the subsequent determination of the species by Steiner (1929). Glaser (1931, 1940) demonstrated that the nematode could be cultured on artificial media. This was a most important contribution, not only in respect to the use of the nematode in artificial control but also for its more general implication that other nematode parasites of man and animals could be cultured axenically apart from the host. The development of methods for large-scale mass propagation quicklj followed (Glaser, 1932); McCoy and Glaser, 1936; McCoy and Girth, 1938). Field experiments (Glaser and Farrell, 1935; Girth et al., 1940), established that artificial introduction of the nematode into sod infested with Japanese beetle larvae would give fair control of the insect, a t least under favorable conditions, and that the nematode would persist for considerable periods following introduction. These experiments were followed by an extensive program of colonization whereby the nematode was introduced into areas with favorable populations at 3X-mile intervals throughout the state of New Jersey. In 1939,161 colonization plots were inoculated, and the program was finally completed in about 6 years. As the 1939 colonization program also included treatment of much larger areas at each location with milky disease as a comparative test of the two agents, and milky disease was found to be far superior, further colonization for control of Japanese beetle in other states was limited to this remarkably successful agent. The large-scale propagation of the nematode was curtailed and, on completion of the New Jersey program, was discontinued. It is most unfortunate that these promising studies were directed against a host that could be attacked by an even more remarkable pathogen. Never-

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theless, the studies of Neoaplectana glaseri show the high potential for control that nematodes possess and serve as a guide to the study of others. Many other species of nematodes that are semiparasites of insects have been found, and some have even greater promise than Neoaplectana glaseri. In many cases, these are accompanied by specifically associated species of bacteria that are extremely virulent pathogens of insects. This association was first demonstrated in another species of Neoaplectana (Dutky, 1937) and I have since found this to be so for a number of species of nematodes of the family Steinernematidae. One of the best examples is the DD-136 nematode-bacterial disease complex of larvae of the codling moth, Carpocapsa pomonella, reported by Dutky and Hough (1955). A more complete report of studies with the complex has since been made (Dutky et al., 1955a). A great deal of information has beeq established about this most interesting disease complex since its initial discovery in 1954, and most of it has become public property through press releases and articles in publication of the Agricultural Research Service (the article in April 1956 number of Agricultural Research is a good example), and by similar releases of agencies of various cooperators. The information has also been disseminated widely by exhibits, talks, and correspondence, and the nematode has been supplied for tests and study to many laboratories in the United States and elsewhere, including Canada, Peru, Chile, Germany, Czechoslovakia, the Netherlands, Egypt, and Japan. Tests with over one hundred insect species, many of them important pests, show that nearly a11 are highly susceptible to its attack. A few are somewhat more resistant, probably because they are less attractive to the nematode. Susceptible species have been found in many ordersLepidoptera, Diptera, Hymenoptera, Coleoptera, Orthoptera, Hemiptera, Hornoptera, and Isoptera. The nematode has so far been described only as a member of the family Steinernematidae. It closely resembles Neoaplectana chresim Steiner (Glaser et al., 1942) in many features, but differs from it sufficiently so that Dr. Steiner considers it to be a new species, possibly not of this genus. The disease is primarily a bacterial septicemia whose vector is a specific nematode. The associated bacterium not only kills the host but also serves as food for the nematodes. It is additionally important because it elaborates an antibiotic that prevents putrefaction of the cadaver and the growth of microorganisms inimical to the development of the nematode, The infective stage of the nematode is the ensheathed second stage larva. The nematode seeks out the host insect, enters usually by way of the mouth parts, exsheaths, penetrates the intestinal wall, and injects the associated bacterium contained in its esophagus into the body cavity of the host. This sets up the septicemia that kills the host. The nematodes search out the insects

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with great rapidity, and the entire process from exposure to death at 30' C. takes less than 24 hours. After the death of the host, the invading nematodes maturate and become adults. If both males and females are present (in this species the sex ratio is 100/100), these mate and give rise to young. The young are born matricidally, that is to say, fertile ova produce embryos within the ovaries of the gravid female. The eggs hatch, and the young feed on the tissues of the mother. They escape after her death as second stage larvae. Some of these (about 80 %) are ensheathed and do not develop further. Others maturate, producing adults that mate and again produce young. Several generations may be completed until the host cadaver is filled with ensheathed larvae. These ensheathed larvae then emerge from the cadaver in search of a new host. At the most favorable temperatures, 25-30' C., the cycle from infective stage to infective stage is 8 days. The cycle lengthens at lower temperatures. If the cadaver is not in contact with free water, the ensheathed larvae may remain inside the partially dried cadavers for at least 2 months without injury. Emergence of larvae from such cadavers begins within minutes after they are in contact with water. The time to death depends on the temperature, and on the degree of exposure. When wax moth larvae are exposed to nematodes on moist filter paper, 50 larvae to 10,000 nematodes, most of the nematodes will have entered the hosts within 2 hours. The death time of these larvae is 16 hours at 30" C., 24 at 25"C., 44 at 19'C., 120 a t 15"C.,and 312 at 9°C. An exposure to fewer nematodes takes somewhat longer, but a single nematode entering the host will produce death. In this case no young are produced. The infective stage larvae are very small, about 25 microns in width and 600 microns in length, and are not visible to the unaided eye except with proper background and light. The adults are quite large. The largest females are nearly 15 mm. long and may contain 1,000 ova. The smallest females are less than a tenth as long and may contain only 16 ova. As the ova are about the same size in the large females and in the small females, the number of ova varies roughly as the cube of the length. Female adults of various size are not present simultaneously, but occur in sequence. The largest females are produced from the invading infective stage larvae, and smaller and smaller ones from subsequent generations. After the initial phase, however, there may be present females of different size groups. The males show a similar sequence in size and are considerably smaller than the females. They are about a fifth as large as the largest females and about a third the size in the case of those corresponding to the smallest females. The infective stage is nonfeeding, can survive for long periods under proper conditions of temperature and moisture, and can be stored for years

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without loss if infectivity. In spite of their delicate appearance, they are remarkably resistant t o shear, and can withstand the stresses encountered in applying them with conventional high pressure spray equipment. They are also resistant to many chemicals including most of the insecticides and fungicides in common use. Because of these facts, the nematodes can be used in conjunction with chemicals for insect control, and they may be applied with the same equipment employed for application of pesticides. The nematode is not resistant to drying and is quickly killed by desiccation. It also requires a moist surface in order to migrate in search for a host. This need for moisture may limit the ability of the nematode to control insects that feed on exposed portions of plants to periods of high humidity and heavy dews. They can live throughout long periods without rain by remaining in the host insect, in the soil, under the bark of trees, or in the stems or whorls of plants where some moisture is available. They are also sensitive to high temperatures and are quickly killed a t temperatures of 42" C. and above. In tests in apple orchards made over the past 4 years, consistently good results have been obtained even with low host populations. The nematode can be introduced by applying them in a spray to the trunks and main branches. Protected by the canopy of leaves, the nematodes have been able to survive even under conditions of extended drought and can produce high mortalities (60% and higher) among codling moth larvae seekin'g cocooning sites. These tests demonstrate that nematodes applied to trees in one brood produced high mortality in that brood within a few weeks, and the same application was still effective against the overwintering brood more than 3 months later. These tests would indicate that nematode treatment is more persistent than chemical sprays used in a similar manner, and more effective than many. Hamilton and Fahey (1958) tested several insecticides against codling moth larvae seeking cocooning quarters. The best insecticide, parathion, gave high kills when worms were exposed to bark surfaces immediately after application (92.4 %), but much lower kills when larvae were exposed to surfaces 13 and 20 days after treatment (57 and 70 %, respectively). Guthion also gave kills immediately after application (92.3%))but control was markedly lower after 13 and 20 days (49 and 36 %, respectively). Tests with the nematode against other insects have also shown promise, but the results are not yet as convincing as those against codling moth larvae. Two methods of propagation of the nematode have been employed. Wax moth larvae grown on artificial media used as a propagation host yield about 160,000 infective stage nematodes per insect, or 1,500,000per gram. It has also been propagated on sterile media inoculated with pure culture

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of the associated bacterium. The maximum yields on these media are about 300,000infective stage nematodes per gram or approximately a fifth of that obtained from insect hosts. The nematodes obtained from the cultures are equal to or superior to those obtained from insects.

Vlll. Summary and Conclusions I have tried to give a summary of each of the several groups of microorganisms that produce diseases and some of the recent progress that has been made toward their utilization for the microbial control of insects. There are representatives of each of the groups that will be put to practical use in the very near future. This is nearly inescapable because of the great variety and number of insect species that in one way or another cause us injury. A great deal of basic facts pertaining to the biology of both the microorganisms and their hosts must be established before maximum use can be made of any of them. There must also be some thorough and extensive studies on the. mode of action of these pathogens. This information will be applicable to both insect pathology and pathology in general. There are no rules that can be applied a priori to assist in the selection of the one most promising representative of any group, and no simple procedure for screening out likely candidates. The best pathogen is not necessarily the one that kills the most quickly or the one most easily propagated. ‘There is the ever present danger of spreading our talents too thinly, spending our time always looking for the new, rather than taking a new and better look at the old. The wide variety of pathogenic microorganisms worthy of consideration and the enormous number of insect species in which they might be sought can easily prevent investigation of the depth necessary to make a real and lasting contribution to the science of microbiology.

REFERENCES Allen, H. W. (1954). Ann. Entomol. 80c. Am. 47, 407424. Angus, T.A. (1954).Nature 173, 545. Balch, R.E.,and Bird, F. T. (1944). Sci. Agr. 26, 65-80. Bergold, G. H.(1943). Biol. Zentr. 63, 1-66. Bergold, G. H. (1950).Can. J . Research E28,5-11. Bergold, G . H. (1958). I n “Handbuch der Virusforschung” (C. Hallauer and K. F. Meyer, eds.), Vol. 4,pp. 60-142. Springer, Vienna. Berliner, E. (1915). Z.angew. Entomol. 2 , 29-56. Biliotti, E.,Grison, P., and Martouret, D. (1956).Entomophaga 1,3644. Bird, F.T. (1950). Can. Dept. Agr., Div. Entomol. Bimonthly Progr. Rept. 6(5), 2-3. Bird, F.T.(1963). Can. Entomologist 86, 437446. Bird, F.T. (1955). Can. Entomologist 87, 124-127. Bird, F.T.(1957). Virology 3, 237-242. Bird, F.T. (1958). Can. J . Microbial. 4, 267-272. Chamberlin, F.S.,and Dutky, S. R. (1968).J . Econ. Entomol. 61,341.

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McEwen, F. L., and Hervey, G. E. R. (1958). J. Econ. Entomot. 61,628-631. Marzke, F . O., and Dicke, R. J. (1958). J . Econ. Entomol. 61, 916-917. Metchnikoff, E. (1880). 2001.Anz. 3, 44-47. Mtiller-Kogler, E. (1958). Naturwissenschuften 46, 248-250. Niklas, 0. F. (1956). Z. Pjlanzenkrankh. u. Pjlanzenschutz 63, 81-95. Niklas, 0. F. (1958). 2. angew. 2001.46, 103-116. Philip, C. B. (1956). Can. J. Microbiol. 2, 261-270. Philip, C. B. (1957). I n “Bergey’s Manual o f Determinative Bacteriology’’ (R. S. Breed, ed.), p. 957. Williams & Wilkins, Baltimore, Maryland. Am. 49.86-93. Richards, A. G., and Smith, M. N. (1956). Ann. Entomol. SOC. Sabrosky, C. W. (1952). Yearbook Agr. ( U . 8. Dept. Agr.) 1962, 1-7. Schuder, D. L. (1957). Proc. Indiana Acad. Sci. 66, 101-102. Smith, K. M., and Xeros, N. (1953). Parasitology 43, 178-185. Steiner, G. (1929). J. Wash. Acad. Sci. 19, 436-440. Steinhaus, E. A. (1947a). “Insect Microbiology,’’ pp. 462-554. Cornell Univ. Press, Ithaca, New York. Steinhaus, E. A. (1947b). J. Parasitol. 33, 29-32. Steinhaus, E. A. (1949). “Principles of Insect Pathology,’) pp. 546-632. MoGraw-Hill, New York. Steinhaus, E. A. (1957). Mimeo. Ser. (Lab.Insect Pathol.) Univ. Calif. No. 4. Tanada, Y. (1953). Proc. Hawaiian Entomol. SOC.16, 167-175. Tanada, Y. (1954). Ann. Entomol. Soc. Am. 47, 553-574. Tanada, Y. (1956). J. Econ. Entomol. 49, 320-329. Thompson, C. G., and Steinhaus, E. A. (1950). Hilgardia 19, 411-445. Thomson, H. M. (1955). J. Parasitol. 41, 1-8. Thomson, H. M. (1958). Can. J. Zool. 36, 499-511. Wallis, R C. (1957). J . Econ. Entomol. 60, 580-583. Wasser, H. B. (1952). J. Bacteriol. 64, 787-792. Weiser, J. (1955a). &skoslov. Parasitol. 2, 181-184. Weiser, J. (l955b). V8stnl.k CeskosEov. zool. spol. 19, 6 5 2 . Weiser, J. (1956). Z. Pjlanzenkrankh. u. Pjlanzenschutz 63, 625-688. Weiser, J. (1957a). (?eskoslov. Parasitol. 4, 365-358. Weiser, J. (1957b). Z. angew. Entomol. 41, 243-245. Weiser, J. (1957~).2. angew. Entomol. 40, 509-527. Weiser, J. (1958). VlistnZk Ceskoslov. zool. spol. 22, 3@12. Weiser, J., and Koehler, W. (1955). &skoslov. Parasitol. 2, 185-190. Weiser, J., and Veber, J. (1957). Z . angew. Entomol. 40, 55-70. White, G. F. (1913). U . S. Dept. Agr., Bur. Entomol. Circ. 16% 1-5. White, G. F. (1919). U . S. Dept. Agr. Bull. 780,59 pp. White, R. T., and Dutky, S. R. (1940). J . Econ. Entomo2.33, 306-309. White, R. T., and Dutky, S. R. (1942). J. Econ. Entomol. 36, 679-682. White, R. T., and McCabe, P. J. (1951). U . S. Dep. Agr. Bur. Entomol. Plant Quarant.

E-816. Wille, H., and Martignoni, M. E. (1952). Schweiz. 2. allgem. Pathol. u. Bakteriol. 16, 470-473.

The Production of Amino Acids by Fermentat ion Processes SHUKUOKINOSHITA Tokyo Research LabOrUtOTy, Kyowa Fermentation Industry Co., Ltd., Tokyo, Japan

I. Introduction.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Possibility of Amino Acid Production by Various Microorganisms. . . . . . . . 111. Glutamic Acid Fermentation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Enzymatic Resolution of DL-Glutamic A cid.. . . . . . . . . . . . . . . . . . . . . . . . . . B. Biochemical Synthesis of L-Glutamic Acid. ........................... C. Fermentative Production of L-Glutamic Acid ......................... IV. Lysine Fermentation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Ornithine Fermentation. . . ........................................ VI. Miscellaneous Amino Acid ntations. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..... References . . . . . . . . . . . . . . . . . .

201 202 203 203 204 205 208 210 211 212

1. Introduction It can undoubtedly be said that microbial production of amino acids has a definite advantage over chemical synthesis because the amino acids produced by the former are, exclusively, the biologically active b-form. Until today, numerous investigations have been reported on the presence of various nitrogenous substances in microbial culture broths. The existence of amino acids in those broths has, naturally, been established. In spite of such historical evidence, amino acid production by the microbial method which will be here discussed is an entirely new field. If a given amino acid could be produced in substantial amounts, in a medium containing sugars and simple nitrogen compounds, by a fermentative procedure, the fermentation might well be called an “Amino Acid Fermentation.’’ The author and his collaborators have been engaged in a survey of amino acid fermentations, and have obtained successful results in several of them. These are glutamic acid, lysine, ornithine, valine, and alanine. Of these, glutamic acid and lysine fermentations have .already been operated on a commercial scale. Intense research activities are being continued on other amino acids, especially the essential amino acids. From all the data obtained so far, it is quite safe to say that the success of the commercial production of various amino acids is merely a matter of time. Today, amino acid fermentation is a rapidly expanding branch of industrial fermentations. 201

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11. Possibility of Amino Acid Production by Various Microorganisms Microbial excretion of nitrogenous substances was first recognized in the last century by Thenard, Pasteur, DucIaux, and others in their studies on yeast. From the beginning of this century, studies on this problem by many investigators yielded further details and were accelerated a great deal by the advancement of paper chromatographic and other biochemical techniques. Reindel and Hoppe (1952) studied yeasts, and Dagley et al. (1950) investigated some bacteria such as Escherichia coli and Aerobacter aerogenes. More recently, Morton and ‘Broadbent (1955) studied the same problem using many fungi. Several species of Streptomyces were also studied by Corum et al. (1954) and Perlman and O’Brien (1958). These investigators found some organic nitrogenous substances in culture media and identified them as free amino acids or peptides by the ninhydrin test or paper chromatography. Unfortunately, since the ultimate goal of these studies was not fermentation on an industrial scale, very few data are available on the quantitative amounts of amino acids produced. Since 1955, qualitative and quantitative studies on amino acid formation by various microorganisms were undertaken by the author in order to examine the possibilities of amino acid fermentation. Several thousands of microorganisms including known type-cultures and newly isolated strains were tested, and a part of this experimental data was published (Kinoshita et al., 1957a). According to the results of these experiments, almost all microorganisms tested were found to be capable of producing some amino acids in their culture broths, although the amount of each amino acid produced was usually so small as to be detected only by paper chromatography. I n general, several kinds of amino acids were present together in the broth, and only in rare cases was a specific amino acid accumulated in a substantial amount. A n excellent glutamic acid producer, Micrococcus glutamicus, which was so named by the author (Kinoshita et al., 1958b) was one of the outstanding ones. It seemed to be of interest whether some relationship might exist between taxonomical classification of those microorganisms such as yeast, fungi, bacteria, and streptomyces and the kind of amino acids produced by each of them. However, up to the present, no data have been obtained which show a positive relationship between the amino acid production pattern and the morphological classification of microorganisms. Generally speaking, there are many kinds of microorganisms which are able to produce the naturally occurring amino acids in an amount from 1 to 2 mg. per milliliter in the culture broth. The most common amino acids which are apt to be found in culture broths are glutamic and aspartic acids, alanine, glycine, serine, valine, and leucine.

AMINO ACID PRODUCTION BY FERMENTATION

203

111. Glutamic Acid Fermentation Since monosodium glutamate (MSG) is in large commercial demand in the food industry as a flavor-enriching chemical agent, more attention has been paid to this amino acid than to others. The proteins of the plant kingdom such as wheat gluten and soybean meal, and also Steffen’s waste (beet sugar molasses) have been the chief raw materials for the production of glutamic acid. Since the demand for MSG in the world is increasing 10 to 15% every year, new methods for MSG production are being sought for the coming years. Especially in Japan and the United States, the largest producers of MSG, intense studies on this problem have been undertaken. Two approaches have been proposed-the chemical synthetic, and biochemical procedures. In the chemical synthesis, the product is always a racemic form, i.e., DL-form. Therefore, its resolution to two optically active isomers must follow. For this purpose, enzymatic resolution of DL-glutamic acid has been studied. On the other hand, the biochemical synthesis of L-glutamic acid, starting from organic acids such as a-ketoglutarate or citrate, using enzyme preparations obtained from various organisms has been developed. But these methods have not been so fully completed that they meet the demands of commercial application. Besides the processes described above, direct fermentative methods starting from carbohydrates and ammonium ions have been studied. A. ENZYMATIC RESOLUTION OF DL-GLUTAMIC ACID In order to obtain the L-isomer from nL-glutamic acid, enzymatic resolution of racemic amino acids seems to be most promising. Various possibilities of DL-amino acid resolution were reviewed by Greenstein (1954), and extensive studies using hog renal acylase were carried out in his laboratory. More recently, they also reported successful resolution using amino acid oxidases (Parikh et al., 1958). Several contributions by Japanese investigators have been made in this field. Michi and Nonaka (1954) studied the application of fungal acylases to the preparation of L-glutamic acid from N-acyl-m-glutamic acid. Workers in the laboratory of Sakaguchi (Izaki et al., 1955; and Sakaguchi et al., 1957) showed that D-glutamic acid oxidase of both bacterial and fungal origin could be used effectively for the resolution of DL-glutamic acid. In this process, D-glutamic acid was oxidized to a-ketoglutaric acid and ammonia, and L-glutamic acid remained intact.

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B. BIOCHEMICAL SYNTHEBIB OF L-GLUTAMIC ACID There are two known chemical pathways for L-glutamic acid formation in living organisms, i.e., transamination by various transaminases and reductive amination by glutamic acid dehydrogenase as shown in Eqs. (1) and (2) : Transamination: a-ketoglutaric acid

+ amino acid .transsminaaeb L-glutamic acid + a-keto acid

(1)

Reductive amination: a-ketoglutaric acid

+ NHiC + TPNH (or DPNH) L-glutamic acid

L-glutamio acid dehydrogenaee

,

+ Ha0 + TPN+ (or DPNf)

(2)

In either case, a-ketoglutaric acid is the common precursor of L-glutamic acid. In fact, when bacterial cells were incubated in synthetic media, it was found that such organisms as Clostridium (Nisman et al., 1947), A . aerogenes (Fowler and Werkman, 1952, 1955), E. coli (Adler et al., 1938; Konikova et al., 1949) and Bacillus subtilis (Konikova et al., 1948) produced a small amount of L-glutamic acid in media containing both a-ketoglutarate or pyruvate and ammonium salts. It was also recognized that E . coli, B . subtilis, Pseudomonas $uorescens (Feldman and Gunsalus, 1950)) Bacillus anthracis (Housewright and Thorne, 1950), and many other bacteria were able to produce some L-glutamic acid in the presence of both a-ketoglutarate and amino acids as amino group donors. For the enzymatic preparation of L-glutamic acid from a-ketoglutaric acid by transamination, Sakurai and Akabori (1956, 1957) and Kato et al. (1957) used bacterial cells of E. coli. In their processes, L-aspartic acid in protein hydrolyzates was employed as the amino group donor. Masuo and Wakizaka (1955) also reported a similar process, in which L-alanine was directly added to the culture broth of a-ketoglutaric acid fermentations after the fermentation was over. Then the transamination reaction between a-ketoglutaric acid and L-alanine was effected by the enzyme of the same organism already present in the broth. The mechanism of L-glutamic acid formation by reductive amination with L-glutamic acid dehydrogenase is more complicated than that by transamination, because, for this reaction to proceed, at least one more dehydrogenase system which will supply the reduced form of coenzyme (TPNH or DPNH) and a hydrogen-donating substrate are needed. Since the coenzyme (TPN or DPN) has multiple relations with other oxidationreduction systems in living organisms, the equilibrium of the L-glutamic acid dehydrogenase reaction to form L-glutamate is apt to be influenced by a variety of other enzyme activities.

AMINO ACID PRODUCTION BY FERMENTATION

205

Otsuka et al. (1957a, b, c) observed L-glutamic acid formation when Pseudomonas ovalis was incubated in a medium containing a-ketoglutarate and ammonium salts, with or without a small amount of glucose. From this result, they suggested that two pathways might exist for L-glutamate formation. One is the reductive amination of a-ketoglutarate and the other is a pathway as shown in Eq. (3):

-

a-ketoglutarate a-ketoglutarate

TCA cycle

+ L-aspartate

b

fumarate

aspartam

L-aspartate

oxdoacetate

+ L-glutamate

(3)

One more possibility may still exist, since Krebs and Cohen (1939) reported that a-ketoglutarate has two roles, one as the precursor of glutamate formation and the other as a hydrogen donor for the reaction. Smythe and Huang (1956a, b) and Huang and Smythe (1957) have also reported processes for glutamate synthesis using various animal tissues, and bacterial and yeast cells. Organic acids such as citric and malic acids, and glucose-6-phosphate were used as hydrogen donors. When citrate was used as a hydrogen donor, L-glutamate was formed without adding a-ketoglutarate. This fact suggests that some reaction or reactions in the oxidation process of citrate are closely coupled with the reductive amination of a-ketoglutarate to glutamate, as clearly verified by Kinoshita et al. (1957~). Enzymatic preparation of L-glutamic acid from a-ketoglutaric acid or citric acid may have some practical importance in industry, because these two acids are available by established fermentation techniques (Lockwood and Stodola, 1948; Koepsell et al., 1955; Sharpe and Cormen, 1957; Masuo and Wakizaka, 1957; and Katagiri et al., 1958).

C. FERMENTATIVE PRODUCTION OF L-GLUTAMIC ACID The biochemical method for the preparation of L-glutamic acid described above consists of at least two steps. Therefore, it is more desirable that glutamic acid be directly produced and accumulated in the culture medium in amounts having industrial meaning. As already mentioned, a number of reports on amino acids in culture broths have revealed that the quantities of amino acids are too small to have some meaning for industry. Such was the case with glutamic acid. The fact that the extracellular accumulation of glutamic acid is generally so small can be accounted for by the following reasons: First, owing to the important function of glutamic acid in nitrogen metabolism, it may be reused for making other amino acids or utilized as a building block for proteins and other cell materials even though it is once produced by organisms. Second, in order to produce glutamic acid by one-step fermentation, two contradictory conditions must be satisfied, i.e., the aerobic oxidation of sugar and the anaerobic reductive amination of a-ketoglutarate which is

206

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obviously the most efficientpath for glutamate synthesis. In fact, Morrison and Hinshelwood (1949) and Dagley et al. (1950) observe that glutamic acid formation in the culture broth of E . coli and A . aerogenes was decreased to a large extent by aerobic conditions. Recently, Kita (1957), in a patent, reported glutamic acid fermentation using Cephalosporium. According to the patent, glutamic acid in the broth is claimed to reach an amount of 2 to 3 gm. per liter. But there are some questions which may be raised concerning this procedure, because the media used are mainly proteinous substances such as soybean meal and distiller’s solubles. Therefore, whether glutamic acid is really synthesized or is merely a degradation product from proteins in the medium is not clear. Asai et al. (1957a, b) observed that a strain of Micrococcus varians and Bacillus megatherium accumulated a few grams of glutamic acid in 1 liter of the culture medium. As these experiments were carried out using chemically defined and protein-free media, it is quite clear that the glutamic acid is a product of biosynthesis from glucose and ammonium ions. In the author’s laboratory, screening tests for finding powerful glutamate producers have been carried out (Kinoshita et al., 1957a). Numerous organisms from various origins were tested, and finally several strains with exceedingly high productivity of L-glutamic acid were obtained. These strains are gram-positive, nonsporulating, catalase-positive, and biotinrequiring cocci. Although small variations in colony-forms on agar plates, color, cell size, and physiological properties are found among these strains, most cells on bouillon agar are coccal to oval in form and very close to the genus Micrococcus. Therefore, a name, Micrococcus glutamicus, was proposed for them (Kinoshita et d . , 1958b). (The cells of these strains sometimes take an elongated form. It is interesting to note that these cells contain metachromatic granules, and septa are demonstrable in the cells with cell wall staining. The cells also contain diaminopimelic acid. These characteristics are known to be common in Corynebacterium or Brevibacterium. A more complete discussion on this problem will appear at some future time.) M . glutamicus can easily produce more than 30 gm. per liter of glutamic acid in the medium under aerobic conditions (Kinoshita et al., 195713). The main ingredients in the medium are glucose and urea or ammonium salts. This yield is high enough to compete with present glutamic acid production costs. The fermentation mechanism of such an abnormal accumulation of glutamic acid is a very interesting problem from both academic and industrial standpoints. A part of the studies concerning this problem has already been reported at the International Symposium on Enzyme Chemistry in 1957 (Kinoshita et al., 1957c) and in other papers (Kinoshita et al., 1958a; Kinoshita, 1958).

207

AMINO ACID PRODUCTION BY FERMENTATION

A brief summary of the results and some additional information will be described below. Glucose is oxidized to form citrate and the citrate is further oxidized to a-ketoglutarate by the TCA cycle. Along the oxidation path from citrate to a-ketoglutarate, two TPN-specific dehydrogenases of this organism, i.e., isocitric and L-glutamic dehydrogenases, are very closely coupled in the presence of ammonium ions. This reaction is schematically shown in Eq. (4). isocitrate

J

(isocitric dehydrogenase) +

a-ketoglutarate

r

1

TPN +J --+ L-glutamate (L-glutamic

+ TPNH

dehydrogenase) (4)

a-ketoglutarate

+ N&+

In M . glutamicus, the oxidized form of TPN which is required for oxidation of isocitrate is repeatedly regenerated by the reductive amination reaction of a-ketoglutarate. Since the glutamic acid dehydrogenase activity of M . glutamicus is so strong compared with that of other microorganisms, the oxidation intermediate of citrate by the TCA cycle is trapped in the form of glutamate. It has also been demonstrated that this organism has both the EmbdenMeyerhof scheme and the hexose-monophosphateshunt as oxidation pathways for glucose. With an appropriate supply of air, the hexose-monophosphate shunt (HMS) dominates over the Embden-Meyerhof scheme and this favors the glutamate formation. Conversely, the Embden-Meyerhof scheme dominates under anaerobic conditions, and in this case lactic acid is accumulated in the medium instead of glutamate. One more important physiological characteristic of this organism is that this organism is a biotin-requiring auxotroph. Although biotin is known as a vitamin of multiple functions, it should be noted that, in the case of M . glutamicus, this vitamin is not merely a growth factor but has a special function on this fermentation. From all the cultural and enzymatic experimental evidence, the vitamin has been revealed to be a key substance which decides the metabolic pattern of M . glutamicus. It may also be safe to say that, with an appropriate supply of biotin, citrate formation proceeds via the path shown in Eq. ( 5 ) .

>+

co;

aoetyl-CoA

pyruvate,

L-malate -+ oxaloacetate

L

citrate

(5)

On the contrary, an excess amount of biotin causes lactic acid formation from pyruvate and subsequent oxidation of oxidized products of lactate. This obviously will decrease the glutamate yield.

208

SHUKUO KINOSHITA

For the complete understanding of the whole mechanism, further studies must be done. However, the most probable scheme for that is shown in Fig. 1.

L- GLUTAM ATE

(DCA CYCLE)

L P o t h in Optimum Biotin A--Path

in

EXCESS

Biotin

FIG.1. Possible pathways of glucose oxidation by Micrococcus glutamicus.

The large accumulation of L-glutamic acid in M . glutamicus culture broth can be effected by the strong L-glutamic acid dehydrogenase activity on one hand, and by the subtle physiological functions of biotin on the other, It is very interesting to note that an incomplete oxidation of glucose is effected under aerobic conditions and results in the subsequent glutamate formation.

IV. Lysine Fermentation The possibility of the microbial production of lysine has been rapidly realized by the findings that lysine is synthesized from a-aminoadipic acid by yeast and Neurospora (Mitchell and Houlaham, 1948; Windsor, 1951) or from diaminopimelic acid (DAP) by E. coli (Dewey and Work, 1952; Davis, 1952). Patents describing processes in which E. coli was used have been issued in the United States (Casida, 1956; Kita and Huang, 1958). According to the methods described in these patents, DAP, once produced, is converted to lysine by DAP decarboxylase under anaerobic conditions.

AMINO ACID PRODUCTION BY FERMENTATION

209

On the other hand, Richards and Haskins (1957) reported on the extracellular lysine formation of some 600 strains of fungi. They found that Ustilago maydis and a strain of Glioclarlium had active lysine productivity. Detailed studies of those strains were done by Dulaney et al. (1956) and Dulaney (1957). They studied the effect of various cultural conditions on lysine production and obtained an average lysine content of the broth in the amount of 400 pg. per milliliter, and ocasionally 700 to 800 pg. per milliliter. In Japan, lysine production has been a topic of great interest, and two directions of study have been followed. Strong activities of diaminopimelic decarboxylase were found in A . aerogenes, Proteus vulgaris, and B. subtilis by Aida and Fujii (1958). Sagisaka and Shimura (1957) reported the enzymatic formation of lysine from a-aminoadipic acid using Torula utilis. Aida and Uemura (1958) obtained 2.5 mg. per milliliter of lysine using a strain of B. subtilis isolated from Japanese sake. They reported that fish solubles was a favorable raw material for lysine production. In the author's laboratory (Kinoshita e l al., 1958c), a number of biochemical mutants of M . glutamicus were obtained using ultraviolet or Cosa irradiation. Among them, five strains had outstanding lysine productivity. Two of these were good enough for the commercial production of lysine. These two strains require biotin and homoserine for their growth. The requirement of homoserine can be replaced by a simultaneous supply of methionine and threonine. The other three mutants require methionine, methionine or homocysteine, and both leucine and isoleucine, respectively, for their growth. The fact that only these amino acid auxotrophs can accumulate lysine is worthy of note, because this fact may be another confirmation of the present knowledge of the biosynthetic pathway of lysine which is schematically described in Eq. (6).

glucose -+ X

t

3

aspartic homoserine + threonine -+ isoleucine

NHa+

I I homocysteine I met hionine

(6)

cysteine --+ cystathione

11

cystine

The lysine productivity of amino-acid-requiring auxotrophs of E . coli and B. sublilis was also tested. In the case of strains of E. coli, lysine productivity was very low, but auxotrophs of B . subtilis accumulated significant amounts of lysine.

210

BHUKUO KINOSHITA

Figure 2 shows a typical lysine fermentation graph. It is not unusual to obtain a fermented broth containing 2.0% (w/v) of lysine. The detailed mechanisms of lysine synthesis in the homoserine requiring mutants of M . glutamicus is not quite clear, but diaminopimelic acid is supposed to be the possible precursor since diaminopimelic acid and DAP decarboxylase activity can be detected in the cells. Various organic acids such as lactate can also be the substrate for lysine production.

5.0 I-

h

-0

24

48 Time

72

go

(hours)

Frci. 2. Chemical change in lysine fermentation (Kinoshita et al., 1958~). Microorganism: Micrococcus glutamicus 613-1 (homoserine-less). Medium composition (per cent) : glucose, 5.0; N H P l , 1.0; NZ-amine, 0.4; KH2PO4, 0.05; K2HP04,0.05; MgS04.7Hz0, 0.025; FeSOc. 7H20, 0.001; MnSOr.4H20, 0.001; Yeast extract, 0.1; CaCOS,0.5. Temperature : 28°C.

V. Ornithine Fermentation L-Ornithine has been known as an amino acid which is a member of the urea cycle or, phrased differently, as an intermediate for the biosynthesis of L-arginine. But it has not been known as a component of natural proteins. The natural occurrence of ornithme is either in its free state and in

AMINO ACID PRODUCTION BY FERMENTATION

211

its simple derivatives or in a polypeptide such as gramicidin S (Meister, 1957). The method of preparation of L-ornithine has been one which utilizes the action of arginase on L-arginine. Owing to the very limited supply of this amino acid, knowledge of its biochemical function is still so scanty that its usage is rather limited to that of a biochemical reagent or a possible prophylactic to ammonium toxicity in animals as reported by some investigators (Greenstein et al., 1955; Bronk and Fisher, 1956). Recently, it was found that some mutants of M . glutamicus were able to produce about 25 gm. per liter of L-ornithine in culture broths (Kinoshita et al., 1957d). These mutants are amino acid auxotrophs which require arginine or citrulline for their growth. The molar yield of ornithine was about 0.36 mole from one mole of glucose. From the recent report by Udaka and Kinoshita (1958a), the biosynthetic pathway of L-ornithine in those organisms has become clear as shown in gl ucoae

1

glutamic acid------(

I

I1

(Transacetylation)

acetylglutamic acid+

I

I+ L-ornithine (7)

a-N-acetylornithine

T

I acetylglutamic acid+ 1r-semialdehyde

Eq. (7). This scheme is very similar to that present in E. coli except that in this scheme the acetyl radical of a-N-acetylornithine is removed by transacetylation instead of hydrolysis as is known in E. coli. In the normal L-ornithine fermentation using these mutants, L-ornithine is a main product, but it is interesting to note that by the addition of an excess amount of arginine, glutamic acid accumulation takes place. This phenomenon is interpreted as arginine inhibition of the phosphorylation reaction of N-acetylglutamic acid (Udaka and Kinoshita, 1958b). This specific inhibition may be a kind of metabolic regulation mechanism known as “feed back” phenomenon.

VI. Miscellaneous Amino Acid Fermentations In addition to the production of the amino acids described in the previous sections, the production, by fermentation, of other amino acids should be developed shortly. For example, the fermentative production of L-valine has been reported by Udaka el al. (1958). Among Enterobacteriaceae such as Paracolobactrum coliforme, E. coli, and A . aerogenes, good producers of L-valine were found. Under some conditions, L-valine in an amount of 7 to 8 mg. per milliliter was obtained.

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SHUKUO KINOSHITA

Fermentative production of L-alanine with Pseudomonas and Streptocolicolor was also studied by Yazaki et al. (1958). Tryptophan, an essential amino acid, was recently reported to be under pilot plant scale production at Eli Lilly and Company (Beesch and Tanner, 1958).

myces

ACKNOWLEDGMENT The author wishes to express his sincere thanks for the kind advice and criticism of Dr. S. S. Rennert, Takamine Laboratory, Clifton, New Jersey.

REFERENCES Adler, E., Hellstrom, V., Gunther, G., and Euler, H. von (1938). 2. physiol. Chem., Hoppe-SeyEer’s 226, 14-26. Aida, H., and Fujii, M. (1968). Oral presentation a t Ann. Meeting Agr. Chem. Sac. Japan, Kyoto, 1958. See summary i n “Symposium on Amino Acid Fermentation, 1958,” pp. 94-96. Agr. Chem. SOC.Japan, Kyoto. Aida, T., and Uemura, S. (1958). Oral presentation a t Ann. Meeting Agr. Chem. SOC. Japan, Kyoto, 1958. See summary i n “Symposium on Amino Acid Fermentation, 1958,” pp. 94-95. Agr. Chem. SOC.Japan, Kyoto. Asai, T., Aida, K., and Otsuka, K. (1957a). Bull. Agr. Chem. SOC.Japan 21, 134-135. Asai, T., Aida, K., and Otsuka, K. (1957b). Hakk8-Kydkaishi 16.371-379. Beesch, 8.C., and Tanner, F. W., Jr. (1958). Ind. Eng. Chem. 60,1341. Bronk, J. R., and Fisher, R. B. (1956). Biochem. J. 64, 106-111. Casida, L. E., Jr. (1956). U. S. Patent 2,771,396. Corum, C. J . , Stark, W. M., Wild, G. M., and Bird, H. L., Jr. (1954). Appl. Microbiol. 2, 326-329. Dagley, S., Dawes, E. A., and Morrison, G. A. (1950). Nature 166,437-438. Davis, B. D. (1952). Nature 169,534-536. Dewey, D. L., and Work, E. (1952). Nature 169,533-534. Dulaney, E. L. (1957). Can. J. Microbiol. 3,467-476. Dulaney, E. L., Bilinski, E., and McConnell, W. B. (1956). Can. J . Biochem. and Physiol. 34, 1195-1198. Feldman, L. I., and Gunsalus, I. C. (1950). J. Biol. Chem. 187,821-830. Fowler, E. B., and Werkman, C. H. (1952). Arch. Biochem. Biophys. 4 1 , 4 2 4 7 . Fowler, E. B., and Werkman, C. H. (1956). Arch. Biochem. Biophys. 66, 22-27. Greenstein, J. P. (1954). Advances i n Protein Chem. 9, 121-202. Greenstein, J. P., Winita, M., Gullino, P., and Birnbaum, S. M. (1955). Arch. BZochem. Biophys. 69, 302-303. Housewright, R. D., and Thorne, C. B. (1950). J. Bacteriol. 60, 89-100. Huang, H. T., and Smythe, C. V. (1957). U. 5. Patent 2,798,839. Izaki, K., Takahashi, H., and Sakaguchi, K. (1955). Bull. Agr. Chem. Soc. Japan 28, 343-346. Katagiri, H., Tochikura, T., and Imai, K. (1958). Japanese Patent Sh8 33-7694. Kato, J., Wakamatsu, H., Kaneo, S., and Hori, S. (1957). Japanese Patent ShB 325707. Kinoshita, S. (1958). Protein, Nucleic Acid, Enzyme (Tokyo) 3 , 247-256. Kinoshita, S., Udaka, S., and Shimono, M. (1957a). J . Gen. Appl. Microbiol. (Tokyo) 3, 193-205.

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Kinoshita, S., Udaka, S., and Akita, S. (1957b). Japanese Patents Sh6 32-8698. Kinoshita, S., Tanaka, K., Udaka, S., and Akita, S. (1957~).Proc. Intern. Symposium Enzyme Chem. pp. 464-468. Kinoshita, S., Nakayama, K., and Udaka, S. (1957d). J. Gen. Appl. Microbiol. (Tokyo) 3, 276-277. Kinoshita, S., Tanaka, K., Udaka, S., Akita, S., Saito, T., and Iwazaki, T. (1958a). Hakk6 KyBkaishi l6,l-11. Kinoshita, S., Nakayama, K., and Akita, S. (1958b). BUZZ. Agr. Chem. SOC.Japan 22, 176-185. Kinoshita, S., Nakayama, K., and Kitada, S. (195%). J . Gen. Appl. Microbiol. (Tokyo) 4, 128-129. Kita, D. A. (1957). U. S. Patent 2,789,939. Kita, D. A., and Huang, H. T. (1958). U. S. Patent 2,841,532. Koepsell, H. J., Stodola, F. H., and Sharpe, E. 5. (1955). U. 8. Patent 2,724,680. Konikova, A. S., Kritsman, M. G., and Yakobson, L. M. (1948). Biokhimiya 13.3941. Konikova, A. S., Kritsman, M. G., Yakobson, L. M., and Samarina, 0. P. (1949). Biokhimiya 14, 223-229. Krebs, H. A., and Cohen, P. P. (1939). Biochem. J.33,1895-1899. Lockwood, L. B., and Stodola, F. H. (1948). U. S. Patent 2,443,919. Masuo, E., and Wakizaka, Y. (1955). I n Report presented at the annual meeting of Agr. Chem. SOC.Japan. (Tokyo, May). Masuo, E., and Wakizaka, Y. (1957). Japanese Patent Sh8 32-1099. Meister, A. (1957). “Biochemistry of the Amino Acids,” p. 37. Academic Press, New York. Michi, K., and Nonaka, H. (1954). Nippon Nbgei-kagaku Kaishi 28,343-346. Mitchell, H. K., and Houlaham, M. B. (1948). J. Biol. Chem. 174, 883-887. Morrsion, G. A,, and Hinshelwood, C. N. (1949). J . Chem. Soc. 380-384. Morton, A. G., and Broadbent, D. (1955). J. Gen. Microbiol. 12.248-258. Nisman, B., Raynaud, M., and Cohen, G. N. (1947). Compt. rend. 226, 700-701. Otsuka, S., Yazaki, H., Nagase, H., and Sakaguchi, K. (1957a). Bull. Agr. Chem. SOC. Japan 21, 69-70. Otsuka, S., Yazaki, H., Nagase, H., and Sakaguchi, K. (1957b). J. Gen. Appl. Microbiol. (Tokyo), 3, 35-53. Otsuka, S., Yazaki, H., Nagase, H., and Sakaguchi, K. (1957~).Hakkb-Kybkaishi 16, 54-62. Parikh, J. R., Greenstein, J. P., Winitz, M., and Birnbaum, 8. M. (1958).J . Am. Chem. SOC.80, 953-958. Perlman, D., and O’Brien, E. (1958). J . Bacteriol. 76, 611. Reindel, F., and Hoppe, W. (1952). Chem. Ber. 86,716-731. Richards, M., and Haskins, R. H. (1957). Can. J. Microbiol. 3, 543-546. Sagisaka, K., and Shimura, K. (1957). Nippon Nogei-kagaku Kaishi 31, 110-114. Sakaguchi, K., Takahashi, H., Izaki, K., and Mizushima, S. (1957). Japanese Patent Sh6 32-8173. Sakurai, S., and Akabori, S. (1956). Japanese Patent Sh8 31-3768. Sakurai, S., and Akabori, S. (1957). Japanese Patent Sh8 32-6787. Sharpe, E. S., and Corman, J. (1957). U. 8. Patent 2,724,680. Smythe, C. V., and Huang, H. T. (1956a). U. S. Patent 2,749,279. Smythe, C. V., and Huang, H. T. (1956b). U. 8. Patent 2,773,001. Udaka, S., and Kinoshita, S. (1558a).J . Gen. Appl. Microbiol. (Tokyo) 4, 272-282.

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Udaka, S., and Kinoshita, S. (1558b).1.Gen.AppE. Microbial. (Tokyo) 4, 283-288. Udaka, S., Tomizawa, K., and Kinoshita, S. (1958). Oral presentation at Monthly Meeting Agr. Chem. SOC. Japan, Tokyo, October, 1958. Windsor, E. (1951). J. Biol. Chem. 192, 607-609. Yazaki, H., Otsuka, S., and Takahashi, M. (1958). Oral presentation at Ann. Meeting Agr. Chem. SOC. Japan, Kyoto, 1958. See summary in "Symposium on Amino Acid Fermentation, 1958," pp. 94-95. Agr. Chem. SOC.Japan, Kyoto.

Cont inu ou s Ind ust ria I Fermentationr PHILIPP GERHARDT AND M. C. BARTLETT' Department of Bacteriology, The University of Michigan, Ann Arbor, Michigan

I. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Classification of Continuous Operations and Proceases . . . . . . . . . . A. Operation and Feed Types. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Process Types. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 219 111. Summary, Predictions, and Experimental Tests of Continuous Fermentation Theory, . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 219 A. Standardization of Mathematical Symbols B. Development of the General Equation.. . . C. Stability of Continuous Fermentation. ... D. Extensions of the General Equation. . . . . . E. Effect of Undesirable Organisms.. . . . . . . . . F. The Oscillation Phenomenon. . . . . . . . . . . . . G. Prediction of Continuous Performance fro

.................... .................... C. Mixed-Culture Fermentations.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ......

237 241 252 255

1. introduction Advances in the field of continuous culture and fermentation have been significant during the past decade, amounting almost to a renaissance. The basis for this may possibly be traced to papers on the theory of continuous culture by Monod (105), Novick and Szilard (114, 115), and others (see Table I), The pioneer work of Utenkov in the Soviet Union and MAlek in Czechoslovakia (23,97) apparently has considerably influenced the investigations in these countries. To a large extent, the upsurge of experimental work in the field has been concentrated in refinements and popularization of special laboratory devices and in theoretical problems of bacterial genetics, physiology, and growth. The extent of these advances and concomitant ones in industrial fermentations may be judged from recent reviews (100, 112), symposia (23, 24, 145), and the extensive bibliographies compiled by Bartlett (10) and kiEica (132). The fact that three major symposia occurred Present address : Abbott Laboratories, North Chicago, Illinois.

215

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PHILIPP GERHARDT AND M. C. BARTLETT

during the year 1958 perhaps best attests to the current popularity and research activity in continuous culture and fermentation. Even before the advent of microbiology as a science, however, the ancient art of fermentation had progressed to continuous production of vinegar in 1670 (102), to yeast manufacture in 1879 (131),and to sewage disposal in 1890 (99). During subsequent years and still today, the industria1 use of continuous fermentation has been restricted almost entirely to such processes, all of which yield bulk products and require relatively crude control. Fermentation science received its greatest impetus with the advent of the antibiotics, and an increasing number and variety of industrial fermentations have emerged which, like antibiotic fermentations, yield fine chemicals and require close biological and mechanical control. It is to these types that questions of future application of continuous fermentation must also be directed. Nonetheless, actual industrial use of continuous fermentations today is restricted not only in the type of process but also in the number of working installations. The Soviet Union apparently leads in this latter regard, chiefly in the production of alcohol and yeast from wood hydrolyzate and sulfite waste liquor (20a). There are several such plants in this country and Canada. Besides sewage and vinegar, other processes in active continuous production are few; for example, in Czechoslovakia, sulfur reportedly (20a) is produced from yeast wastes on a fairly large scale by a continuous process using sulfate-reducing bacteria. But as Herbert (65) has observed, “continuous culture is still a subject that is more talked about than practised . . .” The potential advantages of continuous industrial fermentations are widely known and have been amply discussed elsewhere (10, 43, 73, 100, 142). Donovan (35) recently has defended the case for batch processes. To a considerable extent, the decision to adopt an apparently efficient process may be influenced by intangible factors such as economic competition, growth outlook and risk. However, considerations other than the usual ones of efficiency and productivity are too often overlooked, particularly in decisions on development of a continuous fermentation. MAlek (97) has amply illustrated and repeatedly emphasized that the physiological state of microorganisms in continuous culture may be markedly different from that in the usual batch growth cycle; consequently, entirely different qualitative and quantitative results may occur in continuous fermentations than we may be led to believe from batch results. Moreover, continuous operation may permit wholly new technical advantages unattainable in batch operation, such as the re-use of cells and the spatial separation of cell-growth and product-formation phases of a fermentation. With this background, we might well pose some questions with the expectation of reasonable answers or at least guidelines for future research,

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217

especially as they pertain to applications in industrial fermentations : What methods of continuous fermentation are available and to what types of processes do they apply? How sound is the theoretical basis, what predictions may be reached from it, and are the predictions experimentally substantiated? Where and how successfully have continuous fermentation processes been tested in practice? And what of the future?

11. Classification of Continuous Operations and Processes A. OPERATIONAND FEEDTYPES Continuous fermentations may be classified either on the basis of the manner in which they are operated or the production process to which they are applied. Several types of operation may be recognized: 1. Single-Stage

Single-stage continuous fermentations are those in which the entire operation is completed under steady-state conditions in one vessel, the nutrients being added and the cells and products being simultaneously removed a t the same continuous rate. This method has simplicity as its major advantage, and as will be seen later from its theoretical applicability and successful uses, single-stage operation is especially adapted t o processes involving only cell growth or a product directly associated with cell growth. 2. Semicontinuous Semicontinuous fermentations are a modification of the single-stage, and occasionally multi-stage, operation in which the feeding and withdrawing are accomplished intermittently rather than continuously. This modification, which is intermediate between batch and continuous operation, also has been termed “cyclic-continuous” (51) and “slug-feeding.” For example, when a fermentation batch has reached its maximum yield, a part of the fermentor contents may be harvested and the fermentor recharged with fresh medium to start another cycle. Obviously. as the size of the fresh medium charge is reduced, the time to complete the subsequent fermentation cycle is shortened until eventually this intermittent feeding becomes essentially continuous. This type of operation lessens the problems of continuous control and essentially compromises the features, both good and bad, of batch and continuous operations. 3. Modified Single-Stage

Modified single-stage continuous fermentations employ a fermentor designed to permit the re-use of the cells, either by separating the cells from the product and returning them to the fermentor or by managing to retain

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PHILIPP GERHARDT AND M. C. BARTLETT

the cells in the fermentor while removing only the product. The effluent may be recirculated until the substrate is completely converted to product. This type of continuous operation is aimed toward making most effective use of the medium in biosynthesis of product rather than of cells, and of course is applicable only when a chemical and not the organism is the desired product.

4. Multiple-Stage Multiple-stage continuous fermentations employ a battery of vessels in series, fresh medium being fed into the first, the effluent from it passed to the second, and so on through the series. In this manner, the extent of substrate conversion may be successively increased, resulting in either more efficient use of the substrate or higher yield of the product. Alternatively, the component phases of the fermentation may be separated and individually enhanced; for example, the first stage might be used and arranged to favor growth of the cells while the second and succeeding ones might be used for production of the chemical product. 5. Internal vs. External Control

Internal vs. external control of continuous fermentation has been proposed by Novick (112) as distinguishing the two major types of singlestage continuous culture but could also apply to any of the above types of operation. Externally controlled continuous fermentations [e.g., in the Chemostat (114)] are those in which the steady-state growth rate or fermentation rate is limited below the maximum by the low concentration of some required nutrient in the feed. Internal control [e.g., in the Turbidostat (IS)], on the other hand, is attained by regulating the population of the organism and therefore again limiting the steady-state rate, usually by means of photocell regulation of turbidity. That is, with external control the dilution rate is set at a chosen value and the microbial concentration allowed to find its own level; with internal control the microbial concentration is set and the dilution rate allowed to find its own level. Herbert (64,65) has pointed out, however, that these two types ultimately resolve to the same principle of control, differing primarily in the useful range over which the dilution rates may be controlled. Externally controlled systems function best at very low growth rates, while internally controlled systems operate at complementary but somewhat higher rates. Most continuous industrial fermentations are operated at the maximum growth rate attainable and in this sense may be classified as internally controlled systems. In such fermentations, the rate of cell growth or product formation directly associated with cell growth is not purposely limited by the low concentration of one required nutrient nor even by the reduced feed

CONTINUOUS INDUSTRIAL FERMENTATIONS

219

of a balanced mixture. Rather, excess nutrients are usually supplied, although undoubtedly some unrecognized factor such as oxygen diffusion establishes the steady-state equilibrium. This condition of excess feed must prevail for a multi-stage system to operate and, in this regard, may account for the seeming discrepancy between the theoretical treatment and conclusions by different authors (for example, cf. 53 and 66). On the other hand, the formation of many chemical products of fermentation is based on specific nutritional deficiencies and depends on the existence of an inverse relationship between the growth rate of an organism and the synthetic rate of a metabolic product. Thus, an essential nutrient may be supplied in growth-limiting concentration, yet may still provide optimal conditions for synthesis of a desired product. Synthesis actually may persist for a long time after growth has stopped. An example of such an industrial fermentation is citric acid. A fermentatioh of this type may be well suited for continuous fermentation of the externally controlled or Chemostat type, in which a nutrient component is concentration-limiting. An excellent and detailed study of this situation is provided by Holme (69), using nitrogen-limited glycogen synthesis by Escherichia coli as an example.

B. PROCESS TYPES Industrial fermentations include processes that are not only classic fermentations (e.g., ethyl alcohol production) and aerobic “fermentations” (e.g., acetic acid manufacture) but also “fermentations” in which only cells are the product (e.g., bakers yeast production). The requirements for continuous production of cells and of chemical products may be divergent, especially if the product formation occurs as a separate phase OF has different nutritional requirements than cell growth. Moreover, in contrast to the usual pure culture fermentations, some continuous processes involve mixed cultures and also may be directed to both cell growth and product formation, as in biological waste disposal. Thus, it becomes apparent that continuous fermentation must be evaluated both from the standpoints of the means of operation and of the process features.

111. Summary, Predictions, and Experimental Tests of Continuous Fermentation Theory

A. STANDARDIZATION OF MATHEMATICAL SYMBOLS Whatever the operation or process used for continuous fermentation, certain theoretical principles are involved and should be understood from the beginning if the approach is to be other than empirical. Moreover, a mathematical analysis of this theory permits a rational and quantitative prediction of continuous fermentation performance and, in this way, sim-

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PHILIPP GERHARDT AND M. C. BARTLETT

plifies and delineates experimental tests. As noted above, the recent advances made in the field can to a large extent be traced to the development of a sound theoretical basis. Reflecting the varied training of the workers, a number of widely different techniques and procedures have been applied to the mathematical analysis of continuous fermentation. In many instances this variety in technical approach has also led to the use of different mathematical symbols for a single concept. Such replication has unduly complicated the study of continuous fermentation. Many difficulties may be resolved, however, if it is generally understood that the standard code of symbols adopted many years ago by the American Standards Association and American Institute of Chemical Engineers (123) is generally applicable to this field of study (48,100). Table I lists the description, units, and standard symbols of concepts useful in continuous fermentation theory, and shows the equivaTABLE I SYMBOLB USEDIN MATHEMATICALANALYBBB OF CONTINUOUB FEBXENTATION" _ I

.-.

Description

Eo

E

P

s

I

Operating volume of fermentor Time Retention or holdup time Generation time or doublini time Delay time Concentration of organism Concentration of eontaminant Concentration of mutant Concentration of product Concentration of substrate Growth rate conatant Production rate conntant Dilution rate Yield conntant Saturation conatant Feed rate Mutation rate

-

I n using these aymbola, a series of superscripts and nubacripts has been applied to give more meaning. The superscripts generally refer to process variables and include: c contaminant, m = mutants, o = organism, p = product, and 8 = subatrate. The aubscripts generally refer to operation variables and include: d = delay, LI = generation, r = retention, and u = upaet. Certain other subscripts will alao be used; these are basic mathematical expreaaione that should not need deflnition or are self-explanatory in context. Where sub/superscripta are not wed, the nymbols are considered to have general applicability or obviously per b i n to the organism.

CONTINUOUS INDUSTRIAL FERMENTATIONS

221

lence of these standard terms with those used by other authors. The references cited are representative of the contributions to continuous culture theory and provide the origination of the following derivations.

B. DEVELOPMENT OF THE GENERAL EQUATION 1. Algebraic Representations of Growth in Batch Cultivation In batch cultivation of cells when changes in mass or number are plotted against time, a sigmoid growth curve is usually obtained, because of the interaction of naturally occurring forces. Mathematically, this growth can be expressed as follows:

ax - = kX ae X

Xoe'"' (1b) Also, since the concentration of organisms at the end of a time period is equal to the original concentration multiplied by 2 (since growth occurs by the fission of one organism to form two) to the nth power (the number of generations occurring in the time interval), the following relationships can be expressed : =

x = X02" n=

log

(24

x - log xa log 2

n = logs X

- log2 X o

(2C)

By combining Eqs. (lb) and (2a) the following relationship is derived: 2" =

ek'

(34

n = - ke

In 2

Since the generation time (6,) is the time necessary for doubling the population, i.e., Bs = (O/n),Eq. (3b) can be rearranged to give:

It should be noted that these equations will be applicable even when growth does not occur by binary fission. In such cases, the generation or doubling time will simply be a mathematically determined number used just as half-life constants are in radioactive decay expressions.

222

PHILIPP GERHARDT AND M. C. BARTLETT

2. Applications of Batch Cultivation Equations to the General Equation for Single-Stage Continuous Culture A culture to be grown continuously starts out in the same fashion as a batch culture. Then at some point, usually during the exponential phase, a continuous flow of nutrient medium is started through the system. By feeding into the fermentor a t a constant rate ( F ) and withdrawing the product a t the same rate, a constant volume of fermenting medium ( V ) is maintained in the fermentor. To achieve this result, the contents of the fermentor must be sufficiently agitated to insure homogeneity, and the entering fresh medium must be thoroughly mixed with the fermenting medium in a short time. This has been demonstrated experimentally (33). Derivation of a material balance for organisms, substrate, product, or any one component then leads to the generally applicable rate equation for continuous operations:

+ Growth Output + Accumulation ax FXo + k X V = FX + V d8

Input

=

X o represents the concentration of the component in the entering feed; X is the concentration of the component in the fermentor and therefore in the discharge stream. In other words, the rate a t which a component is fed into the fermentor plus the rate at which it is produced is equal to the rate a t which it accumulates plus the rate a t which it leaves the fermentor. If Eq. (5a) is rearranged and the symbol for dilution rate (0)substituted for F / V , these more widely used forms of the general equation result:

-ae_ - D ( X o - x ) + ICX - -- 0x0+ X ax d0

(k - D )

(5c)

3. Simplified Forms of the General Equation

Two special cases of the general equations immediately come to mind: that for the situation where the desired component is not in the feed and that where it is. If none of the desired component is in the entering stream, as is the case when the organism or a product of the organism is the component in question, X o is zero and the general Eq. (5c) becomes:

ax - -- X ( k - D ) d8

CONTINUOUS INDUSTRIAL FERMENTATIONS

223

which means that in continuous operation where (dXld8) = 0, k = D. Conversely, the retention time (O,), which is equal to 1/D,must also equal l/k. If the conditions are made more specific so that the component under consideration is the microorganism itself, when (dX/d8> = 0 and D = (F/V), Eq. (4)may be combined with Eq. (6a) t o give:

Thus, at steady state the medium flow rate is a function of the generation time (e,) for the fermentor volume used. This relationship was used by Adams and Hungate (2) to develop a very useful method of predicting the time required for a continuous fermentation cycle on the basis of the generation time found from the slope of a batch growth curve. This value of 8, was then substituted in Eq. (7b) and for a particular volume gave the flow required t o maintain conditions of equilibrium. In the second special case of the general equation, if the desired component is a limiting substrate in the entering stream, in continuous operations where (dXs/ldB) = 0, Eq. (5b) becomes:

D(X: - X s )

+ kSXa= 0

(84

Since the k as normally used denotes the production of some substance, its sign must be changed in order to denote utilization. Therefore:

D(X{ - X s ) = ksXs

(8b)

which shows that the dilution rate times the difference in concentration between the entering stream and the vessel contents is equal to the grams/ liter/minute of substrate utilized. Equation (5c) may also be altered to give the equation (which could easily be obtained from Eq. (8b) as well):

D(Xb)

=

X"D

+ k8)

(8c)

which means that all substrate which enters either is utilized or passes out the effluent unchanged. C. STABILITY OF CONTINUOUS FERMENTATION One of the most important predictions of the above theory is that a continuous culture is an inherently stable system. By closer consideration of the behavior of the system, the forces bringing about the stable equilibrium condition ko = D can be demonstrated (100). Since k0x" is the expression for the cells produced per unit volume per unit time, and VX'

224

PHILIPP GERHARDT AND M. C. BARTLETT

is the expression for the substrate used per unit volume per unit time, a yield constant ( Y ) can be defined (104) :

y = -k O P

PX'

With Eq. (5b) written for the case where the component is the substrate and the sign on k changed as in Eq. (Sb), the substitution of kox"/Y for k 8 P gives :

dX8 -= D ( X t do

- X') -

Using Eq. (loa), we can demonstrate the stability of the steady state ko = D. At equilibrium, of course, dX8/de is zero, but if the concentration of cells is disturbed so that x" is less than its equilibrium value, then (x"/Y) ko will be less than D(X: - X'). Since dX8/de will then be greater than zero, X', the substrate concentration, will increase. It was shown by Monod (104) that the growth of cells follows the same general pattern as enzymes with respect to the effect of substrate concentration. Thus, as an approximation :

where K is the Michaelis-Menton constant and ?corn is the maximum growth rate constant. Equation (11) indicates that increasing X a increases ko, in which case ko would become greater than D . A rearrangement of Eq. (6a) gives :

Thus with ko greater than D,x" will increase in the exponential fashion dictated by Eq. (6b). However, as Eq. (10a) suggests, with an increasing x", the growth rate will diminish once again until steady state is reached once more at ko - D . Here dX'/dO must be zero, and Eq. (10a) reduces to:

P = Y(X,' - X * )

(12a)

Similar reasoning will demonstrate the return to steady state that occurs when x" is greater than its equilibrium value. Consider now what happens to a fermentation operating at steady state when the dilution rate is changed. If D is decreased, Eq. (10a) shows that dX'/dtJ is less than zero and X' decreases. Decreased X 8 causes decreased ko, according to Eq. (11). The effect continues until once again ko = D

CONTINUOUS INDUSTRIAL FERMENTATIONS

225

and steady state is re-established a t the new dilution rate. As before, Eq. (12a) prevails. A similar re-establishment of equilibrium will occur if D is increased. However, if D exceeds ito, ,then the cells will "wash out" and X o will decrease to zero in an exponential manner according to Eq. (6b). Another event previously described by Novick and Szilard (115) is that which takes place when the concentration of substrate (Xo") is increased. This also increases X 8 which, from Eq. (ll), increases in turn the growth rate itoand therefore, from Eq. (6b), the concentration of organisms (x"). However, as x" increases, the growth rate decreases until a new stationary state is reached, whereupon ito = D. When this state is reached, the concentration of substrate in the fermentor is again at the level noted before the concentration in the feed was increased. Equation (12a) again is valid, although the values of x" and Xd are a t higher levels than before. These derivations are valid only when the yield Y has one specific and unique value a t each growth rate in the given system. This is essentially true for purely aerobic metabolism, but it is not true in certain other cases (101). For instance, where there are two processes competing for the available substrate the yield will be highly dependent on the substrate concentration and thus on the growth rate and the dilution rate. This is because one of the competing metabolic processes gives a lower yield than the other. If D is changed in the range of growth rates where both types of metabolism are occurring, the changing yield will cause the steady state population to change according to Eq. (10a). Herbert and his associates (64,65, 66) have conducted a systematic and critical test to see how the behavior of various microorganisms in externally controlled continuous culture compared with the theoretical prediction of stability. The method adopted was to set a flow rate and allow the culture to continue for several days, during which time samples were analyzed in several ways for cell counts, cell constituents, and supernatant constituents. The flow rate was then changed and the determinations repeated at a new steady state. A wide range of flow rates was covered in this way, and a typical run lasted for a month or more. In general, the quantitative data obtained agreed well with theory and confirmed the stability of continuous cultures with several organisms. Observations and analyses of exceptions to the stable situation have been made by Finn and Wilson (43)and will be discussed below.

D. EXTENSIONS OF THE GENERAL EQUATION 1. Modijied Single-Stage Fewientation When it is desired to increase the productivity of a continuous fermentor by recycling a portion of the cells produced, Eq. (5c) may again be applied,

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PHILIPP QERHARDT AND M. C. BARTLETT

using the type of analysis first demonstrated by Golle (53). When recirculation is begun so that 8 = 0, then X o is the concentration of cells in the recirculating line, XI is the initial concentration of cells in the fermentor, and Eq. (5c) becomes: k

-D

In

X(k - D) Xi(k - D )

X(k - D> Xdk - 0)

+ DXo = e + DXo

0x0- e(k-D)8

+ DXo

From the above it may be seen that when k as 0 goes to infinity. When k - D < 0 then:

- D > 0, X

(13b) goes to infinity

and : l i m X = - DXo 8-m D - k When k - D = 0, X is indeterminate. However, a solution of Eq. (5c) for the case k = D yields:

X = DX&

+ Xi

(164

Hence, X theoretically goes to infinity with increasing time but in practice will probably reach a new maximum limited by some environmental variable. It is also true that in the case of recirculation:

Y =

kOXO- x," kaF

Equation (9b) may then be used to develop an equation similar to Eq. (10a) :

which, in steady state when dX*/do = 0, gives a more complex version of Eq. (12):

2. Multiple Stage Fermentations It was also shown by Golle (53) that it is sometimes theoretically desirable to operate a continuous fermentation with the effluent of the first fermentor

CONTINUOUS INDUSTRIAL FERMENTATIONS

227

flowing continuously as feed into a second fermentor, with successive overflow through as many vessels as desired. There is an advantage in this method when growth rates higher than practical in a single stage fermentor can be maintained in the first tanks in the series; in this case the total retention time may be as low as in a single fermentor but there will be a more complete use of the substrate. This use, of course, requires that excess feed be supplied to the first fermentor. A means of comparing and predicting the economical advantages of additional stages in a given continuous process of known or assumed cost distribution has recently been described by Deindoerfer and Humphrey (32). In analyzing the theoretical behavior of such a system, Eq. (5a) can be applied to the period during which the second fermentor is filled, giving:

VdX FX1f kzXV = de

X i is the concentration of organisms in the feed coming from the first fermentor, while X and V are the concentration and volume, respectively, of the organisms in the second fermentor a t any time. Equation (18a) can be rearranged and integrated as follows:

When e = er (i.e. when the vessel begins to overflow a t Vz/F), V X = VzXz, and Eq. (18c) becomes: FXlek2"21F = FXi kzVzXz (19)

+

At steady state when V1 = V ? ,(F/V2) =

kl

so that:

The maximum value of X z occurs when kl = kz , since kz cannot exceed Icl . Both are equal only when both are in the exponential growth phase. For this condition :

Xz

=

X l ( e - 1)

Xz

=

1.718X1

Thus it becomes apparent that the concentration of organisms in the second

228

PHILIPP GERHARDT AND M. C. BARTLETT

fermentor can never be more than 1.718 times higher than in the first fermentor at the moment overflow begins. In considering the next step, the continued overflow from the second fermentor after it has achieved steady state, the equations developed for the case of a single vessel with recycling can again be applied. When kz D2 > 0, X 2 goes to infinity as 8 goes to infinity. When kz - D2 < 0, then:

and when k2 - Dz = 0, then:

X2

=

02x18

+ Xi

(16b)

so that X2 goes to infinity with increasing time. Since the first fermentor conforms to the equations developed for the single vessel, and the second behaves as a single vessel with recycling, it is possible also to derive expressions in terms of retention time (e, = ( V / F ) ) for the capacity of the combination:

Rearranging equation (5a) for the case when (dXld8) = 0 and applying it to the second fermentor: v 2- x2 - XI -

Fz I%zXa Equations (22b) and (23) can then be combined to give: 1

=

1

(23)

xzxzxl>

IE; + 6(

In a propagation system when kl = kz,Eq. (%a) becomes:

er=-+-z) L1

Since the concentration of cells is greater in the second tank ( X z )than in the first (XI), it is evident that the retention time for the two tanks in series is up to twice as long as that of a single tank. Thus there is no advantage in using more than one fermentor if the growth rate is the same in each, unless time is not important and a high cell population is. However, if the fermentation is such that a high growth rate can be

CONTINUOUS INDUSTRIAL FERMENTATIONS

229

maintained in the first tank because of the presence of a higher concentration of a limiting substrate, then an advantage can be demonstrated. I n this case kl will be greater than k2, while lcz may equal the growth rate in a single tank. This situation would also occur if there were two substrates in the medium, one capable of supporting a higher growth rate than the second. I n the first tank, the easily assimilable nutrient would be used, and the harder to assimilate nutrient would be left as the limiting substrate in the second fermentor. From Eq. (%a) it can be seen that in this case the retention time for the two fermentors in series would be reduced significantly over that for a single fermentor doing the same job. This would mean, of course, a reduction in total volume for the same rate of product formation. E. EFFECT OF UNDESIRABLE ORGANISMS 1. Contaminants

Contamination potentially is a serious problem in continuous fermentations since the long operating periods for which they are designed make them particularly liable to the occasional introduction of undesired organisms. However, equations have been developed (53) which show that the mere entry of foreign organisms into a continuous culture does not mean the process will fail. For example, Eq. (13b) can again be applied, this time to the case of a single fermentor being contaminated with a foreign organism a t a rate FXOC:

+

Xc((kc- 0) DXO" Xi"(P - 0) DXo'

+

e(kc-~)e

(13c)

Just as before, there are three possibilities with respect to the growth rate of the foreign organisms in the contaminated fermentation. This growth rate (V) can be greater than, equal to, or less than the dilution rate. If k" is greater than D (which equals ko), then Eq. (13c) show$ that X" will increase exponentially with time until the steady-state concentration of limiting substrate is reduced to the point where k" = D. Under these conditions, the growth rate of the original organism kQ will be less than D and its concentration will decrease to zero exponentially according to Eq. (6b). After a period of time the original organism will be entirely replaced by the contaminating organism. An infection by such an organism will result in complete failure of the fermentation. This will also eventually happen in the case where the growth rate of the foreign organism is equal to the dilution rate, and according to Eq. (5c), its concentration increases linearly a t a rate DXOC. On the other hand, if the growth rate of the contaminant is less than the dilution rate, Eq. (13c) shows that its concentra-

230

PHILIPP GERHARDT AND M. C. BARTLETT

tion will approach a limit a t infinite time: limXc = 9-m

DXac D-k"

A contamination by such an organism will become serious only if its rate of entry is extremely high and its growth rate only slightly less than that of the desired organism. Moreover, the situation of a contaminant having a growth rate less than that of the original organism may be brought about as the result of the continuous fermentation. For example, an antibiotic fermentation may inhibit the growth of an otherwise more rapidly growing contaminant and, in contrast to the batch operation, the antibiotic will be continuously present in high concentration. 2. Mutants When mutation occurs in a single vessel in continuous operation, it is governed by a material balance similar t o Eq. (5a), in which 4 is the mutation rate or the fraction of the total population that mutates in each generation and in which Dlln 2 represents generations of the original organism per unit time: Input

+ Growth = Output + Accumulation

Equation (25b) may be rearranged as was done with Eq. (5a) to give:

This equation can be integrated to give an equation similar to Eq. (13b):

By the same line of reasoning that applied before, if the growth rate of the mutant ( k m ) is greater than the dilution rate, then the entire culture will be replaced by the mutant form. If km is equal to the dilution rate,

CONTINUOUS INDUSTRIAL FERMENTATIONS

231

then the concentration (Xm) will increase linearly a t a rate D(+Xo/ln 2). If km is less than the dilution rate, then Xm will approach a limiting value:

Two important conclusions can be drawn: regardless of the value of k”’ the product cannot be entirely free of variant organisms; and if lcm is greater than D, a steadily increasing number of mutants will be encountered. Even though this was used to advantage by Novick and Szilard to study mutation rates (115) and would be useful in obtaining large populations of a desirable mutant, such a mutation might also give a poorer yield of the product. From an optimistic viewpoint, it could also give a higher yield. The result is unpredictable and would have to be determined by experimentation. For the case where lc” = D, x“probably would increase very slowly since the proportionality factor of mutation (6) would undoubtedly be low. However, if selective growth in favor of an undesired type should occur, then k” > D and the fermentation would have to be stopped. The only condition permitting uninterrupted fermentation requires that the growth rate constant of the undesired variant be less than that of the parent organism, i.e., k m < D. The number of these variant organisms in the culture can be predicted from Eq. (27), since the growth rate constant is known. As in the case of contamination, the situation of a mutant having a growth rate less than that of the parent organism may be induced as the result of proper operation of the continuous fermentation. For example, if the operation is adjusted so that the growth rate of the parent organism becomes maximum, then k” may become lower than ko with favorable results. This situation is in contrast to the less-than-maximum ko that prevails in externally controlled systems and usually in internally controlled systems, and may account for the failure to observe increased “strain degeneration” in continuous fermentation practice (11, 74, 117). Experimental tests of this important point are discussed in greater detail in the section on butanol-acetone fermentation.

F. THEOSCILLATION PHENOMENON Finn and Wilson (43) noticed in their work and the work of others (2, 101) that continuous growth of a yeast culture induced a cycling of the population in contrast to the usual stable situation (see above). They accounted for this phenomenon by assuming that some initial disturbance, such as a reduction in the flow rate, caused the number of cells to increase. As each cell manufactured acid, the pH fell, causing the growth rate to decrease. Before long there would be a net washout of cells and decrease in

232

PHILIPP GERHARDT AND M. C. BARTLETT

population, a sequence of events which would result in steady oscillation if there was a time lag in the adjustments of pH and population. In order to describe the behavior of the yeast population, Finn and Wilson integrated Eq. (6a) to give:

In-x2 = ( k - D ) e

x1

assuming X2 to represent a fluctuating value arising from a disturbance applied to the system, and X1 to be the steady population as set initially. Xu was used as an abbreviation for the left side of the equation, and k, for the right hand member, thus allowing the equation to be rewritten as:

x, = k,B ax, - - k,(X,)

(28)

dB

This expresses the fact that k , depends on Xu.Equation (29a) can be expanded in a Taylor series, but for small values of Xuonly the first two terms need to be retained:

where a is a positive constant. Substitution of X2 = X1 shows that ko in the above equation is zero. Hence:

ax, -+ a x , dB

=

0

(29c)

This can be rearranged in the form:

ax, --xu

( 29d )

to show that the upset normally will decay exponentially and that there is no tendency for self-excited oscillations. On the other hand, if there is a time delay in the adjustment so that k, depends upon the value of Xu at some previous time, stable oscillations are a natural consequence. Finn and Wilson, by an abstruse study of the complexitiesinvolved, were able to deduce that in this cycling the logarithm of the population fluctuated sinusoidally. Oscillations in yield have also been reported for two continuous antibiotic fermentations but could be traced to operational failures and recoveries rather than to an inherent characteristic of the fermentation (11).

CONTINUOUS INDUSTRIAL FERMENTATIONS

233

G. PREDICTION OF CONTINUOUS PERFORMANCE FROM BATCH DATA This possibility obviously has far-reaching significance to industrial practice, for it should enable the manufacturer to make a reasonable estimation as to whether a given batch process should be more efficient under continuous fermentation. Most of the growth constants characteristic of an organism can be evaluated from batch culture data, and by means of these, several methods of arriving a t continuous performance data have been derived. A simple method of calculating continuous fermentation cycle times and the level a t which continuous fermentations should be operated was devised in 1950 by Adams and Hungate (2), and has been discussed in connection with Eq. (7b). The method is based on the analysis of growth curves constructed from data from the periodic analysis of batch fermentations and was tested by continuous alcoholic fermentation of fruit cannery wastes. More recently, Luedeking (94) has used instantaneous rate data from batch fermentations to develop equational and graphical methods for predicting the performance of both single- and multi-stage continuous fermentation systems. The theoretical predictions were tested in singlestage continuous lactic acid fermentations a t controlled pH levels. Herbert et al. (66) have derived a formula for comparison of the relative outputs of batch and externally controlled continuous culture. Values of the ratio of continuous output to batch output are affected mainly by the growth rate of the organism and the total batch delay time. The output of a batch culture of course varies throughout the growth cycle but a mean output can be calculated as follows: Consider a batch of medium of initial substrate concentration Xd, inoculated initially with organisms to a concentration X,"; the maximum growth attained when all substrate has been utilized is Xo.Then the total time of one production cycle is: 1 xn e=-ln-+&

h%

xoo

(30)

1 xn where the term - In - is the time needed for the organisms to grow k,

Xno

exponentially at maximum rate from start to finish [a modification of Eq. (16a)], and the second term (&) is the delay time, which includes the initial lag and final retardation phases of growth and the "turnaround" time necessary to take down, sterilize, and reassemble the plant preparatory to a second cycle. The total amount of organisms produced is:

x,"- x,"= Yx:

(31)

234

PHILIPP GERHARDT AND M. C. BARTLETT

This modification of the equation used by Herbert et al. (66) is used with Eq. (30) to give the following expression for the mean output: total organisms produced total cycle time

kmYX'o X1° In - kmOd

xoo+

(32)

Since, from Eq. (12a) :

xo = Y ( X t - x;)

(12b)

(kz)

is the same as k,Y(Xo" - XP). When the maximum output of cells X < is low and may be neglected as would be the goal in practice, the maximum output from a continuous system is: maximum continuous output

=

kmYXoB

(33)

Hence, when the same growth medium is used in both continous and batch fermentations, we have from Eqs. (32) and (33): continuous output batch output

kmYX(

continuous output x," = Inbatch output xoo

+

kmOd

( 34b1

Herbert et al. (66) concluded that "in the majority of cases, therefore, a continuous process would be expected to show a t least a 5- to 10-fold advantage over the corresponding batch process." Their experimental tests gave data that generally agreed well with the predictions and the general conclusion is consistent with a t least one previous comparison (51). Exceptions occurred with several organisms a t low dilution rates; presumably these resulted from the complicating effect of endogenous metabolism, which became proportionally more important as the dilution rate was reduced. This and other physiological differences in continuous cultures (971 may act to upset performance predictions based solely on batch results.

H. PRODUCT FORMATION IN CONTINUOUS FERMENTATION When (dX8/dO) = 0, Eq. (loa) becomes:

0 D(XC - X 8 ) = Xko Y

(35)

As D is increased will also increase until decreasing Y and increasing X ; cause x" to reach a maximum and then decline. If the substrate is not highly valuable with relation to the product, and the cells themselves are

CONTINUOUS INDUSTRIAL FERMENTATIONS

235

the desired product, then the dilution rate which gives this maximum will be economically optimal. However, when a product other than the cells is important, another maximum, the rate of product formation, becomes the governing factor. This rate is determined by material balance and kinetic considerations, as is the growth rate, and similar equations can be used to express the relationships. The material balance expression is based on the yield of product (Y”)from the rate-limiting substrate and is analogous to Eq. (12a):

X”

=

Y”(X0”- xa)

(36)

For processes where product formation is directly related to cell concentration, the kinetic factors are summarized by:

X”

=

k’P

(37)

For some processes such as for several of the antibiotics, product formation is not so related. When the relation is determined, an equation similar to Eq. (37), expressing kP as a complex variable, will probably be applicable. It may be said, then, that under given conditions of dilution rate, environment, and nutrition, the quantities X a , Y’, X”, and k’ will be fixed a t equilibrium levels as defined by Eqs. (36) and (37). In this way the productivity, kpXo,at steady state is established.

I. SUMMARY The derivation of the majority of the equations which have just been reviewed is from one of the forms of the general material balance equation for continuous fermentation:

FXo

+ kXV

=

FX

+ V ax do

(54

These may be said to form the theoretical basis for almost all steady state processes and lead directly to the following conclusions: (1) The growth rate (k’) in a single vessel must be equal to the dilution rate ( D ) , so that a stable equilibrium is retained despite disturbances, provided the growth rate is less than its maximum. This prediction of stability has been confirmed experimentally. (2) If the growth rate in a second fermentor in series, or in a single fermentor with recycling, is greater than or equal to the dilution rate, the concentration of the organism theoretically will go to infinity and in practice to some newly established maximum. This is also the case in a single-stage fermentor if the growth rate of either mutants (k“) or contaminants (k“) is greater than or equal to the dilution rate. In all four of these circum-

236

PHILIPP GERHARDT AND M. C. BARTLETT

stances, however, if the growth rate is less than the dilution rate, a constant level of organisms is reached. Thus, for a single fermentor with recycling:

For a second fermentor in series:

For contamination:

DXco lim X c = e+m D - k" For mutation: 8-w

(3) The population is separately determined at each dilution rate by the yield of cells from substrate ( Y ) ,the concentration of substrate entering ( X : ) , and the concentration of substrate in the fermentor ( X ; ) :

When the fermentation process is single-stage without any modifications, i.e., when X," = 0 and ko = D,the equation is simplified to the more usual form :

xo= Y(X80- X")

(12b) 1

(4) The retention time, defined for one fermentor as 0, = - is for two k10 ' fermentors in series: 1

1

er=Q+p(

x,"- x : x20

)

Thus for example, when k: is greater than k," and k; is equal to the growth rate in a single fermentor, use of two fermentors will minimize the retention time, The principle can be extended to three or more fermentors in series. (5) When oscillations occur, they will normally decay exponentially unless there is some sort of feedback mechanism. In this event a stable pulsation may be initiated and this has been observed in certain fermentations.

CONTINUOUS INDUSTRIAL FERMENTATIONS

237

IV. Continuous Fermentation in Practice A. CELL PRODUCTION 1. Yeast

Mass propagation of microorganisms is the most direct application of continuous culture methods and is especially suited to single-stage operation. Among the first continuous cell-production processes to be attempted (131) and still paramount among the industrial applications, yeast manufacture (along with fermentation alcohol from yeast) is in many ways an ideal process for continuous operation. The situation has been well stated by Maxon (100) : “These industries depend upon a large volume and a low profit margin. Thus, equipment of tremendous capacity operating a t maximum output is required. High efficiency and low waste are necessities. The advantages gained in productivity for a given equipment capacity by continuous fermentation are great. Furthermore, the time lost in cleanup and preparation necessary to batch fermentation is obviated. The difficulties that could arise from contamination or yeast degeneration me not encountered, and no expensive precautions are required to prevent them.” De Becze and Rosenblatt in a 1943 review (30) have discussed several of the earlier described processes for continuous yeast fermentation. More recently, several papers on current experimental work and literature concerning both bakers yeast and food yeast appeared in the Czechoslovakian symposium (23). Accordingly, only a summation and a few illustrative references need be mentioned here. In general, single-stage fermentors have been used, usually of fairly large capacity and equipped for aeration and agitation of the contents. Although the typical deep-culture fermentors have been common and full-scale design figures are available (150), the so-called Waldhof system also has found considerable use. This equipment was developed and extensively used in Germany during World War 11. Afterwards, several detailed reports were made public (68, 137, 138) and the process was installed and operated in a plant in this country (72). The Waldhof fermentor as presently used is unique in that both air and agitation are supplied by a single, hollow-bladed impellor which is positioned beneath a central draft tube; foam circulates down through this tube and out into the turbulent emulsion by means of the centrifugal action of the impellor. A high-power input and a low operating capacity of the total fermentor volume are two limitations of this effective continuous system. Sulfite waste liquor from paper mills has been used extensively in this country and in Europe as a sugar source for yeast production, although wood hydrolyzate, cane or beet molasses, fruit and other agricultural

238

PHILIPP GERHARDT AND M. C, BARTLETT

residues, waste beer from other fermentations, or virtually any other form of fermentable carbohydrate may be employed. Such substrates usually are deficient in phosphorous, in potassium, and in nitrogen, which often is added as ammonia. A number of different yeasts have been produced continuously, depending principally on their intended use and the raw material available. Saccharomyces cerevisim and related strains are commonly used for continuous production of bakers, brewers, and distillers yeast. Torula utilis and similar “false yeasts” are most often used in production for human or animal consumption; moreover, these types are especially adapted to sulfite waste liquor and other substrates in which pentoses are present.

2. Other Microorganisms Quite obviously, organisms other than yeast may be efficiently grown in continuous culture and have present or future industrial potential. Table I1 lists representative genera that have been used in continuous culture studies and provides key references. Although the general principles of the method may be expected to apply to any organism used, nonetheless special modifications will be required to accommodate certain types. Aerobic and facultative organisms require provision for adequate aeration if maximum growth rates are to be attained. Anaerobes, growing only a t a relatively reduced Eh, require exclusion of air, but fortunately their fermentative metabolism provides sufficient GO2 , CH4, or Hz to flush away the small amounts of air that enter the normal closed fermentor. Special problems in safety have entered into systems designed for producing pathogenic bacteria so that, for example, Brucella was propagated continuously under a negative pressure (51). Advantages may accrue from continuous culture of bacteria such as Pneumococcus (41))which remains intact and increases in virulence under these conditions but which lyses as the normal batch growth curve progresses and often loses virulence on subculture. Recently, Harris-Smith et al. (63) were able to maintain the in vitro toxigenicity of Bacillus anthracis only by continuous culture. Sporeforming organisms can be expected to remain in the vegetative state during continuous culture (97) * Increasing attention has been directed t o large-scale production of algae as a potential source of food and fodder (15, 18). Since growth of these plants poses special problems in supplying adequate light for photosynthesis, shallow troughs or tubes have been employed in continuous methods (19, 25, 26, 58, 84, 103, 109). Algae also have been used as a source of oxygen in the continuous treatment of sewage (54, 93, 120, 121). The filamentous fungi not only have been propagated continuously for their biosynthetic products (see later) but also to a limited extent for the

TABLE I1 REPRESENTATIVE GENERAOF MICROORQANISMS GROWN I N CONTINUOUS CULTURE Microorganisms

Selected references

Algae: Ankistrodesmus Chlorella Euglena Nitzchia Scenedesmus Bacteria: Acetobacter Aerobacter Azotobacter Bacillus Brevibacterium Brucella Clostridium Cor ynebacterium Desulfovibrio Escherichia Lactobacillus Mycobacterium Pneumococcus Propionibacteriurn Proteus Pseudomonas Salmonella Serratia Shigella Staphylococcus Streptococcus Streptom yces Fungi : Aspergillus Dermatophyta Ophiostoma Penicillium Trichophyton Mammalian Cells : L Strain human fibroblasts Embryo rabbit kidney Protozoa: Tetrahymena Yeast.: Candida Monilia Rhodotorula Saccharom ycee Torula 239

80 29, 103, 109 93, 153 81 83, 84 67, 152 39, 66, 126 95, 96, 97, 122 50, 63, 105, 110 43 51 37, 111, 117 139 20 21,69,76, 105, 113, 135 94, 159 36, 86, 139, 144 41 74 56, 139 43 44, 70 57 8 134 56, 79, 118,'135 11, 33, 34 128, 136 36 154 1, 11,22, 36,82,89,155 36 17, 52 27 14, 153 137, 138 98 80 12,30,43,101,122,150 42, 72, 137, 138

240

PHILIPP QERHARDT AND M. C. BARTLETT

use of the mycelium itself. The method was employed for producing Aspergillus oryzae for subsequent use as a medium supplement (136). A patent (90) described a continuous process for growing fungi on the outside of a porous tube through which medium was circulated. The industrial potential of growing suspensions of mammalian tissue cells is just beginning to be explored. These cells, properly handled, could immensely facilitate a host of direct and indirect products, including viruses for vaccines, hormones, and enzymes. Some initial efforts to scale-up laboratory techniques to conventional fermentors have been reported (160) and a number of commercial laboratories are known to be active. So far, the cell concentrations achieved are pitifully low and the cost of medium components is prohibitively high. However, research on the nutrition of these cells is burgeoning, and imaginative innovations are beginning to appear and can be expected to dramatically change the industrial potential. For example, it has been shown that certain strains may be adapted to grow in an inexpensive milk medium completely free of the normally required and expensive serum additives (1304. That population densities are not inherently limited to the levels usually attained (e.g., 1 t o 5 X lo6 viable cells per milliliter) is attested by the heavy suspensions obtainable by replacing the medium (38) and by simply growing the cells on solid media (156). There is good reason to expect that two-phase systems for concentrating cells during growth (55, 147) may be applicable. Tissue cell lines are often spoken of as being in “continuous culture,” but this peculiar usage refers simply to continued serial subculture. However, single-stage continuous culture in the usual sense has been achieved in laboratory equipment by Munyon and Merchant a t The University of Michigan and Sayblaski a t Rutgers University (personal communications and brief mention in 17 and 52), and by Cooper at the Medical Research Council in England (27). Munyon and Merchant, for example, were able to maintain L-strain fibroblasts in steady-state growth for periods up to 35 days and with populations of 6 X lo6viable cells per ml. This yield represents about half the maximum usually obtained in batch operation, and the generation time they observed was about twice the usual. Cooper el al. (27) maintained a suspension culture of a transformed embryo rabbit kidney cell continuously for 4 months, Their system produced an average of lo9 (2 gm. wet weight) cells per day in 1 liter of medium from a constant culture volume of 1.5 liters. The mean generation time was 25 hours and sometimes 16 hours or less. Growth rates and cell densities were cyclic. Most importantly, they observed that liquid-phase oxygen-levels for maximum growth-rates were much less than air (and even 14 % oxygen) equilibrium values; oxygen reductions of a few per cent sometimes increased the growth-rate by 50 %.

CONTINUOUS INDUSTRIAL FERMENTATIONS

241

Clearly, we may expect extensive and successful development in this application of continuous fermentation. But surely the quintessence of ingenuity in the continuous propagation of organisms must be owned by zoological laboratories a t The University of Michigan and probably elsewhere. A two-stage continuous culture device generates algae in one tank by means of conventional principles. The overflow from this stage continuously feeds the second, providing live food for a population of fresh water shrimp. Phototactically attracted to a yellow light, the shrimp in turn are removed intermittently and fed either to experiments on phototaxis or to colonies of tropical fish. The way is open, it seems to us, to devise a three-stage continuous guppy generator!

B. PRODUCT FORMATION 1. Ethyl Alcohol

The current picture of continuous alcohol production by means of yeast fermentation is much the same as that for food and fodder yeast. The inherent advantages, equipment, and substrates in the two process types are, for the most part, interconvertible, except that alcohol production requires anaerobic conditions and yeast strains, usually Saccharomyces spp., that efficiently convert sugar to ethanol. An illustrative example in this country is the plant of the Puget Sound Pulp and Timber Company a t Bellingham, Washington (40). The fermentation equipment consists of a battery of eight tanks in series, each having a working capacity of 80,000 gallons and equipped with propeller-type agitators to keep the yeast suspended. Raw material for the fermentation is sulfite waste liquor, which is preparatively steam-stripped of free sulfur dioxide in a continuous counterflow apparatus, vacuum-flask cooled, and limed. Yeast, sulfite-liquor, and supplementary nitrogen in the form of urea are proportioned and pumped into the first tank, the overflow passing continuously from tank to tank until it reaches the eighth tank. A unit volume of liquor is fermented in 20 hours. The effluent from the last tank is fed into continuous centrifuges; from these, the separated yeast is returned to the fermentors and the effluent liquor is passed to distillation .columns for recovery of the alcohol. The plant can handle 600,000 gallons of waste liquor daily, with the production of 6,000 gallons of ethanol. Lignin, methanol, and fuse1 oil are by-products. Operation was put on stream in 1945 and has continued in operation with shutdown only for personnel vacations. It employs only a minimum of manual help. The process suffers from the competitive disadvantage of any fermentation alcohol as compared to petroleum alcohol. However, the plant is strategically located in an area to which petroleum alcohol must

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be shipped and in which there is a plentiful supply of sulfite liquor. In fact, alcohol or yeast production from sulfite waste liquor may be looked upon as a salvage operation in a waste disposal operation. The general characteristics of alcohol fermentations and their modifications in continuous operation have been adequately described by several authors in an excellent book on industrial fermentations (149), in Maxon’s review (lOO),and in portions of the Czechoslovakian symposium (23). The latter source may be of special interest because of its presentation of the literature and of examples of current research in the Soviet Union, which have not heretofore been widely available. With the availability of these comprehensive descriptions of continuous alcohol fermentations, however, it seems that further discussion here would be redundant. 2. Acetic Acid and the Generator Method

Although often not considered more than in passing, the acetic acid fermentation for food vinegar provides the earliest and most widely used example of continuous fermentation. The process is variously known as the generator, quick, German, Boerhave, or Schuzenback process. Although it first came into prominence in 1832 when introduced into Germany by Schuzenback, a generator prototype reportedly (102) had been described and used in France as early as 1670. A comprehensive survey of the generator process and of vinegar manufacture in general has been made by Vaughan (152). Briefly, the quick vinegar process involves the use of a tank filled with a mass of packing medium such as beechwood shavings, on the extensive surfaces of which occurs a sessile growth of Acetobacter organisms. The alcohol-containing vinegar stock is continuously or intermittently distributed over the packing and trickles through the packing against a countercurrent of air, the converted vinegar eventually reaching a collection chamber a t the bottom. Thus essentially the operation is a two-phase (solid organism phase and liquid substrate phase), continuous or semicontinuous aerobic fermentation. There are a number of modifications of the basic method. The generator may be operated as a single-stage, “one-run” fermentation, with or without recycling. Two or even three generators can be operated in series as a multi-stage or “tandem” operation. Although vinegar conversion is usually accomplished in the two-phase generator system, recent work (71) has shown that the fermentation can also be conducted efficiently as a conventional one-phase submerged fermentation, in which the cells and medium are homogeneous. There is no obvious reason why such a submerged aceticacid fermentation could not be conducted continuously, despite the apparent absence of attempts. Further modifications of the generator process

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occur in the feed medium or vinegar stock, which may be composed of any of a number of alcohol-containing natural products, such as fermented apple cider, but may be diluted distilled ethanol. In principle, feeding distilled alcohol represents the complete separation of microbial growth, which is accomplished first, from the fermentation itself; however, in practice nutrient supplements are added to the alcohol solution to maintain the bacteria over a prolonged period of time. Vinegar generators are normally operated continuously over periods of months, the principal cause of termination being the plugging of the generator as the result of slime formed by contaminating Acetobacter xylinum. Continuous acetification of alcohol by the vinegar generator thus represents a crude but remarkable example of the basic method and refinements of continuous industrial fermentation. It might well serve as a superior test system for both theory and practice. The vinegar generator principle has in fact been applied to several other fermentations. For example, the “Fesselhefe” method of making alcohol from sulfite waste liquor (30, 137) used a packed bed to which yeast was bound, and a pulp suspension has also been used to bind yeast in much the same way (4, 77, 78). Gluconic acid (67) and acetone-ethyl alcohol (111) fermentations have also been accomplished by means of a fixed bed of wood shavings. One of the earliest attempts a t continuous antibiotic fermentation employed the equivalent of a shaving-packed generator (5, 22), which will be discussed in detail below. The principle advantage of this type of continuous fermentation appears to reside with the separation and retention of the organisms on the solid phase; the greatest disadvantage, especially in pure-culture, fine-chemical applications, lies with the difficulty in sterilizing and the relatively inefficient use of total tank capacity. 3. Antibiotics

Antibiotics manufacture, currently the most valuable fermentation process, typically consists of continuous recovery and finishing steps but with batch medium-preparation and fermentation. Recent progress (e.g., 49, 124, 151, 157) has made continuous sterilization of medium a real and attractive possiblity. There remains only conversion of the biological operation to make the entire process essentially continuous. Although a number of commercial organizations are k n o b t o have carried steady-state fermentations of antibiotics through varying degrees of development, the published literature still carries only limited reports of progress in this area. Papers on antibiotics other than penicillin are few and short. In 1950 a process patent was awarded to the Distillers Co., Ltd. for a single-stage continuous fermentation method to be used in making streptomycin (34). A fermentor with a 30-liter operating capacity has also

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been described (85) which was modified slightly and reportedly was used for continuous fermentation of unmentioned antibiotics. Recently, Brown from this same company (13) briefly spoke of further results with singlestage continuous streptomycin fermentation in a 3,000 liter vessel. Yields approximately 75 9% of those obtained by batch methods were continuously produced with a 2 to 4-day retention period (i.e., 0.25 to 0.5 volume changes per day). Subtilin has been made by a modified single-stage fermentation whereby a small portion of the preceding run is left in the fermentor as inoculum for the next run (50). Bartlett and Gerhardt (11) have studied single-stage continuous chloramphenicol fermentation, and this work is described below. However, penicillin is the antibiotic to which the literature has principal reference concerning continuous fermentation. The earliest such process to be described consisted of a modified semicontinuous method in which the spent medium was periodically removed from a surface culture and fresh medium added (1). Later a process much like this was awarded a patent (59). Another modified surface culture concerned the growth of the mold on trays suspended in a 10-liter bottle, which contained the medium and served as a fermentor (143). The penicillin was released into the medium where it was removed at intermittent intervals and replaced with fresh medium. Similarly, a tray over which nutrient medium slowly flowed was used in an attempt to devise a continuous fermentation (106); a similar process has been patented by Lilly (90). The organism has also been grown on the outside of a cellophane or porcelain tube through which nutrients were passed (88), and Beatty is reported (132) to have used a ceramic cylinder for the same purpose. A two-phase, single-stage continuous fermentation has also been attempted whereby the medium was passed through a vessel 2 inches in diameter and 4 feet long similar to a vinegar generator (5,22). Using wooden shavings packed so as to be freely exposed to circulating air, it was claimed that this fermentation produced a continuous flow of penicillin. This method also was described in a Swiss patent (142) and was tried on an industrial scale; however, it was not a practical success (132). A more conventional single-stage continuous fermentation for making penicillin hps also been described briefly in an abstract (141) and later in a patent (82) by Kolachov and Schneider. A 30-liter battery jar containing 12 liters of medium (3% lactose, 3% corn steep liquor solids, and 1% CaC03) was used. After 48 hours of initial grmth, a replacement medium was added and fermented broth was removed at a rate of 250 ml. per hour (i-e.,0.5 volume changes per day). The replacement medium was the same as the starting one except that in addition it contained 0.5 % glucose which served to maintain the pH near 7. Phenyl acetic acid as a precursor was

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added a t a rate of 1 gm. every 12 hours. Yields of 500-800 units per milliliter were maintained for 4 days of continuous operation. A patent also has been awarded Liebman and de Becze for a two-stage penicillin process (89). Nutrients were continually fed into a primary fermentor where the mold was said to be propagated a t substantially its maximum rate by only partially fermenting the medium; in a secondary fermentor fed from the overflow from the primary one, the fermentation was completed at lower aeration and a slower fermentation. The level of penicillin in the first fermentor was maintained a t 150-250 units per milliliter. Another patent by Foster and McDaniel (45) is for a modified singlestage fermentation, in which the penicillium mold was propagated for 2 to 3 days under normal aerated submerged conditions with 20% of the fermentor contents being removed just prior to the time that the penicillin concentration reached a maximum. An equivalent amount of fresh medium was then added to the fermentor and the fermenttion continued until it once more approached a maximum, whereupon the cycle was started again. These cycles lasted 5 to 6 hours and were repeated until the withdrawn broth showed a distinct drop in penicillin content. Schenley Industries, Inc., also has been awarded a French patent for a modified single-stage method for making penicillin (140). Work has also been done in Japan on the continuous fermentation of penicillin with 2,000-liter fermentors (155). In one modified single-stage fermentation, at the end of a fermentation the vessel was refilled with medium, sterilized, and cooled; its contents were then transferred to an actively fermenting tank where the contents were mixed and then split evenly between the two fermentors. Another modified single-stagefermentation was simpler in that a part of the fermentor contents was removed a t intervals and new medium added. A typical two-stage fermentation was also investigated in which a constant flow of medium was fed into a primary fermentor with the overflow from this vessel undergoing final fermentation in another vessel in series with the first. Unfortunately, this Japanese paper is largely descriptive of the procedures and contains little data on the fermentation results. Recently completed investigations by the authors on single-stage continuous fermentation of chloramphenicol and penicillin provide data from pilot plant experiments with two contrasting processes, and consequently are described below in some detail. The results have been published as a thesis (lo), and currently in a journal (11). A pilot plant of 20-liters working capacity was designed and constructed to be as simple as possible and potentially capable of being scaled-up to existing production equipment. Features of the assembly included the use

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of needles and rubber diaphragms for feed lines and sampling, an uirproduct effluent pipe to maintain the liquid level in the fermentor, a backpressure regulating value and entrainment separator on the effluent system, alternately sterilizable air filters, and heat seals to control back growth of contaminants. The medium was transferred by air pressure from a batch sterilizer to a magnetically stirred, sterile hold tank and from there was continuously metered by a peristaltic-action pump into the aerated and agitated fermentor; the crude product simultaneously was air lifted from the fermentor, separated, and collected. After successful sterility and batch fermentation trials, the system was first tested in producing chloramphenicol, a process selected because of its parallel growth and antibiotic formation, minimum pH changes, and favorable growth characteristics. I n tests with a dilution rate of 1.0 volume change per day, the continuous fermentation showed an initial drop from the higher batch antibiotic yield and then allowed maintenance of 75 to 125 pg. per milliliter, about one quarter of the maximum batch yield. Lower feed rates of 0.5 and particularly 0.25 volume changes unexpectedly and unexplainably seemed to lower the yield, although the pH and solids were relatively unaffected. Experiments were conducted which indicated that this lower yield was not attributable to strain degeneration. I n contrast to the chloramphenicol batch process, penicillin is characterized by pH changes, a thick mycelial suspension, antibiotic activity primarily against gram-positive bacteria, and a relatively stable product. Penicillin is also characteristically formed after mycelial growth has approached its maximum, although this was not observed in these batch runs. Tests with a dilution rate of 1.0 volume change per day washed out the penicillin within 4% days. At dilution rates of 0.5 volume changes per day, penicillin yield levels of 1,000 units per milliliter were maintained except when operational variables had an adverse effect. Substitution of glucose for lactose in the feed medium adversely affected yields. Both of the continuous fermentations were maintained for periods up to 17 days. However, contamination with extraneous microorganisms was common in the trials and represented the most important limiting factor to the success of continuous operations. I n addition, during penicillin fermentations the accumulation of mycelium inside the fermentor and within pipes and valves appeared to be an inherent and ultimate limitation of the continuous fermentation. Production estimates on a daily production basis indicated that chloramphenicol batch and continuous operations were roughly comparable, while penicillin continuous fermentations were up to twice as productive as the batch operation.

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It was concluded that these single-stage antibiotic fermentations are feasible and that further development could improve their efficiency. The current general status of continuous antibiotic fermentation is difficult to assess from the scant published literature and patents. Personal discussions with representatives from commercial organizations at best only allow speculation, especially since two persons from the same group will paint an entirely different picture of their findings. However, the general feasibility of producing antibiotics by continuous fermentation seems reasonably well established, and inherent limitations to usable periods of steady-state fermentation appear more theoretical than real. Despite predictions of greater contamination problems, aseptic operation has been accomplished for periods in excess of 2 weeks with pilot-plant equipment for antibiotic production (11) and for periods of several months with a pilot plant for production of bacterial cultures (39). Experience has proven that contamination of antibiotic fermentations occurs more often early in the batch cycle or in the start-up period of continuous operation rather than later, when the growth rate of the organism is maximum and the antibiotic concentration is high; consequently, contamination should be experienced less in continuous than in batch operation. Moreover, the advantages of larger and permanent equipment, of accumulated experience with a given process, and of possible integration with continuous sterilization should considerably lessen the incidence of contamination in the industrial scaleup of continuous antibiotic fermentation. In a similar contrast of theory and practice, the often-predicted overgrowth of mutants has not been experienced. Perhaps the most important limitation of the duration of continuous production with mycelial organisms will be the accumulation of growth on surfaces inside the fermentor and within pipes and valves, the resulting congestion eventually stopping operation. Wall growth of cells in various laboratory devices has been partially controlled by coating the walls with silicone and adding a detergent to the medium (471,adding abrasives such as glass beads to scrub the walls (117), and even employing “windshield wipers” (3, 110). However, these devices have apparently not been tested with mycelial systems, and a fully effective measure remains to be found. The comparative productivity of batch and continuous antibiotic processes apparently is still in favor of the conventional method. Certainly no commercial organization is known to have converted to continuous operation despite research work and patents. One published comparison (11) was promising in that, with very little development, roughly equal or possibly double productivity was attained by continuous as compared to batch operation over a 2-week period. It seems likely that further devel-

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opment of the continuous process could make its daily yield considerably more favorable, and this prediction is backed by theoretical calculations (66).Moreover, as discussed in the introduction of this review, the relative practicability of the two types of operations depends on a number of features other than yields. Admittedly, we are biased in our optimism for the future of continuous antibiotic fermentations and consider the development almost inevitable. It has been pointed out, and we agree, that the current choice between the undeveloped continuous and the established batch operation is analogous to that in the early 1940's between bottle surface-culture and deep-tank fermentation. The process with inherent advantages usually will succeed, the timing being determined by competitive forces. Competition, particularly in the established major antibiotics, is increasing to the point of forcing a re-evaluation of continuous processing. Several commercial producers already are known to have accepted the challenge, and an active period of research and development in this area is sure. 4. Other Chemical Products There remains to be considered several other chemical products that have been studied in continuous fermentation. The rather limited diversity of these is indicated by the tabulation of products and key references given in Table 111. Among the earlier fermentations in this group to be studied in the laboratory and attempted on a plant scale was lactic acid formation by Lactobacillus spp. The product has an attractive potential today as a valuable and versatile intermediate in the plastics industry, and the fermentation is well suited to possible continuous operation because it is a simple biochemical conversion and occurs under highly restrictive growth conditions of low pH and high temperature. A single-stage continuous fermentation of milk whey to produce lactic acid was studied by Whittier and Rogers in 1930-1931. Their original laboratory studies (135) were extended and adapted to production equipment (159). Five-thousand gallon wooden tanks were operated continuously for periods of weeks, producing 80 % of the theoretical (batch) yield with a 24-hour replacement time (i.e., 1.0 volume change per day). The fermentation was carried out a t 43°C. and low pH, using Lactobacillus bulgaricus ; special provisions for asepsis were not taken. Despite its apparent success and relatively advanced development, the process was abandoned because side reactions could not be avoided as easily as in the batch process (119). Recently, Leudeking (94) has employed the lactic acid fermentation by Lactobacillus delbruckii as a model to compare theoretical aspects of batch kinetics with continuous operation. Theoretical design equations and graph-

CONTINUOUS INDUSTRIAL FERMENTATIONS

TABLE I11 REPRESENTATIVE CHEMICAL PRODUCTS STUDIED CONTINUOUS FERMENTATION Product Acetic acid Acetone-butanol Acetone-ethanol Antibiotics: Subtilin Chloramphenicol Penicillin Streptomycin Ethanol Gliiconic acid Glycogen Hydrogen sulfide Itaconic acid Lactic acid Oxygen Sulfur

Organism

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IN

Selected References

Acetobacter Clostridium Bacillus

152 37,74,117, 158 111

Bacillus Streptomyces Penicillium Streptomyces Saecharomyces Acetobacter Aspergillus Escherichia Desulfovibrio Aspergillus Lactobacillus Euglena, Chlorella Desulfovibrio and Chlorobium

50 11 11,22,45,82,89,155 13, 33, 34 23, 40, 100, 149 28, 67, 125a 127, 128, 148 69 20, 161 125 94, 135, 159 54, 93, 120, 121 20

ical methods were developed for predicting from batch data the performance of both single- and multi-stage continuous fermentations under both steady-state and transient conditions. The predictions were tested in singlestage continuous lactic acid fermentations at controlled pH levels. The continuous fermentation of butanol and acetone by Clostridium spp. was described as early as 1932 in a U. S. patent by Wheeler and Goodale (158). Rieche et al. (133) in Germany patented a continuous process for butanol and acetone production from sulfite waste liquor, and a similar process was described in the Soviet Union (91, 92). All three were multistage operations. A detailed report on this type of fermentation appeared recently in the Czechoslovakian symposium under the authorship of Dyr, Protiva, and Praus (37). They employed CEostridium acetobutylicum, a potato-glucose medium, and single-, three-, four-, or five-stage continuous fermentors. Although we found it difficult to be certain of comparative data for the various types of operations, one tabular summation of their data indicated that approximately equal yields of neutral solvents were achieved during continuous and after batch operation. The batch fermentation was completed in 100 hours, and the total holdup time of the four-vessel continuous fermentation was 30 hours. The ratio of solvents produced (butano1:acetone:ethanol) was 4.5:2.7:2.8 in batch operation and 5.2: 2.7:2.1 in continuous operation. Their experiments and theo-

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retical analyses were aimed toward the separation of the propagation stage from the solvent production stage, and this aim was at least partially accomplished in the multi-stage systems. Dyr et al. also sought to determine the extent to which cultures degenerated during continuous culture; the butanol fermentation offers an especially suitable test system since it has long been recognized that these organisms frequently lose their fermentative ability upon repeated vegetative subculture. Nowrey (117) earlier had conducted continuous butanol fermentations for 2 weeks (about 650 generations) without restrictive strain degeneration; a low cell (and probably solvent) concentration and a pH above 5.5 probably were instrumental in maintaining strain stability. These findings on the stability of clostridia when held in continuous culture have also been presented and discussed in a recent note by Finn and Nowrey (42a). Although Dyr el al. (37) surprisingly were able to maintain fermentative activity of their cultures through 40 vegetative subcultures in potato mash, they observed gradual degeneration of the culture during continuous fermentation when solvents accumulated. It was for this reason that they attempted to achieve cell propagation in a first fermentor and solvent production in later stages. However, in the same symposium Jerusalimskij (74) reported that “. . . degeneration due to ageing is not unavoidable. We cultivated Cl. acetobutylicum in a continuous flow of stable and suitable medium for some 200 days. During that period more than 4000 vegetative generations were formed, corresponding to 400-600 transfers on normal, non-exchanged media. In spite of this prolonged vegetative multiplication the bacteria fully maintained their capacity of normally fermenting and of sporulation. Therefore the degeneration of a .culture under normal conditions of cultivation can be explained by unsuitable changes of the media.” Thus, in assessing the work from these three laboratories, we conclude that strain degeneration is not necessarily a consequence of prolonged continuous culture. This is a conclusion that is important for successful application of continuous culture methods. Moreover, an even more useful observation than this negation also was made by Jerusalimskij (74) and warrants confirmation and application. Not only did the population of Clostridium acetobutylicum not undergo strain degeneration, but it became increasingly resistant to butanol toxicity. To quote directly again, “The original culture withstood only 0.8% of butanol: after 21 days of growth in the presence of 0.6 % butanol, it withstood up to 1 % of butanol. This increased resistance to butanol was unstable and ceased after the cells had changed to spores, and these proliferated in the absence of butanol. In the course of further adaption the

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resistance to butanol increased still further and became constant. In the course of 200 days the bacteria were able to withstand more than 2.5% of butanol and this level of resistance was then transferred through the spores to further generations. Thus the resistance began t o show a hereditary character.” Since the ability of an organism to produce large amounts of a desirable product may be associated with a high degree of tolerance to the product, it follows that increased productivity in continuous fermentations in some cases might be achieved by a similar adaptation, phenotypic or genotypic, in the original strain population. Adaptation of yeast to specialized media such as sulfite liquor has been shown in several instances to increase the efficiency of the continuous fermentation (61, 62, 75), and such adaptation of microbial populations, probably by mutant selection, is not uncommon. In addition to lactic acid and butanol-acetone, only a few other products have been reported to be studied in continuous fermentation. A gluconic acid process proposed by Porges et al. (128) was essentially a modified, semi-continuous operation. The mycelium in a rotary drum fermentor was allowed to rise to the top after completion of a fermentation cycle. The lower 80 % of the charge was then removed, replaced by new medium, and a new cycle begun with a resulting shorter fermentation period. As many as 15 successive fermentations were carried out in this manner. Subsequently, Porges et al. (127) modified the method by separating the mycelium of the Aspergillus by filtration or centrifugation. Hermann and Neuschul described (67) a two-phase generator process for continuously converting a 25% glucose wort to gluconic acid by means of a pure culture of Acetobacter gluconicum. This (125a) and a similar process by Currie and Carter (28) were patented. With the currently increased demand for sodium gluconate and the favorable growth and biosynthetic characteristics of the fermentation (148), development of a modified single-stage continuous gluconate process would appear likely of success. Other continuous fermentations of some interest include generator-type processes for producing itaconic acid (125) and acetone-ethanol (111). Although it has no immediate industrial use, intracellular glycogen formation in Escherichia coli grown in continuous culture has been extensively investigated by Holme (69); this study is instructive as a model of the continuous production of an “overflow metabolite” accumulating as the result of the limitation of a required nutrient, in this case nitrogen. Of the various chemical products studied in continuous fermentation, citric acid is notable by its omission. The demand for this acid is stable and large, the conditions of the fermentation largely preclude contamination, and the conditions limiting efficient production are reasonably understood.

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Continuous citric acid fermentation offers a good likelihood of success, and increasing competition for the market probably will force development in this area. C. MIXEDCULTURE FERMENTATIONS The most notable examples of mixed-culture continuous fermentations occur in municipal sewage treatment plants, where two principles of continuous processing are widely used. The first method, the so-called trickling filter process (7, 60) was first used in this country in 1890 (99) and still is the method of choice for towns with a population of less than 10,000 people. In this process, raw liquid sewage is distributed over a bed of rocks on which a mixed flora of microorganisms is maintained. The sewage is aerobically digested as it flows downward through the layers of stone; aeration is achieved by the natural or forced flow of air upwards countercurrent to the flow of sewage. In principle, this method is a two-phase, single-stage, continuous operation analogous to vinegar generators. The second example of continuous sewage fermentation occurs in the activated-sludge process (7), which was developed in 1914 (6, 46). In this method, raw sewage is continuously fed into one end of a long and vigorously aerated horizontal tank, and the digested material is continuously withdrawn at the other end. During subsequent settling, some of the sludge, a biomass consisting of a variety of microorganisms, is withdrawn and reintroduced as a massive inoculum with the incoming sewage. In principle, this method may be described as a modified single-stage continuous fermentation. After one of these or similar aerobic processes, sewage solids usually are passed to anaerobic digestion tanks. These often are operated in a semicontinuous fashion; that is, a certain proportion (usually > i o or ?,is) of the fermenting sludge is removed daily and an equal quantity of raw sludge is added. In addition to the digested sludge, which sometimes is further processed for use as a fertilizer or soil extender, the effluent gas represents a product of the fermentation. This gas is rich in methane and often provides the sole source of heat and power for a sewage treatment plant. Further use of sewage sludge as a fermentation raw material for producing hydrogen sulfide (and potentially elemental sulfur) has been extensively investigated by the Department of Scientific and Industrial Research at Teddington, England. The results have recently been summarized by Butlin (20). Investigations were begun on a laboratory scale and then tried in a 50-gallon pilot plant. Like the usual sludge digestion, the process was operated semicontinuously but probably could be made fully continuous. Raw sludge was enriched with 5 % CaSOc.2Hz0. Initially the enriched sewage was inoculated with crude mixed cultures of sulfate-reducing bacteria and

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then repeatedly subcultured until a population of greatly increased activity was developed. Digestion of the enriched sewage eventually was maintained at 30°C. with turnover periods of 10 to 20 days for as long as 3 years. The sulfide produced was swept out by a continuous stream of normal sludge gas (e.g., 70% CHI plus 30% COZ)and collected over acidified saturated brine solution. Yields varied somewhat with the nature of the sludge, but normally lay between 1.0 and 1.5% (i.e,, 100 tons of sludge yielded 1 to 1.5 tons of sulfur) in the case of a 10-day turnover period. The extensively available and large quantities of largely wasted raw sewage make it a fermentation substrate that could be further exploited, and the British group have set an excellent example of its potential use. Hydrogen sulfide formation by organisms such as Desulfovibrio desulfuricans also has been taken advantage of and conducted as a continuous fermentation in a petroleum-purifying process patented by ZoBell (161). Complex sulfur compounds occur in crude petroleum hydrocarbons and often are undesirable in the final product. ZoBell’s process provides for their conversion to easily removed reduction products by means of a multiplate bubble-cap column in which crude hydrocarbon plus hydrogen flows upward and countercurrently against the downward flow of the aqueous culture of bacteria. The gaseous reaction products pass out at the top and subsequently are separated. A somewhat similar process has been patented by Taggart (146), in which gaseous hydrocarbons are continuously converted to the corresponding fatty acids and esters through the oxidative action of Bacillus spp. Both of these processes are examples of two-phase (i.e., aqueous bacteria and gaseous hydrocarbons), single-stage continuous fermentations, although several equilibrium steps are involved in the reactions so that in a sense the operation also may be classified as multi-stage. They are unique in that, unlike the fixed bed of catalyzing bacteria in a vinegar generator, the bacteria occur in a liquid phase and flow countercurrent t o the gas-phase substrate. We have reserved for last a most remarkable example of continuous fermentation that occurs as a natural sulfur-forming process in remote Cyrenian lakes. It was first examined in 1950 by Butlin and was described by him (20) in the recent Czechoslovakian symposium. We see no way of improving on his lucid and fascinating account: “The appearance of the sulphur-producing lakes was striking. The main body of water in brilliant sunshine reflected a vivid milky blue, though a bottle sample was virtually colourless with a slight haze, Bordering the blue was an uneven band of red gelatinous material, stretching in some places several yards from the banks in shallow water. Bulbous formations could be seen in this red material and a few red masses were floating in the water. There was a strong smell of hydrogen sulphide. A deposit of finely divided

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sulphur, 15-25 cm. in depth, covered the bottom of the lakes, and large lumps of calcium sulphate were also found there. The temperatures of the water in the lakes varied within the range of 30-34°C. and in each case the water was overflowing into the surrounding desert, showing that the lakes were continuously fed by warm springs. The fourth lake, producing no sulphur, contained none of the red material. . The water was saturated with calcium sulphate and contained various other mineral salts, but only traces of organic materials. Numerous sulphate-reducing bacteria were present. The red gelatinous material bordering the lakes consisted of masses of coloured photosynthetic bacteria, mainly Chromatiurn (red) and Chlorobium (green). All these organisms are obligate anaerobes. Blue-green algae, fish (Cyprinodon), and a few aerobic bacteria were also present. Our observations, supported by many experiments carried out later with mixtures of pure cultures of the organism concerned, led to the conclusion that most of the sulphur was produced by a combination of two microbial reaction: (1) sulphate-reducing bacteria converted sulphate to sulphide which (2) the colored photosynthetic bacteria oxidized to sulphur. At first it was not at all clear how the sulphate-reducing bacteria obtained energy for the reduction of sulphate, since the organic content of the water was very low. Experiments with mixtures of pure cultures of sulphate-reducing bacteria and of Chlorobium and Chromatium in suitable culture media with no carbon source other than NaHCOa showed that Chlorobium or Chromatiurn, which can satisfy their own carbon requirements by photosynthesis from COz , are also capable of providing suitable organic materials for the sulphate reducers. Thus, for the production of sulphur in the lakes, only inorganic compounds were necessary, with sunlight as the primary source of energy. It is possible, of course, that the continuous supply of organic matter (however small) from the springs, as well as any hydrogen evolved by microbial action from the bottom of the lakes, contributed to the final result. Also, atmospheric oxidation of sulphide was undoubtedly responsible for some of the sulphur, but only for a very small amount, as shown by the small quantities of sulphur produced in the fourth lake mentioned above. . . . In its simplest form we can consider it (the microbial reaction) as proceeding according to the following sequence: Chlorobium Desulphovibrio sulphide sulphur in near autoSulphate Chromatium trophic conditions, using suniight as the primary source of energy. The process is not, of course, strictly continuous. It is complicated by darkness, climatic conditions, adventitious addition of organic matter (foliage, animal and bird excrement etc). Also, Chromatiurn is able to carry the oxidation of sulphide, through sulphur, to sulphate, and thus part of the process becomes continuous in another, cyclical, sense. . . . We have been able to reproduce

..

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these reactions in the laboratory using mixtures of pure cultures of sulphatereducing and photosynthetic sulphide-oxidizing bacteria in batch processes. Investigations using modern techniques of continuous culture would not only be most interesting, but might well be profitable.’’ With the last conclusion, we fully agree! How better to forecast the future of continuous industrial fermentations?

ACKNOWLEDGMENT Support for preparation of thin review and for our experimental work came from the Rackham School of Graduate Studies of The University and from Eli Lilly and Company.

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The large-Scale Growth of Higher Fungi RADCLIFFE F. ROBINSON AND R. S. DAVIDSON Batlelle Memorial Institute, Columbus, Ohio

I. Introduction.. . . . . e . . ......................................... 11. History of Mushro 111. Research in the IV. Development of Deep-Vat Fermentation Methods ....................... V. Food Yeasts an VI. Mushrooms in Submerged Culture.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VII. Problems t o be Met in the Large-Scale Production of Higher Fungi in Submerged Culture.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . .... .......

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1. Introduction In the field of microbiology no group of organisms offers a greater challenge to the researcher or a greater opportunity to the food technologists than the higher fungi. Of the more than 37,500 species of fungi, of which 2000 are estimated to be edible, few have been studied thoroughly from a standpoint of their commercial potential. Less than 25 species of higher fungi are accepted widely as food, and only a very few species have attained the level of an item of commerce. However, these plants must be considered as one of the world’s greatest natural resources since they have the ability to transform nutritionally valueless substances into high fat and protein foods. Many fungi can be grown in large masses on cheap carbohydrate material such as molasses, vegetable waste, potatoes, and sulfite waste liquor from the wood-pulping industry. Other fungi are being used in the beer and whiskey industries; still others have been developed for fat production. It has been estimated that 1 acre of concrete-enclosed pond used for the propagation of fungi could produce fat equivalent to that yielded by 25 acres of a vegetable oil crop. Proteins produced by fungi offer important industrial possibilities. Fungi can be produced in quantity within a few hours or days. Their efficiency in protein production from a given quantity of carbohydrates is about 65 % in comparison with about 20 % for pork, 15% for milk, 5 % for poultry, and 4% for beef. The production capacity of an acre of land has been estimated to be ten times greater for fungus proteins than for meat proteins. 261

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Many scientists have visualized the fungi as an answer to the food problem in an overpopulated world. Nutritional potentialities of fungi for this purpose are great; however, the need for new food sources may be a century away. Meanwhile, the higher fungi should be recognized for their immediate potential contributions: new and flavorful food, high-protein nutritious products, and economical advantages in food production. The fungi are often divided by mycologists into two groups: higher fungi and lower fungi. Bessey (1907) placed all the higher fungi in a phylum called Carpomyceteae, i.e., fruit-producing fungi, referring to spore fruit production. Other mycologists have defined the higher fungi as those having a conspicuous and fleshy sporocarp, e.g., members of the Hymenomycetes and Gasteromycetes among the Basidiomycetes, and Helvellales, Tuberales, and Pezizales among the Ascomycetes. A less complicated definition of higher fungi and the one here used places in the Ascomycetes, Basidiomycetes, and Fungi Imperfecti in this group and includes only the Phycomycetes in the lower fungi (Bessey, 1950). The mushrooms, particularly the mushroom of commerce, Agaricus bisporus, are the best known higher fungi. The term mushroom is most frequently applied to the gill-bearing fungi as characterized by Agaricus, but in the broadest sense include the “boletes,” with pores replacing the gills; the “puffballs”; the “morel,” with a spongy reproductive structure; and even the subterranean “truffles.” Boletes, morels, and truffles may occasionally be obtained on the market as special luxury items, but Agaricus has become the mushroom of commerce. Agaricus can be cultivated, while practically none of the other desired higher fungi have been grown successfully. The fruiting bodies or sporophores of the rarer fungi which are frequently sold canned must be gathered from the wild. Few people in North America eat mushrooms for their food value. These fungi are eaten because of their flavor, and are used in soups, gravies, and omelettes or as a condiment or garnish for meats and other foods. I n Europe, mild-flavored as well as piquant mushrooms are considered an important food source. European countries have been plagued for centuries by food shortages; consequently people have been more prone to make use of such natural products as mushrooms. The Europeans are considerably more “mushroom-conscious” even flavorwise than Americans, and the per capita consumption is much greater in Europe. The cultivation of the mushrooms started in Europe in the 17th century, or possibly earlier. Interest in the product has become world wide and has developed into a multimillion-dollar business. Changes in the methods of mushroom growing have been so few and gradual that the process today is substantially the same as that described in 1904 (Duggar, 1904). Many attempts have been made to adapt some of the edible mushrooms

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other than Agaricus to cultivation, but such attempts have met with only limited success. The possibility of growing the mushroom mycelium as well as, or instead of, the sporophore was apparently not seriously considered until about 1943. At this same time large-scale production of penicillin by the submerged-culture of Penicillium species was begun. Success in growing fungi other than mushrooms stimulated research on the production of mushroom mycelium in submerged culture by several investigators. Because of its commercial importance, Agaricus bisporus naturally received first attention, and the research resulted in several patents. But, it was not an unqualified success; commercial production was not achieved. While there was no difficulty in growing the mycelium, it did not have the full flavor of the fresh mushroom and so could not be marketed as an acceptable food product. The production of “morel” mycelium in submerged culture was more successful. The mycelium thus produced has the flavor of the morel, and the process has been experimentally engineered to full-scale production in 2000-gallon fermenters. The product, which has the appearance of grains or pellets, has passed taste and cooking tests and appears to be on its way to public acceptance and commercial production. Possibly the main detriment to acceptance is the public’s lack of familiarity with the natural morel flavor. The success with the morel gives promise of additional success with other higher fungi. Although each species has its own nutritive requirements, as long as the mycelium can be grown on laboratory media it can probably be adapted to growth in submerged culture.

II. History of Mushroom Culture Edible fleshy fungi were undoubtedly one of man’s earliest foods, and considerable folklore, profuse in mystery and superstition, has been built around them (Rolfe and Rolfe, 1928). The Greeks and Romans consumed many species of edible fungi and probably were familiar with Agaricus campestris, although they could not cultivate it (Buller, 1915). The Japanese and Chinese learned several centuries ago the technique of cultivating a tree-inhabiting mushroom called the Shii-take (Cortinellus berkelyanus). Millions of pounds annually are raised today on logs cut for the purpose (It0 and Imai, 1925). The “Padi-Straw” mushroom Volvuriu volvacea has been cultivated for many years in southern China and has been introduced into Malaya and the Philippines. This mushroom is able to compete more successfully with other fungi on undecomposed straw than our common cultivated mushroom, but neither the Padi-Straw nor the Shii-take are grown in Europe or North America. The first attempts to cultivate or increase the production of Agaricus

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campestris were by the application of animal manures to areas where

inoculating material had been strewn. Early records show that the practice of gathering wild spawn and planting it in prepared horse manure occurred in the 17th century (Lambert, 1938). The French botanist, Tournefort, in 1707, was one of the first to describe the primitive methods then used to raise mushrooms. At first mushrooms were cultivated in the open. The utilization of the extensive cave system under the city of Paris marked one of the most significant developments in the mushroom history. The removal of limestone from deposits under and surrounding the city to construct the early buildings in Paris was so extensive that many miles of galleries and tunnels were available. The subterranean climatic conditions were well suited to mushroom culture, and the industry grew rapidly. By 1900 it was estimated that there existed approximately 1500 miles of mushroom beds winding through the caves, These beds were built entirely on the floor and were merely ridges of composted manure about 2 feet wide a t the base and about 1% feet high. Beds of this type are seldom used today, except in England, and have been almost entirely replaced by benches or trays. These are much more economical in terms of space and time, and make it possible to control disease and insect pests more easily. From France, mushroom culture spread to other European countries, particularly England and Germany. The first mushroom growers in America apparently were English and French gardeners who had been associated with the industry in Europe. Using European methods, they established a thriving business on Long Island to supply the New York market. About 1885 several greenhouse men in the Kennett Square area of Pennsylvania began to grow mushrooms under benches as a secondary greenhouse crop. This enterprise was so successful that special houses were constructed with the specific purpose of growing mushrooms under controlled conditions. This move was also an immediate success. Encouraged by the Kennett Square group, mushroom growing eventually became an industry estimated to be worth more than 45 million dollars, exclusive of enterprises depending on mushroom growing, such as trucking, mushroom canning, and supply dealing. With the extensive production facilities in the Kennett Square area, Pennsylvania produces about 37,000,000 pounds of mushrooms per year, almost five times as many as New York, the next most productive state, and nearly one-third of the entire United States production. The bulk of the industry is concentrated in the East, largely because of historical and not geographical reasons, but the industry has spread and about half of the states now produce some mushrooms. Sales of fresh mushrooms have not increased in recent years, but production of canned mushrooms has increased from 2,500,000 pounds in 1929

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to 18,000,000 pounds in 1946. The amount used for soup has grown from 1,000,000 pounds in 1937 to more than 24,000,000 pounds today. The immediate future of the mushroom industry is not predictable. At present mushrooms are generally considered a luxury food, and prices are extraordinarily variable from year to year with relatively high production costs. The domestic canned product, which is a substantial part of the industry, has had increasing competition from foreign goods. The industry in this country has had the advantage of excellent research but suffers from high labor costs and market fluctuations.

111. Research in the Mushroom Industry Prior to around 1900, research on mushroom production was a carefully guarded secret of the individual grower. Investigational work at that time could scarcely be called research, but it resulted in some information gathered by a trial-and-error method. At about the turn of the century, the United States Department of Agriculture and several agricultural experiment stations, notably the Pennsylvania Station, became interested in the mushroom industry. Data were collected, and detailed bulletins on improved techniques for growing mushrooms were distributed. Today, information has been published by these agencies on every phase of mushroom growing, and an understanding of the various aspects of mushroom cultivation has resulted which is unquestionably superior to that in Europe (Kligman, 1950). The method of spawn preparation was formerly the individual supplier’s or mushroom grower’s secret, and for hundreds of years the technique was without significant improvement. The primitive method consisted simply of digging up clods of earth in soils known to contain mushroom mycelium and encouraging further growth by filling the holes or trenches with manure. In this way a spawn maker could keep a stock of original spawn in the field. The French spawn makers in many cases planted spawn in a special bed of composted manure and when the spawn had permeated the bed, the entire mass was broken up and flaked. The English modified the technique somewhat by inoculating spawn into “bricks” of horse manure, cow manure, and loam. The bricks when ready were broken up for use in prepared beds. Brick spawn, as it was called, was popular in this country prior to 1920. Needless to say, the mushrooms produced from such spawn were not always uniform. Moreover, insects and undesirable fungi were often unsuspectingly transmitted to the growers’ beds. In about 1890, spawn of known origin, which was developed from spores and inoctlated into sterilized media, was placed on the market in France. This pure culture spawn commanded a very good price until a method for germinating spores was published in a Cornell University Bulletin in 1901.

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Published at about the same time was a method of making pure culture spawn by culturing tissue from a mushroom cap and growing it on an appropriate medium under sterile conditions. As a result of these findings, the technique of producing high quality spawn was mastered. In 1920, spawn was first put up in sterilized bottles of horse manure, tobacco stems, or rye grain. This represented an important improvement because it assured the maintenance of a pure, viable culture. Spawn growing is now well standardized. There are only a few large spawn makers in the United States. The business is highly specialized and requires skilled personnel and technical equipment. The main responsibility of spawn suppliers at present is to select and supply tested mushroom strains. Composting of manure for use in mushroom growing was started in the 18th century. In composting, which is necessary for satisfactory mushroom growth, the more soluble and readily decomposible food materials are used up by bacteria; when bacterial growth has slowed down, the mushroom spawn can hold its own against other competing microorganism. The process of composting horse manure has changed but little since it was first used for growing mushrooms. However the machine age has displaced the horse in industry and the manure supply has steadily diminished as a consequence. At present practically all available manure is obtained from racing stables. The mushroom industry some years ago saw “the handwriting on the wall” and sponsored research on the development of synthetic composts. In recent years the chemical and biological factors of composting have been studied and are more clearly understood. This study has permitted a more realistic approach to the development of synthetic composts, which are necessary to supplement the existing manure supply. A practical method for making a synthetic compost was published in 1923 in a report from the Rothamstead Experiment Station (Hutchinson and Richards, 1923). This compost was not developed particularly for the mushroom growers, however, and several years passed before it was adapted to their use. Since then considerable additional research has been done both in Europe and in this country. In developing synthetic composts it was originally postulated that the various components could be assembled in the synthetic compost heap from the known analysis of the inorganic composition of manure and of mushrooms. Hence it was held that the basic ingredients could be straw, fodder, cobs, oat hulls, or any other material just so long as the theoretical ratio of nitrogen, potassium, phosphorus, and secondary and minor ‘elements existed. Had this view been entirely correct, the problem would have been

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simplified and all composts of equivalent chemical composition would be of equal value. Experiments did not bear out this hypothesis, however. Plant residues have, in addition to their chemical structure, different physical qualities that greatly affect their composting properties. It was also determined that the chemical form in which the nutrients, particularly nitrogen, were available was very important. Therefore, it became necessary to evaluate the physiological role of each ingredient separately. Experiments showed that straw was not merely a filler in the compost but was an important source of carbon and that straw well wetted with urine had a production capacity equal to manure composed chiefly of droppings. However, straw alone as a carbon source in synthetic compost was too loose and interfered with the heating of the pile. On the other hand, corn fodder alone was too dense. A mixture of the straw and corn fodder was superior to either by itself. Excess nitrogen diminishes the yield, and the nitrogen-carbon ratio must not be too extreme. Because a nitrate or ammonium salt alone cannot supply all the required nitrogen, a protein concentrate, such as low quality cereal grain, must be incorporated. Brewer’s grains are satisfactory, and a smaller amount is necessary because of their higher protein content. When one-third or even two-thirds of a mixture is Synthetic, the yield is comparable with that of pure manure, and mushrooms may be grown on the synthetic product alone. However, no wholly synthetic substrate has proved to be better than the natural compost. Another improvement or modification, which was started in the United States about 1915, is known to mushroom growers as the “sweat out.” Composting manure, as it is placed in the beds, lacks uniformity for the growth of spawn. Some may be more favorable for the growth of contaminating molds. This condition is corrected by the sweat out prior to inoculating it with spawn. In this process the temperature of the compost is raised to 130°F. in the relatively shallow trays. This temperature is easily reached in tight houses by means of natural fermentation in the beds. Detrimental overheating is prevented by ventilation adjustments. An additional source of heat, usually steam, may be used in the rare cases where the beds fail to heat properly by themselves. This pasteurization requires seven to ten days. No attempt is made to cool the houses until the temperature has begun to drop of its own accord. The “sweat out” is generally considered to kill the insect pests and nearly all the molds of importance. For this reason, the use of fumigants, other than live steam or steam and formaldehyde, is not a regular practice with most growers at present. A relatively new culture innovation which shows promise for the industry

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is the (‘two-zone” system. This system consists of running the spawn and cropping the mushrooms as independent operations in different houses. To achieve this, movable trays rather than stationary beds are used. The advantages are several, including decreased cost because of the increased number of crops possible. As soon as the final crop has been harvested, a house within a period of hours may be emptied, cleaned, and filled with fresh trays already sweated and spawned. The beds are thus practically always producing and, with refrigeration during the summer, five or six crops a year can be grown. On the permanent-bed system and without refrigeration, usually two and not more than three crops are obtained annually. Disease control is also more efficient with the two-zone system for, if necessary, a diseased tray can be quickly removed before the infestation spreads throughout the house. Moreover, the spawn is grown under such highly controlled conditions that infestation by insects is more readily prevented. Also, since the trays are closely packed together during the sweat-out before spawning, a good sweat-out is more certain. One of the most recent phases of commercial mushroom production is that of mushroom genetics. In this country all the cultivated varieties of mushrooms belong to the single species Agaricus bisporus, a derivation from the wild Agaricus campeslris, but the species contains several varieties or strains that are quite readily distinguishable. A “brown” mushroom was the first to be cultivated. It was vigorous and of good flavor but had a brown scaly cap and was superseded by a “cream” mutation. In 1927, a clump of pure white, smooth-capped mushrooms appeared spontaneously in a bed of the cream variety, and all the white mushrooms on the market today have been propagated from this cluster, Neither the “brown” nor the “cream” varieties are now cultivated to any extent. New mushroom strains are ordinarily obtained by isolating single spores and growing the mycelium from each one separately. Up to one-fifth of the spores cultured will produce sterile or nearly sterile mycelium, but among the remainder certain differences will become manifest. There will be differences in yield, size, color, and quality and in many other respects. Naturally, the constant objective of the spawnmaker is to secure a strain which will yield more mushroom of higher quality. New strains are constantly being produced and some appear on the spawn market. Nevertheless, the process of obtaining new strains by mutation from single spores is a tedious one. Following the lead in other fields, the possibility of using X-rays and mutant-inducing chemicals to speed up mutations in the mushroom is now being investigated. Until recently, serious losses resulted from the invasion of the mushroom house by various insects and pests. Although such losses have by no means

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been entirely eliminated, they have been reduced markedly owing to improved entomological and pathological research. At one time the control of mushroom flies was difficult; however, this situation was changed almost overnight with the advent of DDT. Because there has been a tendency for some insects to build up a resistance against DDT, other insecticidal preparations are constantly being studied. The battle against mushroom pests has been won, a t least temporarily, except for the lack of an effective agent against mites. Most of the miticides ordinarily used for control in greenhouses are not recommended for use on mushroom beds because of their toxicity to the developing crop. Sanitation is still the most important method of control. Several relatively new preparations show promise as miticides for this use. Our knowledge of control of the various diseases of mushrooms and mushroom beds has increased greatly. Unfortunately no one fungicide or treatment is the best for all diseases, but the newer fungicides are increasingly effective. The older sprays such as lime sulfur and Bordeaux have been replaced by certain chlorinated phenol compounds and dithiocarbamates. No fumigant has yet replaced formaldehyde in the houses, but a combined formaldehyde and steam fumigation has been found to be more effective than either alone. Research has made the mushroom industry less of a gamble than formerly.

IV. Development of Deep-Vat Fermentation 'Methods Deep culture methods have really been used a long time. The gallic acid discovered by Scheele (1787) probably came from mold fermentation (Van Tieghem, 1867a, b). Certainly gluconic acid [observed in bacteria in 1880 (Boutroux, ISSO)] was prepared by mold fermentation (Molliard, 1922) and citric acid goes back to 1893 (Wehmer, 1893; Karow and Waksman, 1947). Submerged culture production of all three acids (Prescott and Dunn, 1949) has been common for some years and the early technology was probably based on the earlier Amylo process (Wehmer, 1914; Calmette, 1902). Development of shake culture (Kluyner and Perquin, 1933) and revolving drums (Takamine, 1914) eventually resulted in modern deep culture technology [history to 1949 by Hromatka and Ebner (1949)l in which aeration (Stefaniak et al., 1946) baffles, agitation (Hixon, 1946; Rushton, 1946; Mack and Uhl, 1947; Finn, 1954), and temperature (Moyer and Coghill, 1946) play important roles.

V. Food Yeasts and Molds in Submerged Culture Yeasts were undoubtedly the first fungi propogated by deep-vat fermentation methods (Frey, 1930). Modern yeast production is of three types: for special purposes, such as baker's yeast; as a by-product in the fermen-

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tation industry; and as a means of using waste products to an economic advantage. Food yeast makes a satisfactory supplement to a diet deficient in animal protein or B vitamins (Sure, 1950) and can be grown on surplus carbohydrates (Basaca, 1953; Reiser, 1954; Wiley et al., 1951). At present a number of installations are producing food yeast from waste or low grade food products (Gilbert and Robinson, 1957; Thaysen, 1945; Saeman et al., 1945). Torulopsis utilis is usually the species of choice, however, Torula pulcherrima, Monilia candida, and Candida arborea have been used. Most yeasts are low in cystine and molds might be a better fungi for human food production providing they are equal to food yeast in other respects. The use of molds for this purpose has received very little consideration.

VI. Mushrooms in Submerged Culture The idea of growing mushrooms for their mycelium rather than their sporophores developed because of, or a t least was stimulated by, the success of deep-tank fermentation in the antibiotic field. Research was started almost simultaneously by several investigators, and it was soon found that the mycelium of some strains of mushrooms was adaptable to this method of cultivation (Humfield, 1948). Agaricus bisporus, because of its commercial acceptance and the ease with which it is propagated, was naturally the first species to be tried. The technique offered the promise of largescale, low-cost production of a mushroom-flavored food material, and the possibility of utilizing quantities of low-cost growth media such as citrus press water or cannery wastes. The suggestion was also made that vatgrown mycelium might be sprayed on mushroom trays after the sweat-out in lieu of spawn and thereby save much of the labor that was used in planting regular spawn. Early investigators found that the mycelium would grow on many media; however, it had certain requirements for sugar and other essential nutrients. There was considerable difference in the strains of Agaricus bisporus as far as their adaptability to submerged culture was concerned. Humfield (1950-1951) isolated and tested more than forty strains and found three particularly well adapted to growth in an agitated, aerated liquid medium. Block and associates (1953) cultivated successfully Agaricus blazei, a southern species which can grow at a higher temperature than A . bisporus. Joseph Seuecs (1957) since 1946 has screened many species of mushrooms for their ability to grow in submerged culture and has been granted several patents in connection with submerged-culture techniques. He reduced the number of adaptable species to a relatively few, but among these were several outstanding agarics, a morel (Morchlla), and a few members of the

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genus Boletus, which in nature is believed to exist only in symbiosis with roots of certain tree species. Szuecs established requirements for a species that he considered had to be fulfilled before industrial production by deep-vat fermentation could be possible: (a) The mushroom should have prolific growth characteristics when grown in liquid media to shorten the production cycle. (b) The selected isolate should be genetically stable, through countless transfers. ( G ) Preferably, it should have a certain defensive ability toward cultural contamination. (d) The mushroom should have convenient nutrient requirements; should be able to grow on low-cost raw material, and should have the ability to synthesize amino acids from simple inorganic ammonium compounds, i.e., autotropic for nitrogen. ( e ) The mycelium when grown in submerged culture should not produce secondary spores. (f) It should form organized tissue in submerged culture with as desirable an appearance as possible. (9) The mycelium should have a firm texture and not be fibrous or tough. (h) The mycelial mat or tissue should be palatable and of characteristic piquant flavor. (i) It should be digestible. ( j ) The vat-produced tissue should not be toxic. Several strains of Agaricus bisporus met most of these requirements. They grew rapidly in the aerated liquid media, and production was very satisfactory. Good yields were obtained on fruit and vegetable wastes, with certain additions to increase production, and also on synthetic media containing dextrose and inorganic salts. Satisfactory growth was obtained with several sources of nitrogen including ammonia, urea, peptone, monosodium glutamate, a mixture of amino acids, and other nitrogen compounds. Most of the' sugars investigated, which included, hexoses, pentoses, and disaccharides, gave good growth. Soluble starch and dextrin were also suitable sources of carbohydrates. Apparently Agaricus bisporus could utilize a wide range of carbohydrates for its mycelial growth, excluding sodium carboxymethyl cellulose. The protein, mineral, and vitamin content of the Agaricus mycelium compared favorably with those of commercially grown mushrooms. The strains of Agaricus bisporus used produced in submerged culture numerous secondary spores similar to those first described by Kligman (1942). These, and the small clusters of mycelium developing from them,

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were not objectionable if the mycelium were to be centrifuged and pressed. They present a problem, however, if a uniform nodular product is desired. The most serious problem in mycelial production of Agaricus bisporus was that the mycelium did not develop a fresh-mushroom flavor. Humfield and Sugihara (1952) found that unless growth was prolonged beyond the point of total sugar utilization, the product, although essentially the same nutritionally, was quite flavorless. Even a t its best, the flavor of the Agaricus mycelium was not that of the fresh sporophore. The production of a true mushroom flavor still remains the primary problem before commercial submerged production of Agaricus bisporus becomes practical. In appraising the flavor of their A. bisporus mycelium, Humfield and Sugihara conservatively say “. . . the flavor is designated as mushroom-like pending more extensive tests.” Fundamental questions that remain to be answered are whether the true Agaricus mushroom flavor is a product of only the specialized cells of the fruiting body and cannot be produced by the vegetative mycelium or whether precursors normally found in soil or manure but not in the laboratory media are required for synthesis of the flavoring compounds in the mycelium. One of the mushrooms screened by Szuecs was a Morchella which he had collected in an orchard a t Yonkers, New York, in April, 1947. The Morchella tissue was cultured but, unfortunately, the sporophore was not preserved for accurate identification by a taxonomic mycologist. Szuecs was later provided with a culture of Morchella hortensis Boud. by Professor Paul Haudoroy of Lausanne, Switzerland and, as the cultural characteristics of both the American and European specimens appeared to be identical, Szuecs considered his “morel” to be Morchella hortensis even though this species had never been described from the United States. The ease with which “morel” mycelium will grow on many media has been known for many years (Yvoire, 1889). The spores are easy to collect and will germinate in less than 24 hours, The mycelium grows vigorously. No one, however, has had more than sporadic success in the production of “morel” sporophores. It would seem that a fertilization phenohenon is one of the requisite factors for fructification. Each individual strain of mycelium developed from a single spore is presumably self sterile, and fertility can be exhibited only between certain compatible strains. Since some fungi closely related to Morchella have this characteristic (Dragton, 1934), it is quite possible that future research will show the same to be the case for Morchella. Until a fructification is produced, however, the identity of the Szuecs Morchella will remain uncertain. This species or strain, whatever its identity, proved to be ideal for submerged culture conditions. It had many desirable characteristics not possessed by some of the other mushrooms in culture and appeared to be

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outstanding in its potential for commercial production. Szuecs, after several years work in studying nutritional characteristics and improving cultural techniques, chiefly on a laboratory scale, was granted a patent (Szuecs, 1956) that gave him broad protection on the submerged growth of any “morel” or other member of the “Helvelaceae” in aerated agitated liquid media. A subsequent patent (Szuecs, 1958), extended this coverage to include the submerged aerobic growth of the mycelium of all edible mushrooms. The main limiting factor to the commercial use of Agaricus bisporus mycelium was its lack of flavor, but the Morchella mycelium has the piquant morel flavor regardless of the stage a t which it is used. Morchella mycelium also has an omnivorous character and can grow on an extremely wide range of nutrient materials including various kinds of molasses, a material on which Agaricus campestris grows but does not thrive. Morchella mycelium in addition has the ability to synthesize from an entirely inorganic source, except for sugar, mycelial tissue with a high vitamin content (see Table I) and with from 20 to 45 % protein (dry matter basis). It can synthesize entirely the growth-promoting substances which it requires, a cultural characteristic not shared by most other mushrooms. Dr. James Y. P. Chen, Professor of Pharmacology at Marquette University School of Medicine, made a thorough investigation of the toxicity of the Morchella mycelium produced in submerged culture. Extrapolating from experiments with rats, he found that a person would have to consume 60 pounds of the mycelium per day before there would be any ill effects. The product has been approved as a food by the Pure Food and Drug Administration of the USA. In any size container or vat, production of morel mycelium is achieved in approximately 72 hours but, unlike yeast fermentation, which can be a continuous process because new media may be inoculated with a portion TABLE I VITAMINS I N MORCHELLA MYCELIUM Vitamin

Micrograms/gram dry weight

Thiamin Riboflavin Niacin Pyridoxine Pantothenic acid Biotin Folic acid Vitamin B12

3.92 24.6 82.0 5.8 8.7 0.75 3.48 O.OO362

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of the old, the production of “morel” mycelial pellets must be a batch process. For a 72-hour cycle, the media must be inoculated or “seeded” with minute young pellets previously cultured from homogenized mycelial fragments. The stock culture is maintained on nutrient agar slants and the content of one slant culture when homogenized in a “blendor,” will serve to inoculate eight 250-ml. Erlenmeyer shake flasks. The flasks containing 100 ml. of liquid nutrient media are agitated for 72 hours on a rotary shaker set a t a speed of about 100 r.p.m. With lower agitation the mycelium is inclined to clog or grow together to form a few large aggregates; a t higher speed, the mycelium will not nodulate well. After 3 days of agitation at room temperature, the liquid shake culture is transferred to the succeeding stage of inoculum preparation which is a 7-liter aerated culture bottle. In commercial production, inoculating culture material is placed in a 5-gallon container and homogenized with a high-speed impeller, which is slowed down after homogenization of the mycelium in order that initial nodulation can take place. At present morel mycelium is being grown commercially in 100-gallon and 2000-gallon vats for research purposes. Morel mycelium is removed from the vats as spherical nodules varying in size from to 1 inch in diameter. The mycelial nodules are dewatered to bring the moisture content down to 90%, approximately that of fresh mushrooms (Anderson and Fellers, 1942). In the most efficient runs the mycelial nodules range in size from to inch in diameter. The product can be canned, fresh frozen, dried as pellets, powdered, or converted to a liquid flavor concentrate. In taste-panel tests with meat, preference was shown for the fresh-frozen nodules, but the dehydrated nodules also retained the characteristic morel flavor. Although the morel mycelium now produced is an acceptable product, additional work is still in progress with strains of identified Morchella species. This research has shown many differences between species and strains of species, not only in cultural characteristics but in the appearance, quality, and flavor of the product. One species or strain of morel may he superior for marketing as a fresh frozen product, another for drying and powdering for soup, and the more piquant for a flavor concentrate. Each species of Morchella has its own distinct flavor, and this flavor can be recognized and distinguished in the mycelium. It is possible that in the future the gourmet will be able to obtain Morchella products in any preferable form as well as choose the species for which he has a preference. Considerable research has been conducted on the cultivation of truffles. So far as is known, no one has successfully induced the truffle (Tuber) fungus to produce a fruiting structure in culture or in field cultivation. Satisfactory growth of mycelium can be obtained in submerged culture,

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but the mycelium lacked the flavor of the sporocarp. Small truffle mycelial nodules can be produced in shake flasks, but in larger aerated carboys an agglomerated mass of mycelium develops rather than discrete mycelial nodules or pellets. The truffle mycelium sporulates during the fermentation process, similar to Agaricus species rather than forming mycelial pellets, as Morchella. Undoubtedly other edible species of higher fungi can be adapted to growth in submerged culture, and some of these may prove to be of economic significance.

VII. Problems to be Met in the Large-Scale Production of Higher Fungi in Submerged Culture

The fact that morel mycelium can be grown on a commercial scale with an acceptable flavor far from terminates the need and desire for further research on the production of vat or submerged cultures of higher edible fungi. The development of any commercial process usually points out many new avenues of research on the same process, suggests potential new uses of the basic concept, and stimulates a desire in the researcher to understand the process. The ease with which morel mycelium can be propagated in commercialsized deep-vat fermenters suggests that the additional problems to be met in large-scale production of mycelium of the higher fungi will be most likely more biological than mechanical. The main engineering and instrumentation difficulties appear to have been overcome in the development of volume production of antibiotics, and there remains in this category, for the most part, only the problems connected with the processing of the product after removal from the fermenter. Dewatering of the mycelium offers little or no problem in small-scale production but must be considered in commercial production. I n a 2000gallon fermenter with 1500 gallons of media, the yield is estimated to be 136 to 2 tons of fresh product. The water must be removed from this volume of material without modifying the texture or changing the flavor, which is sensitive t o high temperatures over long periods of time. Several methods of dewatering mushroom mycelium have been investigated. In centrifuging, a Tolhurst basket centrifuge was used for 10 minutes a t 1179 r.p.m. per minute. At the end of this period a cake approximately 2 inches thick was obtained which had a moisturg content of 74% by weight. Although the centrifuge was an excellent dewatering device, the centrifugal force exerted on the mycelial pellets ruptured many of them and the product recovered from the centrifuge was as a dense cake. This method of dewatering might be practical only if the dried mycelium is to be powdered for soup or flavoring. The dewatering of mushroom spheres with a Byrd-Young rotary vacuum

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filter has been studied. It was found that the Byrd-Young filter would not work efficiently owing to the ball-like shape of the mushroom pellets. These more or less spherical bodies would not cling to the vacuum filters, and the vacuum was ineffective because of the spaces between the spheres. Inclined screens have also been used in the recovery process, and from very limited tests this method showed some promise. In dewatering by any procedure, however, too much pressure causes the mycelial spheres to felt together. This is particularly undesirable where the product is to be frozen as firm rounded pellets. Washing of the mycelium was greatly facilitated when the inclined screens were used. Since the residual culture liquor has a flavor quite distinct from that of the mycelium, the mycelial spheres must be washed sufficiently to remove this extraneous flavor. The optimum amount and method of washing are still problems for research. The drying temperature is important in the quality of dried morel mycelium. A drying temperature of over 120°F. produces an inferior product. Results of laboratory trials indicate that it is preferable to dry at about 110"F. Several methods of drying have been studied, including the Louisville steam-tube rotary drier, the Proctor and Schwartz drier, and the GATX drier. No one method has yet been adapted as superior to the others, and research is still being conducted. The optimum temperature for the growth of Morchella has not yet been determined, but this must be known to insure the most efficient production. Laboratory and pilot-plant fermentation runs were more efficient at temperatures below 70"F. than a t higher temperatures. Contamination, always a problem in vat fermentation, seldom occurred at the lower temperatures. An acceptable formula for the production of morel mycelium has been developed, but studies are necessary to establish the most productive and the most economical. The fermentation process in the production of the mycelial spheres requires about 72 hours. During this time the liquid clears and a gradual decrease in the pH occurs until about 0.25% of the sugar remains, after which the pH rises again. It has been found that the best time to harvest the crop is just before this rise, because autolysis is initiated in the spheres beyond this point. The time of harvest may be determined very accurately with each nutrient formula, but for the best production the time of harvest must be redetermined a t each formula change. The flavor of the mycelial product depends upon the fermentation formula, and improvements or a t least modifications in flavor may be possible through additional formula research. The morel mushroom has a distinctive flavor well known to gourmets, but is not the mushroom flavor familiar to the general public. Modification of the flavor might be desirable for certain uses of the mycelium. More recent studies indicate that various species and strains of species of Morchella have different flavor and textures. Much research lies ahead to evaluate the new products obtained with these

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species, and of course with each species or strain, the media, time of harvest, and optimum methods of harvesting and processing must be determined. A variety of new taste products could arise from such efforts. The effect of nutrient media and harvest procedures on taste development are challenging problems and point back to the Agaricus and truffles, which t o date have not developed their characteristic flavor in the mycelial product. Recent investigations have shown that truffle mycelium on agar slants develop the truffle odor. It is probable that a clearer understanding of the basic problems of flavor biosynthesis must be acquired before success can be obtained with other desirable higher fungi. The use of deep-vat fermentation production of the higher fungi has been considered as a backlog against a future need of high protein food for the world; however, current uses of this process are also evident. The yeast, molds, mushrooms, and other fungi have the ability to produce many chemicals as a function of their metabolic processes. Some of the chemicals produced are miscellaneous nitrogen compounds including alkaloids, glucosides, and saponins. Others may be materials such as carotenoids, auxins, hormones, enzymes, antibiotics, and vitamins. Some of these are so complex that no means of synthesizing them chemically has yet been found or even tried. Tyrosinase, 5-hydroxytryptophan1 and muscarin are among the many compounds that may be obtained from the higher fungi. These materials are interesting per se; however, their use as intermediates for the preparation of other organic chemicals might be of even greater significance in fundamental studies of biosynthesis. Heretofore, the evaluation of many of the chemically significant fractions of the nonedible fungi have not been investigated because specimens of such fungi have been obtainable only in minute and sporadic quantities from the wild. With the advances which have been made in the deep-vat production of higher fungi, our knowledge that the mycelium can carry the chemical qualities of the sporophore, and our research on the effect of media on mycelial biosynthesis, a new role for the higher fungi may be found in the industrial chemical field. The economic importance of the higher fungi will not skyrocket overnight because of this potential usage, for years of research lie ahead, and many problems not yet apparent will have to be solved. The higher fungi should be recognized, however, as one of our greatest natural resources, a resource whose importance has begun to expand but whose potential we have not even started to realize.

REFERENCES Anderson, E. E., and Fellers, C. R. (1942). Proc. Am. SOC.Hort. Sci. 41, 301. Basaca, M. G. (1953). Philippine J . Sci. 81, 75. Bessey, C. E. (1907). Univ. Nebraska Studies 7 , Pt. 1, 1-99. Bessey, E. A. (1950). “Morphology and Taxonomy of Fungi.” Blakiston, Philadelphia, Pennsylvania.

278

RADCLIFFE F. ROBINSON AND R. 6. DAVIDSON

Block, S. S., Steams, T. W., Stephens, R. L., and McCandless, R.F. J. (1953).J . Agr. Food Chem. 1,890. Boutroux, L. (1880). Compt. rend. 91,236. Buller, A. H. R. (1915). Trans. Brit. Mycol. SOC.6, 21. Calmette, A. (1902). German Patent 129,164. Dragton, F. L. (1934).Mycologia 26, 46. Duggar, B.M. (1904).U . S. Dept. Agr. Farmers’ Bull. Mo. 204. Finn, R.K. (1954).Bacleriol. Revs. 18,254. Frey, C. N. (1930).Ind. Eng. Chem. 28, 1154. Gilbert, F. A.,and Robinson, R.F. (1957). Econ. Botany 11, 126. Hixon, A.W. (1946).Ind. Eng. Chem. 36, 488. Hromatka, O.,and Ebner, H. (1949).Enzymologia 13,369. Humfield, H. (1948). Science 107, 273. Humfield, H.(1950-1951). Yearbook Agr. ( U . S. Dept. Agr.) p. 242. Humfield, H., and Sugihara, T. F. (1952).Mycologia 44,605. Hutchinson, H . B., and Richards, E. H. (1923).J . Ministry Agr. (Engl.)28, 398. Ito, S., and Imai, S. (1925). Bolan. Mag. (Tokyo) 39, 320. Karow, E.O., and Waksman, S. A. (1947).Ind. Eng. Chem. 39,821. Kligman, A. M. (1942).Am. J . Botany 29, 304. Kligman, A.M. (1950).“Handbook of Mushroom Culture.” J. B. Swaynne, Kennett Square, Pennsylvania. Kluyner, A. J., and Perquin, L.H. C. (1933).Biochem. 2.266,68. Lambert, E . B. (1938).Botan. Rev. 4,396. Mack, D. E.,and Uhl, V.W. (1947).Chem. Eng. 64, 119. Molliard, M. (1922).Compt. rend. 174, 881. Moyer, A. J., and Coghill, R.D. (1946).J . Bacleriol. 61, 57. Prescott, S. C. and Dunn, C. G. (1949). “Industrial Microbiology.” McGraw-Hill, New York. Reiser, C. 0. (1954).J . Agr. Food Chem. 2, 70. Rolfe, R. T., and Rolfe, F. W. (1928).“The Romance of the Fungus World.” Lippincott, Philadelphia, Pennsylvania. Rushton, J. H. (1946).Can. Chem. Process Znds. 30, 55. Saeman, J. F., Bubl, J. L., and Harris, E. E. (1945).FIAT Rev. Ger. Sci. Final Rept. 49s. Scheele, K . (1787). CreZ1’s Ckem. Ann. 1,3. Stefaniak, J. J., Gailey, J. J., Brown, C. S., and Johnson, M. J. (1946). Ind. Eng. Chem. 38, 666. Sure, B. (1950).Arkansas Univ. Agr. Expt. Sta. Bull. 493, 62. Seuecs, J. (1956).U. S. Patent 2,761,246. Seuecs, J . (1957). Personal communication. Szuecs, J. (1958).U. S. Patent 2,850,841. Takamine, J. (1914).J . Ind. Eng. Chem. 6, 824. Thaysen, A. C.(1945).Food 14, 116. Van Tieghem, P. E. L. (1867a).Ann. sci. nat. Botan. et biol. ubgbtale. [5]8,240. Van Tieghem, P. E. L. (1867b).Compt. rend. 66, 1091. Wehmer, C. (1893). Compt. rend. 117, 332. Wehmer, C. (1914).I n “Handbuch der technischen Mycologie” (Lafar, ed.) Val. 6, p. 319. Fischer, Jena. Wiley, A. J., Dubey, G. A., Lueck, B. F., and Hughes, L. P. (1951).Znd. Eng. Chem. 43, 1702. Yvoire, M. (1889). Bull. SOC. d’acclim. (France),No. 18.

AUTHOR INDEX Numbers in parenthesis are reference numbers and are included t o assist in locating the reference where the authors' names are not mentioned in the text. Numbers in italics refer to the page on which the reference is listed.

A Abe, Y., 2, 18 Abelson, P. H., 35, 46 Abraham, E. P., 1, 8, 18, 19, 239(1), 244 (I), 866 Adams, 5. L., 223(2), 231(2), 233(2), 866 Adler, E., 204, 818 Aida, H., 209,818 Aida, K., 206, 818 Aida, T., 209, 8f8 Akabori, S., 204, 813 Akatsuka, T., 89, 180 Akita, S., 202,205,206,209, 813 Albans, J. W., 104, f88 Albert, A., 75, 83 Alberti, C. G.,96, 108, 109, 114 Alcorn, S. M., 78, 83 Alexander, A. E., 129, 140 Alexander, P., 61, 70 Alicino, J. F., 25, 46 Alikhanian, S. I., 14, 15, 18 Allen, E. E., 239(136), 240(136), 869 Allen, H. W., 183, 184, 198 Allen, K. A., 97, 11.2 Allen, 0. N., 82, 84 Allgeier, R. J., 90, 98, 101, 116 Allred, R. C., 129, 137 Almon, L., 98, 118 Alper, T., 61, 62, 70, 7f Altman, J., 77, 83 Anderson, A. W., 62, 70 Anderson, H. W., 76, 77, 78, 83 Anderson, E. E., 274,877 Anderson, P. A., 247(3), 866 Anderson, R. C., 103, 118 Andreasen, A. A., 244(141), 869 Andreev, K. P., 243(4), 866 Angus, T. A., 179, f98 Ansbacher, S., 90,07, 111 Anslow, W.H., 95, fdl Aplington, S. P., 136, 138

Aquarone, E., 9, f8 Ardao, M. I., 126, 138 Ardern, E., 252(6), 866 Arita, M., 101, 118 Ark, P. A., 78, 83 Armitage, J. B., 88, 90, 118 Arnstein, H. R. V., 24, 25, 26, 27, 28, a, 31,32,33,36, 37,38,41,42,43, 46,46 Aronovsky, S. I., 239(128), 249(128), 251 (128)) 869 Asai, T., 5, f?O, 206,818 Asakawa, M., 76, 84 Asano, M., 145, 147, 173 Ashe, L. H., 239(111), 243(111), 249(111), 251(111), 868 Aso, K., 101, 118 Austin, F. R., 99, 180

B Babbitt, H. E., 25217)) 866 Bachelder, S., 77, 83 Bacher, F. A., 95, 11.9 Backus, M. P., 25, 46 Bacq, Z. M., 61, 70 Badgley, M. E., 186, 199 Bahler, M. E., 136, 138 Baila, D. L., 94, 118 Baird, K. A., 166, 171 Baker, E. B., 58, 70 Baker, H., 92, ff6 Baker, R. O., 165, 178 Baker, S. J., 103, flS Balch, R. E., 191, 192, 193, 198 Baldwin, G. C.,54, 70 Ball, S., 90, 95, fdf Balwit, J. S., 55, 78 Balzam, N., 145, 149, 154, 171 Bangham, D. R., 165, 171 Banhidi, 2. G., 96, ll4 Barber, F. W.,94, 118 Barber, M., 131, 137 Barchielli, R., 99, 118

279

280

AUTHOR INDEX

Bardi, U., 110, 112 Berna, K., 239(98), 268 Barker, H. A., 88, 89,91, 94,99, 106, 108, Bernhauer, K., 89,90,94,95, 96,99, 105, 109,111,112 107, 108, 109, 110, 111, 112, 113, 114, Barnabie, O., 128, 137 116 Barnes, E. M., 9, 18 Bessell, C. J., 93, 112 Barnes, F. W., 239(8), 266 Bessey, C. E., 262, 277 Baron, L. S., 239(44), 266 Bessey, E. A., 262, 277 Baron, S., 240(130a), 266, 269 Besson, S., 136, 139 Barr, M., 128, 138 Bide, A. E., 25, 47 Barrett, J. M., 96, 108, 109, 119 Biffi, G., 137, 138 Barron, E. S. G., 126, 158 Bilford, H. R., 243(49), 266 Bartigia, I. L., 154, 173 Bilinski, E., 209, 212 Bartlett, M. C., 215, 216(10), 231(11), Biliotti, E., 193, 198 232(11), 239(11), 244(11), 245(10, l l ) , Binkley, F., 31, 36, 45 247(11), 249(11), 266 Binkley, W. W., 55, 73 Bartos, D., 128, 131, 137, 138 Bird, F. T., 190, 191, 192, 193, 198 Basaca, M. G., 270,277 Bird, H. L., Jr., 202, 212 Batchelor, 'F. R., 46 Bird, H. R., 89, 118, 120 Bates, C. J., 54, 71 Bird, 0. D.,90,93,97,99, 105,111,119 Birnbaum, S. M., 168, 171, 172, 203, 211, Batzer, 0. F., 58, 70 211,213 Baumann, C. A,, 165, 171 Bsumler, J., 127, 138, 139 Black, F. L., 63, 67, 70 B s y m , A. P., 300,119 Blanchard, F. A., 78, 83 Blankmeyer, H. C., 243(151), 269 Bean, H. S., 132, 138 Beard, J. W., 164, 171 Blayney, J. R., 152, 154, 173 Beard, P. J., 239(21), 266 Block, S. S., 270, 278 Beaven, G. It., 88, 112 Bloomfield, N. J., 126, 139 Becher, E., 89,105,107,108,109,110,112, Blumberger, K., 94, 111, 112 113, 11.4, 116 Bockelmann, J. B., 9, 20 Bodian, D., 164, 171 Bechhold, H., 131, 138 Bogorad, L., 77, 83 Beckett, A. H., 132, 138 Beesch, S. C., 4, 6, 8, 12, 18, 76, 86, 2f2 Bohrer, C. W., 62, 71 Boley, A. E., 95, 112 Beguin, S., 179, 199 Behrens, 0. K., 25, 28, 31, 32, 33, 46, 46, Bolle, A., 128, 138 Bonde, R., 81, 83 47 Bonicke, R., 137, 138 BBik, E., 7, 12, 13, 15, 17, 18, 19 Bellamy, W. D., 49, 56, 59, 60, 63, 67, 70, Bonnefoi, A., 179, 199 Bonnett, R., 88, 112, 116 r i , 72 Bonventre, P. F., 62, 71 Belt, M., 93, 116 Borensztaijn, D., 97, 98, 112 Benedict, R. G., 76, 86, 98, 101, 116 Borett,i, G., 96,99, 108, 109, 110, 112, 114 Benjamin, J. C., 97, 98, 116 Borg, A. F., 129, 140 Bennett, E. O. , 129, 138 Boruff, C. S., 99, 120 Benyesh, M., 63, 67, 70 Borzani, W., 9, 18 Beran, K., 239(12), 266 Bosshardt, D. K., 93, 120 Berger, H., 133, f b 8 Bontroux, L., 269, 278 Bergold, G. H., 190, 192, 198 Bovey, F. A . , 55, 70 Bergstrom, S., 157, 165, 172 Boxer, G. E., 25,39,46,95, 104,112, 113 Berky, J., 37, 47 Boyd, B. R., 162, 163, l7f Berliner, E., 178, 198 Boyle, A. M., 76, 83 Berman, D., 93, 112

28 1

AUTHOR INDEX Bradley, J. E., 103, 115 Bradley, S. G., 16, 18 Braendle, D. H., 16, 18 Branion, H. D., 98, 116 Brasch, A., 50, 67, 70,73 Bray, R., 103, 11.9 Brian, P. W., 76, 77, 83 Bricker, H. M., 97, 98, 115 Briggs, G. M., 88, 92, 111, 115, 115, 120, 121

Brighenti, L., 128, 137 Brink, C., 88, 113 Brink, N. G., 87, 88, 89, 91, 98, 104, 115, 119

Brinker, W. O., 67, 71 Broadbent, D., 202, 213 Brockman, J. A., 88, 121 Brogle, R. C., 65, 72 Broncova, B., 98, 120 Bronk, J. R., 211, 212 Brook, A. J., 92, 115 Broquist, H. P., 88, 121 Brown, C. S., 269, 278 Brown, F. B., 89, 90, 113, 121 Brown, J. G., 76, 77, 83 Brown, R. A., 145, 146, 150, 151, 156, 174 Brown, W. E.,3, 6, 8, 20, 244(13, 85), 249(13), 255, 257 Brownell, L. E.,65, 66, 70,71 Browning, I., 239(14), 265 Brunel, J. , 238(15), 255 Brunings, K. J., 90, 115 Bryant, J. C., 240(38), 256 Bryson, V., 218(16), 239(17), 240(17), 255 Bubl, J. L., 251(61), 257, 270, 278 Buchanan, J. G.,88,90, 113 Buller, A. H. R., 263, 278 Burger, M., 239(42, 98), 256, 268 Burkholder, P. R., 93, 113 Burks, R. E., 58, 70 Burlew, J. S.,238(18, 19), 266 Burmester, B. R., 162, f7f Burnet, F. M., 161, 162, 163, 171 Burris, R. H., 10, 19, 24, 46 Burroughs, R., 162, 172 Burt, A. M., 239(27), 240(27), 256 Burton, M. O., 96,97,08, 100, 113 Burton, W. G., 65, 70 Busset, R., 60,72

Butlin, K. R., 216(20a), 239(20), 249(20), 252(20), 253(20), 256

C Cade, A. R., 129, 158 Cain, J. C., 90, 115 Cain, R. F., 62, 70 Calam, C. T., 37, 46 Caldwell, J. H., 90, 97, If2 Calhoun, K . M., 98, 101, 118 Calkins, D. G., 89, 90, 96, 99, 111, 114, 119

Callaham, J. R., 220(123), 268 Callow, S. D., 8, 18 Calmette, A., 269, 278 Cammarata, P. S.,38, 46 Campbell, A. H., 8, 18 Campbell, C. J., 145, 146, 150, 151, 156, 174 Campbell, W. L., 50, 71 Cannon, J. R.,88, 90, 112 Caputo, V. A., 56, 70 Carr, E. M., 59, 72 Carter, H. E., 25, 33, 45 Carter, R. H., 249(28), 251(28), 256 Carvajal, F., 4, 18 Carver, J. S., 90, 97, 118 Cary, C. A., 89, 115, 116 Casida, L. E., J r., 38, 46,208,212 Catron, D. V., 165, 172 Ceran, L. E., 51, 70 Chaiet, L., 103, 113 Chain, E., 1, 8, 18, 19, 239(1), 244(1), 255 Chain, E. B., 8, 18, 20 Chalmers, J. N . M., 104, 121 Chamberlin, F. S., 191, 198 Charles, A., 79, 85 Chiang, M. C., 25, 46 Chiao, J. S., 93, 97, 11.9 Chick, H., 124, 158 Chilton, T. H., 220(123), 868 Chipault, J. R., 59, 70 Chow, B. F.,97, 113 Christensen, E., 65, 78 Chu, E., 155, 172 Churchill, B. W., 67, 71 Cicconi, M., 124, 138 Clark, E. C., 191, 199

282

AUTHOR INDEX

Clark, L. B., 54, 70, 238(109), 239(109), 668

Clark, P., 58, 70 Clark, T. F., 239(128), 249(127, 128), 251 (127, 128), 869 Clarke, G. D., 96, 98, f l 5 Clauss, J. R., 59, 76 Cleary, J. P., 239(21), 666 Clifton, C. E., 239(21, 22), 243(22), 244 (22), 249(22), 666 Clubb, M. E., 32, 33, 36, 38, 41, 46 Coates, M. E., 88, 92, 93, 94, 111, l f 5 Cobb, N . A., 193, 199 Coelho, F. P., 103, 118 Coffin, D. L., 161, l7f, 176 Coghill, R. D., 23, 25, 47, 269, 678 Cohnn, M. S., 38, 47 Cohen, G. N., 204, 615 Cohen, P. P., 38,46, 205, 6fS Cohendy, M. M., 142, 145, 147, 149, 154, 171 Coleman, D. L., 165, f7l Colingsworth, D . R., 4,60, 98, 101, 118 Collet, R. A . , 60,75 Colovos, G. C., 49, 56, 67, 70, 7f Combs, G. F.,89, 92, f f 5 ,if8 Cook, L. G., 68, 69, 7f Cook, 1’. M., 238(25, 26), 266 Coolidge, W. D., 50, 71 Cooper, E. A., 125, 128, 131, 137, 138 Cooper, P. D., 137, f58,239(27), 240(27), 666 Corcoran, J. W., 103, 104, 115, 260 Cords, F., 97, 98, 116 Corman, J., 205,613 Correa, R., 96, 116 Gorse, J., 25, 33, 4, 46, 47 Corti, U. A., 104, 117 Corum, C. J., 202, d f 6 Covert, A. S., 244(85), 667 Craig, J. T., 25, 26, 46 Crawhall, J. C., 29, 31, 42, 46 Crossan, D . F., 80, 85 Crosse, J. E., 80, 85 Crowdy, S. H., 77, 78, 82, 85, 8.4 Currie, J. N., 249(28), 251(28), 666 Cury, A., 92, 116

Cuthbertson, W. F. J., 88,92,93,95,115, fdi

Cutter, L. A , , 244(85), $67

D Dack, G. M., 60, 7f Dacquisto, M. P., 155, f75 Daft, F. S., 88, 111, f f 5 Dagley, S., 131,134, 138,202, 206, 6 f 6 Darken, M. A., 98, 100, 115 Darpoux, H., 76, 84 Das Gupta, P. N., 127, 131, 158 Davey, V. F., 24,46 Davidson, 0. W., 78,86 Davidson, S., 54, 7f Davies, D . S., 133, 158 Davies, M. K., 111, 115 Davis, B. D., 93, 97, 115, 208, 6 f 6 Davis, D., 77, 84 Davis, E. A., 239(29), 666 Davis, E. V., 240(160), 660 Davis, F. L., 124, 158 Davis, J. M., 191, 199 Dawbarn, M. C., 93, f f 6 Dawes, E. A., 131, 138, 202, 206, P f t Ihwson, R., 111, 115 Day, E. A., 58, 7f Day, M. F., 190, 199 Day, W.H., 8, 19 Dean, A. C. R., 133, 158 de Becze, G. I., 237(30), 239(30,89), 243 (30), 245(89), 249(89), 666,668 Dedrick, J., 239(29), 366 De Haan, P. G., 220(31), 666 Deindoerfer, F. H., 227(32), 666 Dekker, J., 76, 84 Delabarre, Y., 103, I16 Dellweg, H., 89, 105, 108, 109, 110, f f 5 , 114,116 De Long, C. W., 29,30,32,33,34, 36,37, 4 ~ 4 7 Demain, A. L., 24, 27, 29, 30, 32, 33, 36, 39, 40, 43, @ Denbigh, K. G., 222(33), 239(33), 249(33), 666

Denkewalter, R. G., 90, 111, f17 Dennis, R. C., 65,66, 70

283

AUTHOR INDEX

Denny, C. B., 62, 71 Denton, C. A., 89, 118 Deromedi, F., 129, 130, 139 deRosa, A. F., 89, 116 Deshpande, V. N., 28,43, 4.6 Deutsch, H. F., 165, 171 DeVries, P. H., 67, 7f De Vries, W. H., 89, 90, 96, 97, 116, 118 Dewey, D. L., 208, 212 Dewey, D. R., II,52, 7f Dewey, M., 239(8), 266 Dhande, G. E., 76, 86 Dicke, R. J., 182, 200 Dickerman, G. K., 237(137,138), 239(137, 138), 243 (137), ,969 Diller, V. M., 78, 83 DiMarco, A., 96, 108, 109, 110, 112, lf4, 137, 138 Dion, H. W., 89,90,96,99,111, 114,119 Doetsch, R. N., 134, 135, 139 Dollar, A. M., 98, 99, 119 Donovan, J. R., 216(35), 866 Donovick, R., 104, 119 Dose, K., 56, 70, 72 DoskoEil, J., 3,7, 8, 12, 15, 18, 19 Doty, D. M., 58, 70 Doudney, C. O., 61, 71 Dougherty, K. M., 238(41), 239(41), 866 Dowden, P. B., 193, 199 Dowing, J. F., 101, f l 6 Downing, J. F., 90,97, 112 Dowler, W. M., 80,84 Doyle, F. P., 46 Dragton, F. L., 272, 278 Drake, M. P., 60, 71 Drews, W., 8, 19 Dryden, L. P., 89, 113 Dubey, G. A., 270, 278 Dubos, R. J., 166, 171 Duche, J., 239(36), 266' Dufrenoy, J. R., 77, 84 Duggan, D., 62, 70 Duggar, B. M., 262, 878 Dulaney, E. L., 100,106, 114, 209,219 Dunegan, J. C., 76,80,84 Dunn, C. G., 8,20,50, 71, 269,278 Duran-Reynals, F., 161,163,171, 172 Dutcher, J. D., 28,47

Dutky, S. R., 177,178,180,183,185,186, 187,191,195,198,199,200 du Vigneaud, V., 31,37,46,46,156,172 Dvonch, W., 98, 116 Dworschack, R. G., 98, 116 Dye, M. H., 80, 84 Dyr, J., 10, 19,239(37), 249(37), 250(37), 266

E Eakin, R. E., 92, 105, 120, 181 Earle, W.R., 240(38), 266 Ebner, H., 242(71), 267, 269, 278 Edelhausen, J. H., 89, 121 Edwards, J . P., 25, 46 Edwards, M. A., 124, 139 Egan, R. W., 163, 174 Ehrenvaard, G., 37, 38, 46 Ehrlich, P., 131, 138 Ehrwein, A., 136, 139 Einola, S., 128, 138 Eisler, M., 131, I38 Elberg, S. S., 166, 172 Elbowicz, Z., 105, 114 Elkin, M., 88,181 Ellis, B., 88,112, 114 Elsworth, R., 219(66), 220(66), 225(66), 233(66), 234(66), 239(39), 247(39), 248 (66), 266, 267 Elvehjem, C. A., 90, 117 Emerson, H., 82, 86 Emerson, R. L., 10, 19, 24, 34,46 Emery, W. B., 93, 95, 11.6, fa1 Endo, T.,2, 18 Eppstein, S. H., 5,20 Epstein, M., 87, 97,121 Erb, N. M., 11, 19 Ergorova, A. A., 134, 138 Ericcson, E. O., 241(40), 249(40), 866 Ericson, L. E., 96, 99, f14, 120 Erlandson, A. L., 128,138,139 Erlenmeyer, H., 127, 138, 139 Ervin, R. F., 143,144,145,146, 149,150, 151,152, 154,156,170,173, 174 Esslinger, J., 58, 70 Ettinger, M. B., 134, 135, 188 Evans, J. B., 62,66, 73 Evans, J. S., 89, 90, 97, 116 Evans, V. J . , 240(38), 266

284 Evans, W. C., 135,140 Everett, P. M., 25,39,46

AUTHOR INDEX

Formal, S. B., 239(44), 266 Formanek, S.,244(85), 267 Forss, D. A . , 58, 71 F Foster, F. L.,Jr., 52,71,145,17.9 Fahey, J . E., 197,199 Foster, J. C., 93,95,114,112 Fahnoe, F., 51,70 Fox, M. R. S., 111, 113,116 Fox, M.S.,247(47), 166 Falk, C. R., 136,138 Fantes, K. H., 88, 95,98,103,107,108, Foster, J. W., 8, 10,19,25,46, 245(45), 109,111,114,112 249(45),266 Farkas, G. L., 81,86 Fowler, C. B., 126,138 Farrant, J. L., 190,199 Fowler, E.B., 204,21.2 Fowler, G. J., 252(46), 266 Farrell, C. C., 194,199 Fox, H., 194,198 Farrell, L., 25,46 Farrell, M. A., 37,47 Fram, H., 50,71 Feeney, R. E., 239(50), 249(50), 266 Franklin, A. L., 87,88,93,97, 216,121 Frana, J.,193,199 Feeney, R. J., 90,113 Frazier, W.C., 10,19 Feirer, W.A . , 133,189 Freeman, L. O., 131,138 Feldman, L.I., 204,218 French, C. S., 239(29), 266 Fellers, C. R.,274,277 Felton, L. D., 164,172,238(41), 239(41), Frey, C. N.,269,278 Fricke, H.H., 89,116 266 Fencl, Z., 239(42, 98),256, 268 Friedemann, U., 67,73 Friedrich, W., 89, 90,94,95,96,99,105, Fenner, F., 161,I72 108,109,110,112, 116 Fernandea, H., 11, 19 Friend, J. N., 76,81, 84 Fest, W. C., 178,199 Fries, N., 239(154), 260 Filfus, J., 94,116 Finn, R. K., 8, 19,216(43), 220(43), 225 Frigerio, N.A . , 125,133,139 (43), 231(43), 239(43), 250(42s), 266, Frobisher, M., 128,138 Froquet, L.,10,19 269,278 Frost, D. V., 89,116 Fisher, C. V., 129,138 Fujii, M., 209,2i2 Fisher, R . A., 90,94,98,102,114 Fujimoto, S.,128,138 Fisher, R. B., 211,d l 8 Fitz-James, P., 178,179 G Fleming, A., 23,46 Gaden, E. L., Jr., 8,19,220(48),866 Fletcher, C . M., 239(1), 244(1), 266 Gailey, J. J . , 269,878 Flett, L.H., 132,238 Gale, A. J., 52,7f Florestano, H. J., 136,138 Florey, H.W., 8, 19,239(1, 139), 244(1), Gale, E.F., 132,138 Gallagher, F. H., 243(49), P66 266,269 Ganapathi, K., 26,28,43,46 Florey, M. E., 8, 18 Ganguly, S.,100,107,I16 Fogg, A. H., 128,133,1.98 Gant, D. E., 90,94,If$, 121 Foley, C., 76,86 Folkers, K.A., 31,47,87,88,89,90,91,98,Gardner, A. D., 239(1), 244(1), 266 104,108,109,110,111, 113, 214,217, Gardner, D., 78,89 Gardner, F. H., 166,I72 119,190,122 Garey, J. C., 101,116 Forbes, M., 155,i72,17.9 Ford, J. E., 88, 90,91,92,93,94,95,96, Garibaldi, J. A . , 4,5, 19,90,96,97, 98, 99, 101, 102, 116, ff7, 118, 239(50), 97,99,108,109,110,111,11.9, I14 244(50), 249(50), 866 Ford, J. H., 82,86

285

AUTHOR INDEX

Garrett, M. E., 80, 83 Garrison, L., 25, 46 Garrison, W. M., 56, 71 Garrod, L. P., 131, 138 Gasparetto, G., 96, 114 Gastrock, E. A., 249(127), 251(127), 269 Gates, L. W., 126, 139 Geerts, S. J., 105, 122 Gentile, F., 98, 122 Gerhardt, P., 217(51), 231(11), 232(11), 234(51), 238(51), 239(11, 51, 52), 240 (52, 147), 244(11), 245(11), 247(11), 249(11), 256, 266, 269 Gershenfeld, L., 129, 130, 139 Ghione, M., 96, 108, 109, 110, 114 Giffee, J. W . , J r . , 60, 71 Gilbert, F. A., 270, 278 Gill, R. J., 97, 98, 116 Gillespie, R. E., 158, 172 Gillies, N . E., 62, 70 Ginsberg, V., 90, 117 Girth, H. B., 193, 194, 195, 199 Glaser, R. W., 194, 195, 1.99 Glimstedt, G., 147, 154, 172 Glister, G. A., 25, 46 Goddard, A. E., 131, 137, 138 Godzeski, C., 37, 38, 46, 47 Goldberg, M. W., 25, 47 Goldblith, S. A., 49, 50, 54, 56, 59, 60, 62, 65, 66, 70, 71, 72 Goldschmidt, E. P., 38, 46 Goldschmidt, M. C . , 24, 46 Golle, H. A., 219(53), 220(53), 226(53), 229 (53), 256 Gomberg, H. J., 66, 71 Goodale, C. D., 249(158),260 Gooden, E. L., 185, 186, 187, 199 Goodman, R. N., 76, 80, 84 Gordon, H. A., 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 159, 160, 170, 172, 173, 174 Gordon, I., 67, 70 Gordon, M., 25, 26, 46 Gotaas, H. B., 238(54, 93, 120, 121), 249 (54, 93, 120, 121), 266, 268 Gottlieb, D., 76, 83 Gould, B. S., 125, 133, 139 Gould, S. E., 66, 7 i Graff, S., 240(55), 266 Graikoski, J. T., 62, 65, 66, 70, 71

Grainger, A., 25, 46 Grant, P. T . , 24, 25,26,28,29,31,32,37, 38, 4 Grau, F. H., 29, 33, 46 Gray, P. H. H., 134, 139 Gray, R. A., 76, 77, 79, 80, 82, 84 Graziosi, F., 239(56), 267 Green, N., 95, 103, 114 Greenberg, D. M., 37, 47 Greene, R. D., 92, 116 Greenfield, R. B., 100, 119 Greenspan, G., 90, 96, 97, 100, 101, 104, 118

Greenstein, J. P., 168, 171, 172, 203, 211, 818, 213 Gregory, J., 93, 113 Gregory, M. E., 97, 105, 116 GrBgr, V., 10, 19 Grison, P., 179, 193, 198, 199 Groninger, H. S., 63, 71 Groschke, A. C . , 89, 116 Gross, G., 89, 95, 96, 105, 116 Gross, L., 162, 172 Grossmann, H., 9, 19 Grosso, J. J., 81, 84 Grove, J. F., 78, 82, 83, 84 Groves, K., 89, 118 Grubb, R., 157, 175 Grubb, T. C., 124, 189 Grundy, A. V., 65, 72 Guenther, K. R., 239(57), 257 Guest, H. L., 127, 1.40 Guida, V. O . , 96, 116 Guiteras, A. F., 132, 138 Gullino, P., 211, 212 Gummert, F., 238(58), 257 Gundersen, K., 134, 135, 139 Gunsalus, I. C., 204,212 Gunther, G., 204, 212 Gurney, D. M., 104, 121 Gustafson, F. G., 65, 71 Gustafsson, B., 142, 144, 145, 146, 148, 152, 156, 157, 165, 172 Gyorgy, P., 155, 172, 173

H Hadot, J. J., 136, 139 Hahn, H. J., 127, 139 Haines, R. B., 125, 138 Haines, W. J., 25, 33, 46

286

AU'I?HOR INDEX

Halbrook, E. R., 97,98, 116 Hale, C.W., 39, 46 Hall, B., 93, 119 Hall, H. H., 97,98, 101, 116, 119 Hall, I. M., 184, 186, 193, 199 Haller, G., 244(59), 867 Halliday, R., 165, 178 Halliday, W. J., 26, 27, 29, 33, 43, 46 Halpern, P. E., 35, 47 Halvorson, H. O., 252(60), 867 Hamdy, M. K., 134, 139 Hamilton, D. W., 197, 199 Hamilton, J. M., 80,84 Hammon, W. McD., 162, f78 Hampil, B., 128, 139 Hanks, J. H., 240(156), 860 Hannan, M. L., 251(61, 62), 867 Hannan, R, S., 49, 54, 65, 70, 71 Hannay, C. L., 178, 199 Hanson, C. T.,4, 19,90,98,101, 116, 116 Hanus, J., 11, 19 Harashima, K., 89, 180 Hargrove, R. E., 16, 19, 98, 99, 101, 116, 117 Haring, R. C., 132, 138 Harm, W., 133, 139 Harriman, H., 60, 71 Harris, D. A,, 4, 80 Harris, E. E., 261(61, 62, 75), 867, 270, 878 Harris, N. D., 133, 139 Harris, S., 239(118), 868 Harris, T. N., 220(79), 239(79, 118), 867, 868 Harris-Smith, P. W., 238(63), 867 Harrison, E., 93, 118 Harrison, G. F., 88,92, 111, 113 Harrison, R. W., 152, 154, 173 Hartman, A. M., 89, 113, 116 Hartman, P. A., 165, 178 Haskins, R. H., 99,116, 209, 813 Haslewood, G. A. D., 131, 137 Hassett, C. C., 65, 71 Hastings, A, B., 88, 181 Hausmann, K., 88, 105, 116 Hayakaya, S., 136, f39 Hayashi, J., 89, 180 Haydon, D. A , , 128, 139 Hayduck, F., 8, 19

Haynes, W.C., 97, 98, 116 Hearon, M., 126, 138 Heathcote, J. G., 94, 106, 116 Heatley, N. G . , 8 , 19,239(1),244(1),866 Heep, I). M., 76, 83 Heger, E. N.,4,80,90,102,119,249(125), 251 (125), 669 Heimpel, A. M., 179, 199 Heinle, R. W., 87, 97, 181 Heinrich, H. C., 88, 109, 116 Hejmova, L., 239(98), 868 Hellstrom, V., 204, 812 Hemming, H. G., 76,78, 82, 83, 84 Hemphill, D. D., SO, 84 Henderson, D. W., 216(24), 866 Hendlin, D., 93, 98, 101, 102, 116, 188 Herbert, D., 216(65),218(64,65), 219(66), 220(64, 65, 66), 225(64, 65, 661, 233 (66), 234(66), 248(66), 267 Herbert, M., 37, 46 Hermann, S., 239(67), 243(67), 249(67, 125a), 251 (67, 125a), 667,869 Herold, M., 3, 4, 7, 8, 12, 13, 14, 15, 17, 18, 19, 80 Hervey, A,, 89, 93, 94, 119, 120 Hervey, G. E. R., 193, 800 Hess, V. F., 5, 80 Hester, A. S., 90, 416 Heuser, G. F., 89, 113 Hickey, R. J., 4,8, 80,242(149), 249(149), 869 Hidaka, Z., 80, 84 Higuchi, K., 25, 46 Higuchi, T., 128, 139 Hilborn, M. T., 81, 84 Hilgetag, G., 249(133), 869 Hill, D. C., 98, 116 Hill, H. H.,90, 97, 98, 118 Hine, D. C. 93, 116 Hinshelwood, C. N., 133,1$8,14O,206,813 Hinz, C. F., 101, 116 Hirai, T., 76, 77, 84,86 Hixon, A. W., 269, 878 Hockenhull, D. J. D., 26, 28, 35, 37, 46, 98, 102, 103, 181 Hodak, K., 98, 116 Hodge, H. M., 90, 98, 101, 116, 116 Hodgkin, D. C., 88, 110, 113, 116, 117

287

AUTHOR INDEX

Hoevet, B., 97, 105, 118 Hoffmann, C. E., 93,116 Holderby, J. M., 237(68, 72), 239(72), 867 Holdsworth, E. S., 90,93, 96,97,99,105, 108, 109, 110, 111, 113, 114, 116, 116 Holiday, E. R., 88, 118 Hollaender, A., 51, 60, 71 Holland, A., 90, 111, 117 Holme, T., 219(69), 239(69), 249(69), 251 (69), 867 Hong, S. C., 145, 147, 173 Hoogerheide, J. C., 98, 116 Hoover, S. R., 89, 104, 116 Hoppe, W., 202, 813 Hori, S., 204, 818 Horodko, J., 239(70), 867 Horowitz, N. H., 35, 46 H o g k e k , Z., 4, 19 Hotchin, J. E., 162, 163, 178 Hougen, 0. A., 220(123), 868 Hough, W. S., 195, 199 Houlaham, M. B., 208,813 Housewright, R. D., 204, 91% Hovanesian, J., 125, 133, 139 Howard-Flanders, P., 61, 71 Howat, A. G., 104, 181 Hromatka, O., 242(71), 267,269,878 Hoy, J. M., 194, 199 Hsu, P. T., 92, 118 Huang, H. T., 205, 208, 818, 113 Huber, W., 50, 67, 70, 73 Hudec, M., 7, 13, 17, 18 Huebner, R. J., 162, 178 Huff, D. E., 25, 46 Huff, J. W., 93, 180 Hughes, K. M., 189, 190, 193, 199 Hughes, L. P., 237(72), 239(72), 867, 270, 878 Huhtanen, C. N., 94,118 Humfield, H., 270, 272, 878 Humphrey, A. E., 227(32), 866 Hungate, R. E., 223(2), 231(2), 233(2), 866 Hurenkamp, B., 95, 189 Hum, W. J., 137, 140 Hutchins, A., 50, 71 Hutchinson, F., 54, 55, 61, 7f Hutchinson, H. B., 266, 878

Hutner, S. H., 92,93,94,96,108,109,110, 11.4, 116 Hutz, B., 178, 199

I Iacocca, V. F., 220(70), 239(79), 167 Iawi, S., 105, 117 Iijima, S., 145, 147, 158, 173 Ijichi, K., 4, 5, 19,90,96,97,98,99, 101, 102, 116, 117, 118 Ikeda, K., 103, 180 Imai, K., 205, 818 Imai, S., 263,878 Inamine, E., 28,29,30,31,32,36,37,47 Inskeep, G. C., 237(72), 239(72), 867 Ireland, D. M., 95, 103, 11.6, 181 Isaacs, P. J., 51, 70 Ishida, T., 239(155), 245(155), 249(155), 860 Isomura, N., 143, 145, 147, 158, 173 Itagaki, K., 5 , 19 Itaya, J., 167, 173 Ito, M., 101, 118 Ito, S., 263, 878 Iwamoto, K., 89, 102, 116, 180 Iwazaki, T., 206, 813 Izaki, K., 203, 818, 813

J Jackson, C. J., 239(34), 243(34), 249(34), 266 Jackson, It. W., 76,86,98, 101, 116 Jackson, T., 216(73), 867 Jackson, W. G., 89,90, 97, 116 Jacobs, S. E., 133, 139, 239(76), 867 Jacobs, W. L., 11,19 Jacobus, D., 155, 173 Janicki, J., 89, 90,95, 96, 97, 98, 99, 105, 107, 116, 117 Jannes, L., 98, 117 Jarvis, F. G., 26, 46 Jasewicz, L. B., 89, 104, 116 Jayko, M. E., 56, 71 Jenkins, D. W., 65, 71, 194, 199 Jennings, M. A., 8,19,239(1), 244(1), 666 Jensen, H. L., 134, 135, 139 Jerusalimskij, N. D., 231(74), 239(74), 249(74), 250(74), 867 Johansson, K. It., 155, 160, f73

288

AUTHOR INDEX

Johnson, A. W., 88, 90, 112, 113, 118 Johnson, E. A., 88, 112 Johnson, M. C., 251(75), 257 Johnson, M. J.,24,25,26,@, 47,225(101), 231(101), 239(101, 122), 258, 269,278 Jones, K. L., 98, 117 Jones, R. G., 25, 33, 45, 46, 47 Jordan, R. C., 239(76), 857 Jukes, T. H., 87,88,89,90,92,93,97,118, 117, 119, 121, 155, 172 Julita, P., 110, 112 Julliard, M., 100, 117

Ketchum, B. H., 239(81), 257 Kilburn, R. E., 63, 72 King, J. D., 132, 1-40 Kinoshita, S., 202,205, 206,209,211,212, $18, 214 Kirily, Z., 81, 85 Kishimoto, H., 145, 147, 167, 173 Kita, D. A., 206, 208, 213 Kitada, S., 209, 213 Klarmann, E. G., 123, 126, 130, 131, 139 Klebe, H., 93, 103, 104, 128 Kleiderer, E. C., 25, 33, 45 Klemmer, H. W., 82, 84 K Kligman, A. M., 265, 271, 278 Kabelik, J., 129, 139 Kluyner, A. J., 269, 278 Kaczka, E. A., 88,89,90,91,111,113,117 Knapp, F. W., 63, 71 Kahlson, G., 156, 178 Knight, 5. G., 10, 19, 34, 38, @ Kalabina, M., 134, 139 Knowlton, J. A., 52, 72 Kaleja, E., 98, 117 Koaze, Y., 76, 84 Kaljufnyj, M. J., 243(77, 78), 257 Kobayashi, R., 145, 147, 158, 173 Kamikubo, T., 97, 104, 105, 117, 121 Kocher, V., 96, 98, 104, 117 Kamitsuka, P., 161, 174 Kockovi-Kratochvilovi, A., 8, 19 Kamper, J., 88, 116 Kohler, H., 76, 84 Kamper, M. J., 110, 117 Koehler, W., 200 Kan, B., 62, 71 Koepsell, H. J . , 205, 213 Kaneo, S., 204, 212 Koffler, H., 10, 19, 24, 38, 46, 47 Karel, M., 54, 65, 71, 72 Kojima, H., 102, 117 Karow, E. O., 269, 278 Kojima, S., 137, 139 Karush, F., 220(79), 239(79), 257 Kolachov, P. J., 237(150), 239(82, 150), Kashio, T., 167, 173 243(49), 244(82, 141), 249(82), 268, Kasik, D., 105, 119 257, 259 Katagiri, H., 5, 19, 205, 212 Komarova, L. I., 8, 19 Kon, S. K., 88, 90, 91, 92, 94, 95, 96, 97, Kato, J., 204,212 99,106,108,109,110, 111,113,114,117 Kato, K., 39, 46 Katznelson, H., 4, 19, 76, 84 Kondo, Y., 239(155), 245(155), 249(155) Kautsky, H., 239(80), 257 260 Konikova, A. S., 204, 213 Kautsky, H., Jr., 239(80), 267 Kay, B., 54, 71 Koniuszy, F. R.,87, 88, 89,98, 113,119 Kayser, F., 136, 139 Konovalov, S. A., 8, 19 Keitt, G. W., 76, 84, 85 Kornberg, H. L., 38, 47 Kekwick, R. A., 165, 172 Kotkova, M., 239(95), 258 Kelliher, M. G., 52, 71 Kowaltl, J., 103, 118 Kelly, B. K., 39, 46 Kramer, N., 134, 135, 139 Krasilriikov, N. A., 76, 81, 84 Kelly, L., 98, 122 Krasna, A. I., 103, 117 Kelner, A., 60, 71 Krassilstschik, I. M., 180, 199 Kemp, C., 98, 115 Krassowska, L., 239(70), 857 Kempe, L. L., 62, 65, 66, 67, 70, 71 Krauss, R. W., 238(84), 239(83,84), 257 Keppie, J., 238(63), 257 Kravitz, E., 128, 132, 139, 140 Kertesz, Z. I., 59, 72

AUTHOR INDEX

Krebs, H. A , , 38, 47, 205, 213 Krieg, A , , 183, 185, 192, 199 Kristensen, H. P. @., 97, 117 Kritsman, M. G., 204, 213 Kroll, C. L., 6, 8, 80, 244(85), 267 Krasilnikov, N . A , , 15, 19 Krupka, L. R., 80, 84 Kuchenmeister, G . F. H., 123, 239 Kudo, R. R., 183, 199 Kuehl, F. A., Jr., 89, 91, 104, 113, 117 Kulkarni, N . B., 76, 86 Kuprianoff, J., 49, 72 Kuroda, T., 126, 139 Kurylowicz, W., 97, 98, fl2 Kurz, W., 100, 117 Kuiika, J., 239(86), 267 Kuster, E., 142, 147, 154, 172

L Lacey, J . C., 58, 70 Lach, J. L., 128, 139 Lahann, H., 88, 116 Lally, J. A , , 93, f l 4 Lambert, E. B., 264, 278 Langlykke, A . F., 11,20,97,104,218,119 Lanius, B., 89, 116 Lapidus, M., 89, 1f6 Larkin, F. E., 93, 117 Larner, J., 158, 172 Larson, L. M., 25, 33, 46 Lascelles, J., 37, 47 Lataste-Dorolle, C., 60, 7 2 , 73 Lang, E. P., 64, 72 Lawrence, C. A , , 128, 138, 139 Lawton, E. J., 55, 56, 59, 60, 67, 7 0 , 72 Layton, L. L., 97, 113 Laznikova, T. N., 102, 118 Lea, D. E., 51, 60, 61, 72 Leben, C., 76, 84, 86 Le Corroller, Y., 179, 199 Lederberg, J., 16, 18 Ledingham, G . A., 99, 116 Lee, S. B., 4, 6, 8, 12, 19, 20, 22 Lees, K . A , , 93, 95, 112, 1 1 4 , 121 Legallais, F. Y., 240(130a), 269 Legg, D. A., 4, 19 Lehman, A. J., 64, 7 2 , 79 Lehr, H., 25, 47

289

Leigh, H. M., 5, 20 Lemieux, R. U., 99, 116 LeMoigne, M., 178, 199 Lennette, E. H., 164, 172 Lens, J., 88, 89, 105, 121, 122 Leopold, J., 4, 19 Levadie, B. T. H., 90,97, 228 Levin, I., 136, I40 Leviton, A., 16, 29, 96, 97, 98, 99, 101, 216, 217 Lewis, C. J., 165, 172 Lewis, J. C., 4, 5 , 19, 90, 96, 97, 98, 99, 101, 102,116,117,118 Lewis, L., 96, 99, 124 Lewis, R. W., 244(88), 268 Lewis, U. J., 90, 117 Licciardello, J. J . , 66, 72 Lichtman, H., 90, f f 7 Liebmann, A. J., 239(89), 245(89), 249 (89), 268 Liggett, R. W., 24, 47 Likely, G . D., 89, 113 Lillie, R. J., 89, 118 Lilly, C. H., 240(90), 244(90), 268 Lindenfelser, L. A., 76, 86, 98, 126 Lindsey, J., 88, 116 Linstedt, S., 157, 165, 172 Lishka, R. J., 134, 135, 138 Littman, F. E., 59, 72 Litwack, G., 79, 84 Liu, C., 161, f 7 1 , 172 Liu, C . H., 165, 172 Lloyd, J. T., 92, 113 Lochhead, A. G., 4, 19, 96, 97, 98, 100, 114

Locke, E. G., 237(137, 138), 239(137, 139), 243(137), 269 Lockemann, G., 124, 149 Lockett, W. T., 252(6), 266 Lockhart, E. E., 65, 66, 72 Lockinger, L. S., 239(14), 266 Lockwood, J. L., 80, 84 Lockwood, L. B., 205, 219 Lodge, R. M., 128, 133, 148 Logotkin, I. S., 249(91, 92), 268 Lokvenc, F. A., 14, 17, 20 Louin, M., 59, 72 Low, R. J . , 266 Lucas, E. H., 244(88), 268 Luckey, T. D., 144, 145, 146, 149, 150,

290

AUTHOR INDEX

151,152, 153, 154, 155, 156, 170, 178, 173, 174 Ludwig, H. F., 238(54, 93, 120, 121), 249(54, 93, 120, 121), 866, 868 Ludwig, L., 105, 116 Lueck, B. F., 270, 278 Luecke, R. W., 89, 116 Luedeking, R., 233(94), 239(94), 248(94), 249 (94), 868 Lugones, Z. M., 90, 98, 118 Lumb, G. D., 132, 1-40 Lundberg, W. O., 59, 70 Lundy, H. W., 126,127,139 Lynch, V., 238(93,120,121), 249(93,120, 121), 868

237(23), 238(97), 239(96, 97,98), 242 (23), 249(23), 866,868 Mamalis, P., 88, 119 Mann, M. J., 25, 46 Mannering, G. J., 145,146,150,151,156, 174 Margreiter, H., 29, 32, 33, 46 Marnati, M., 110, 118 Marquardt, R. R., 251(61, 62), 867 Martens, R. A., 65, 71 Martignoni, M. E., 186, 800 Martin, E., 37, 47 Martouret, D., 179, 193, 108, I90 Marzke, F. O., 182, 900 Masuda, K. 239(155), 245(155), 249(155),

M

Masuo, E., 204, 205, 813 MatelovB, V., 4, 14, 1 9 , 80 Matrishin, M., 87, 97, 98, f i 0 Matrone, G., 168, 173 Matsuda, T., 90,96,98, 118 Matsui, C., 76, 84 Matsuki, M., 102, I17 Matsumoto, R., 89, 180 Maurice, P., 128, 139 Maxon, W. D., 215(100, 145), 216(100), 220(100), 223(100), 225(101), 231 (101) , 237 (loo), 239 (101), 242 (loo), 249 (loo), 868, 869 Meader, P. D., 133, 139 Meakin, L. R. P., 239(39), 247(39), 866 Meffert, M. E., 238(58), 867 Meissner, B., 134, 135, 1% Meister, A., 211, 8fS Meister, P. D., 5, 80 Melnick, J. L., 63, 67, 70 Melnykowycz, J., 155, 160, f73 Menge, H., 92, 118 Menkova, K. A., 81, 84 Metchnikoff, E., 180, 800 Meyer, C. E., 96, 97, 118 Meyer, H., 127, 139 Michi, K., 203, ,913 Middlebeek, A., 88, 105, 188 Migliacci, A., 96, 99, 108, 109, 110, 112, 114 Mikata, M., 89,103, 107, 180 Miki, T., 89, 120 Millar, R. L., 78,80,86

McAdams, W. H., 220(123), 868 McCabe, A. B., 55, 73 McCabe, P. J., 178, 800 McCabe, W. L., 220(123), 268 McCandless, R. F. J., 270, 878 McCarty, K. S., 240(55), 866 McCoy, E. F., 4, 80 McCoy, E. E., 194, 195, 199 McCombie, J. J., 96, 97, 114 McConnell, W. B., 209, 818 McCormack, R. B., 92,97, 116, 118 McDaniel, L. E., 104, 118, 245(45), 249 (45), 866 MacDonald, R. E., 240(147), 860 McEwen, F. I,., 193, 800 McGinnis, J., 89,90,96,97, i16, 118 McGlohon, V., 90, 99, 111, 119 Mack, D. E., 269, 878 Mackay, J. H. E., 76,81,84 Mackay, M., 88, 116 McLaughlin, M. M., 155, f73 McLean, D. M., 162, 175 McLimaus, W. F., 240(160), 860 McManus, I. R., 38, 47 McMillen, W. N., 89, 116 McPherson, J. F., 108, 109, 110, 180 Macura, J., 239 (95), 868 Maddock, A. G., 103, 118 Mahn, G. R., 52, 78 Makarevich, V. G., 102, 118 Maki, B., 99, 118 MAlek, I., 6, 80, 215, 216(97), 234(97),

260

~

291

AUTHOR INDEX

Miller, A. A., 55, 72 Miller, G. A., 39, 46 Miller, I. A., 103, 120 Miller, I. M., 103, 104, 108, 109, 110, 120 Miller, P., 37, 47 Millett, M. A., 59, 78 Mills, J. A , , 88, 90, 113 Milner, H. W., 239(29), 266 Milner, J., 239(34), 243(34), 249(34), 266 Miner, C. S., 105, 118 Minghetti, A., 99, 112 Mingioli, E. S., 93, 115 Minogata, M., 103, 118 Mirimanoff, A., 128, 138 Mirzabekian, R. O., 81, 84 Misato, T., 76, 84 MigeEka, E., 7, 13, 17, 18 Mitchell, C. A., 216(102), 242(102), 268 Mitchell, H. K., 208, 213 Mitchell, J. W., 9, 80, 76, 80, 84, 86 Mituya, A., 238(103), 239(103), 868 Miyakawa, M., 143, 145, 147, 158, 167, 173 Miyawaki, T., 104, 105, 117 Miyazawa, S., 94, 120 Mizuhara, K., 239(155), 245(155), 249 (155), 260 Mizumo, G. R., 59, 70 Mizushima, S., 203, 215 Molliard, M., 269, 278 Mollin, D. L., 103, 113, 118 Monod, J., 215, 220(105), 224(104), 239 (105), 268 Mooney, F. S., 94, 106, 116 Moor, W. A , , 244(106), 268 Moore, C. N., 50, 71 Moore, D., 61, 71 Moore, J. E., 29, 36, 41, 47 Moore, W. A., 134, 135, 138 Morgan, B. H., 49,59,62,67,72 Morgan, B. S., 76, 84 Morgan, R. R., 239(111), 243(111), 249 ( I l l ) , 251(111), 268 Moring-Claesson, I., 37, 47 Morris, D., 46 Morrison, G. A., 131, 158, 202, 206, 818, 213 Mortimer, D.C., 25, 47 Morton, A. G., 202, 213

Moser, H., 220(107, 108), 268 Mouton, R. F., 60, 72, 78 Moyer, A. J., 23,25, 47, 269, 278 Muller, K. O . , 81, 84 Muller, Z., 13, 19 Muller-Kogler, E., 186, 200 Muller, W. H., 76, 86 Mulli, K ., 97, 105, 106, 116, 118 Mundel, O., 90, 98, 118 Munk, V., 11, 19 Murano, H., 80, 84 Murdock, H. R., 104, 118 Murphy, L., 98, I28 Murray, H. C., 5, 20 Musilek, V., 15, 20 MusilkovB, M., 14, 17, 20 Myers, J.,238(109),239(29,109),266,268

N Nagase, H., 205, 213 Naimski, K., 239(70), 267 Nakayama, A., 89, 120 Nakayama, K., 202, 206, 209, 211, 213 Napier, E. J., 76, 80, 86 Natti, V. J., 80, 86 Nayler, J. H. C., 46 NeEhsek, J., 3, 4,7, 8, 14, 17, 19, 20 Nehemias, J. V., 65, 66, 70, 71 Nelson, H. A., 89,97,98, 101,116,118 Neu, J., 239(36), 866 Neujahr, H. Y., 96, 104, 105, 118 Neumann, K., 137, 139 Neuschul, P., 239(67), 243(67), 249(67), 251(67), 267 Ney, P. W . , 96, 121 Nicholas, D. J. D., 99, 118 Nickell, E. C., 59, 70 Nickerson, J. T. R., 66, 72 Nickson, J. J., 51, 78 Nielson, N., 100, f f 7 Nienow, I., 77, 78, 85 Niklas, 0. F., 186, 800 Nishikawa, Y., 76, 77, 84, 86 Nisman, B., 204, 215 Niven, C. F., 49, 56, 62, 66, 70, 7 2 , 73 Nonaka, H., 203, 213 Nordon, H. C., 62, 70 Norman, A., 157, 165, 172, 173 Norman, A. G . , 77, 86

292

AUTHOR INDEX

Norris, L. C.,89,93, 11.9, 118 North, R. A,, 129,140 Northrop, J. H., 239(110, lll), 243(111), 247(110), 249(111), 251(111), 268 Novick, A . , 215, 218(112, 114), 220(112, 113,114,115,116), 225(115),231(115), 239(113), 268 Nowakowska, K.,96,99, 105, 108, 109, 117,118 Nowrey, J . E., 231(117), 239(117), 247 (117), 249(117), 250(42a, 117), 266, 268 Nnmerof, P., 25,26,46,103,118 Nunheimer, T. D., 4,21 Nunome, K., 90,96,118 Nuttal, G. H. F., 142,145,146,147,153, 173 Nygard, J. C., 52,71 Nyunoya, T,, 238(103), 239(103), 268

Page, A . C., Jr.,87,88,89,90,97,119,121 Page, J. E., 88, 114 Paine, J. R., 163,174 Paladino, S.,8, 18,20 Pan, H., 59,72 Pan, 5. C., 26,46 Parikh, J . R., 203,213 Parker, L. F. J., 88,90,94,112, 113, 114, 121 Parks, 1’. E., 155,175 Patel, hf. D., 134,138 Patel, M.K., 76,86 Patte, F., 95,118 Patki, S.J., 132,138 Patton, S., 58, 71 Pawelkiewicz, J . , 89,90,95,96,97,98,99, 105,106,107,108,109,110,116,fi7, 118 Peck, R . L., 31,47 0 I’edziwilk, F., 107,116 O’Brien, R., 25,47,100,102,119,202,213Peeler, H. T., 93,118 O’Callaghan, C. H., 98,107,108,109,111,Pegler, H. F.,88, 92,93,if3 Pehany, E.,105,118 114 Pepinsky, J. B., 104,116 O’Connell, I>. J., 105,I22 Perlman, D., 3,6,7,8, 11, 16,80, 24,25, Ogborn, C . A . , 239(118), 2G8 Oguni, Y . , 90,104,118, 121 46, 47,96,97,100,102,104,107,108, O’Kelly, R.J., 128,140 109,118, 119,202,913 Olive, T.R.,248(119), 2.58 Perlstein, D., 129,130,139 Olson, J . A., 38,47 Perquiri, L. H. C., 269,278 Opton, E.M . , 63,67,70 Perry, J . G . , 220(123), 268 Ordal, E.J., 126,129,130,f39,140 Peterson, A , , 98,119 Ordanik, M., 98,if8 Peterson, B. H., 93,119 Orland, F. J.,152,154,173 Peterson, G. E., 5,16,20 Orr, M.I,., 129,l4O Peterson, R.C., 90,99,111, fig Ortiz, L.O.,111, 116, f2l Peterson, W .H., 25,46,93,97,113 O’Sullivan, P., 93,113 Petrides, P., 94,111, 112 Oswald, W.J., 238(54, 93,120,121), 249 Petrow, V.,88, tl2,i f 4 (54,93,120,121),266,268 Petty, M.A., 87,97,98,101,102,119 Otey, M. C., 168,171,172 Pfeifer, V. F.,4, 20, 90, 102, 119, 243 Otsuka, K . , 206,218 (124), 249(125), 251(125), 668,269 Otsuka, S.,205,212,613, 214 Pfiffner, J. J., 89,90,96, 99,111,11.4, 119 Ott, W. H., 88, 118 Philip, C. B., 186,200 Otto, R. H., 98,100,f90 Owen, S. P., 239(122), 268 Phillips, B. P., 147,154,173 Phillips, L. S.,133,140 P Pichon, P., 10, 19 Pagano, J. F., 90,96,97,100,101,104, Pickering, V.L., 77,84 Pickworth, J.,88, 116 118

293

AUTHOR INDEX

Pierce, J., 87, 88,89, 90,97, 117, 119,121 Piret, E. L., 252(60), 267 Pirt, S. J., 239(126), 269 Plastridge, W. N., 126, 128, 131, 140 Plaxco, J. M., 137, 140 Pleasants, J. R., 149, 152, 153, 156, 173 Poe, C. F., 128, 140 Pollard, E. C . , 60, 61, 63, 67, 70 Pope, H., 98, 119 Popova, L. A., 14, 80, 107, 121 Porges, N., 89, 104, 116, 239(128), 249 (127, 128), 251(127, 128), 269 Pornish, G., 62, 70 Porter, J. W. G., 88,90,91,92,95,96,98, 99, 108, 109, 110, 111, 113, 114, 119, 155, 173 Potter, C., 190, 199 Pouchol, J., 136, 139 Powell, E. O., 220(129, 130), 269 Powell, R . S., 55, 72 Pramer, D., 77, 78, 79, 81, 83, 84, 86 Prasad, N., 98, I21 Pratt, R., 77, 84, 244(143),269 Praus, R., 239(37), 249(37),250(37),266 Prescott, G . C . , 82, 86 Prescott, S. C . , 8, 20, 269, 278 Prescott, G. W., 238(15), 266 Preston, W. H., Jr., 76, 80, 84, 86 Price, F. P., 59, 72 Pridham, T. G . , 76, 86, 98, 119 Principe, P. A., 102, 119 Princivalle, M., 93, 119 Pritchard, H., 93, 119 Privett, 0. S., 59, 70 Proctor, B. E., 50, 54, 59, 60,62, 65, 66, 71, 7 2

Prosen, R . J., 88, 116' Protiva, J., 9, 20, 239(37), 249(37), 250 (37), 266 Provasoli, L., 93, 94, 116 Pryce, J. M., 133, 138 Pulvertaft, R. J . V., 132, 140 Purohit, S. N., 65, 66, 70 Puziss, M., 34, 46 Pyrova, K., 98, 120

Q Quilter, A. K. J., 98, 102, 103, 121

R Rabek, V. T., 105, 119 Rabson, A. S., 240(130a), 269 Rachele, J. R., 156, 172 Radouco-Thomas, C., 60, 7 2 , 73 Rahn, O . , 132, 140 Rainier, W., 216(131),237(131),269 Rajewsky, V. B., 56, 72 Ramachandran, K., 35, 46 Ramsey, H. A., 168, 173 Randles, C. I., 134, 139 Ranfte, J. W., 52, 72 Rangaswami, G., 76, 86 Rapps, N. F., 129, 1-40 Rasmussen, R. A., 99, 120 Rautenktejn, J. I., 4, 20 Ravel, J. M., 92, 120 Rawinski, P., 60, 73 Raynaud, M., 204, 213 Redfield, A. C., 239(81), 257 Reed, J. M., 62, 7 1 , 72 Rees, C . W., 147, 154, 173 Reeves, W. C., 162, 172 Reichstein, T., 88, 90, 120 ReilIy, H. C., 4, 20 Reindel, F., 202, 213 Reineke, L. M., 5, 20 Reio, L., 37, 38, 46 Reiser, C. O., 270, 278 Ressler, C., 156, 172 Rettger, L. F., 126, 128, 131, 140 Reyniers, J. A., 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 159, 161, 170, 172, 173, 174

Reynolds, L. I., 144, 174 Rhodes, A., 76, 80, 86 Richards, A. B., 136, 138 Richards, A. G . , 179, 200 &%a, J., 215(132), 139(98), 244(132), 245(140), 268, 269 Richards, E. H., 266, 278 Richards, M., 209, 813 Rickards, J. C., 95, 104, 112, if3 Rickes, E. L., 87, 88, 89, 97', 98, 99, 113, 118, 119

Rieche, A., 249(133), 869 Riker, A. J . , 82, 84 Robbins, W . J . , 89, 93, 94, 119, 120

294

AUTHOR INDEX

Roberts, M. H., 132, 1.40 Robertson, J. H., 88, 116 Robinson, A. E., 132, 138 Robinson, F. M., 88,96,108,109,110, 114 ,

Sakao, H., 103, 107, I20 Sakurai, S., 204, 213 Salle, A. J., 127, 140 Saluste, E., 37, 38, 48 120 Samarina, 0. P., 204,813 Robinson, K. C., 78, 82, 84 Sanders, A. G., 8, 29 Robinson, R. F., 270, 278 Sanfilippo, A., 108, 109, 110, 114 Robison, R. S., 78, 81, 86 Sanwnl, B. D., 82, 86 Saudek, E. C . , 4 , 7 , 1 0 Rogers, H. J., 239(134), 269 Rogers, L. A.,239(135,159), 248(135,159), Saunders, A. P., 98, 100, 120 249(135, 159), 169, d60 Savage, G. M., 252(60), 267 Savage, M. C., 239(139), 269 Rogowskaya, C., 134,139 Sawada, Y., 96, 121 Roholt, 0. A , , 28, 31, 36, 37, 47 Scalf, R. E., 237(150),239(150), 269 Rolfe, F. W., 263, 878 Rolfe, R. T., 263, 878 Schaffer, J. M., 128, 129, 1.40 Rolinson, G. N., 27, 46,47 Scheele, K., 269, 278 Schenk, A., 179, 199 Rose, N . R., 163, 174 Schilling, E. L., 240(38), 266 Rosen, W. G . , 77, 86 Rosenblatt, M., 237(30), 239(30),243(30), Schindler, O., 88, 90, 120 866 Schmid, 0. J., 97, 105, 106, 118 Rosenblum, C., 103,104,113, 117,120,122 Schmidt, W. H., 25, 47 Rosengren, E., 156, 172 Schneider, W. C . , 239(82), 244(82, 141), Rosenutein, C., 136, 140 267, 269 Roth, W., 127, 158, 199 Schneidl, W., 249(82, 133), 269 Rothberg, H. D., Jr., 11, 20 Schockman, G. D., 155, 172 Rothrock, J. W., 77, 84 Schoog, M., 128, 140 Routien, J. B., 98, 100, 120 Schottelius, M., 145, 154, 174 Roy, S. C., 100, 107, 116 Schrodter, H., 78, 86 Rubin, M., 89, 120 Schuder, D. L . , 193, 200 Rudkin, G. O., 95, 120 Schultze, M. O., 168, 274 Ruf, E. W., 239(136), 240(136), 269 Schumann, G., 76, 86 Ruger, M. L., 102, 116 Schwartz, S., 103, 120 Rushka, J., 239(144), 269 Schwrtrze, W., 216(142), 244(142), 269 Rushton, J. H., 269, 278 Schweigert,, B. S., 58, 70 Ryer, R., 3rd, 60, 71 Scott, J. P., 125, 1.40 Scruggs, W., 155, 17.4 S Sebek, 0. K., 26, 47 Sabet, K. A., 80, 86 Sen Gupta, N. N., 134, 140 Sabrosky, C. W., 189, 800 Senkus, M., 25, 26, 46 Sacksteder, M. R., 144, 159, 174 Seno, N., 89, 120 Saeman, J. F., 59, 72, 237(137, 138), 239 Serjak, W. C., 8, 19 (137, 138), 243 (137), 869 270, 878 Sermonti, G., 16, 20 Sagisaka, K., 209, 213 Sfat, M. R., 8, 19 Sagihara, T. F., 5, 19 Shakleton, M. C., 98, 119 Sahashi, Y., 89, 103, 107, 120 Shapiro, R. L., 132, 158 Saito, T., 206, 215 Sharpe, E. S., 205, 215 Sakaguchi, K., 5, 20, 203, 205, 818, 213 Sakai, H., 76, 84, 89, 93, 94, 98, 99, 103, Sheets, W. D., 134, 199 Shemin, D., 103, 104, 115, 120 107, 118, 180

AUTHOR INDEX

295

Sherrer, E. L., 134,139 Smith, M. N . , 179,800 Shibazski, I . , 136,f4O Smith, V. R., 165,171 Shigeo, F., 133,140 Smyth, H.F., 124,131, 140 Shimizu, W., 125,140 Smyth, R. D., 88, 99,106,118 Shimomura, T., 76,77,84,86 Smythe, C. V., 205,211, 213 Shimono, M., 202,206,212 Snell, N. S., 4,19, 90,96,97,98,99,101, 102,116, 117,118 Shimura, K . , 209,213 Shiotsu, S.,2,18 Snook, G. F., 88, 114 Shirasawa, H., 143,158,173 Sobolov, M., 99,120 Shive, W., 92,93,120 Solomons, I. A., 90,113 Shoemaker, C. B., 88, 116 Somerson, N.L., 43,47 Shonk, C . E., 95,111 Sondheimer, E., 80, 84 Shorb, M.S., 88, 92,118, 120 Soper, Q.F., 25,33,46,46,47 Shotwell, 0. L., 76, 86 Sorkin, E., 96,98,117 Shternov, V. A., 126,130,131,139 gorm, F., 5,20 Shull, G. M., 6,8, 12,18,98,100,120 Southcott, B.A., 93,96,108,109,110,121 Shulman, S.,163,174 Spada-Sermonti, I., 16,10 Shunk, C. H., 88,114 Spalla, C., 96,108,109,110,112, ff4 Shultz, A . R . , 55,72 Sparrow, A. H., 65,72 Shute, C. C. D., 129,140 Spaude, S., 89,105,116 Sichova, O., 105,119 Speedie, J. D , , 11,20 Sikyta, B., 8,213 Sperber, E., 37,&’ Silcox, H.E., 6,8,10 Spielman, M. A., 28, 47 Simek, A., 98,120 Spilman, W . , 239(44), 866 Simon, H., 93,1.82 Spivey, M.R., 111, 121 Simpson, J. R . , 135,140 Spoehr, H.A., 239(29), 266 Singer, E.A., 107,112 Sprinson, D. B., 103,117 Singh, K . , 25,47 Sribney, M., 58,70 Siu, R.G.H., 49,64,67,72 Stafford, W. H., 88,90,112 Sjostrom, A. G. M., 96,99,114, 180 Stapleton, G. E., 60,61,71,72 Skaggs, L. S., 65,72 Stark, D. K., 164,174 Skeggs, H. R., 93,120 Stark, W. H., 101,116,237(150).,239(136), &coda, J., 5,80 239(150), 240(136), 243(49), 268, 269 gkola, V., 14,20 Stark, W. M., 202,21% glechta, J.,4,8, 20 Starkey, R. L., 78,81,86 Smiley, K. L., 99,110 Stauffer, J. F.,25,46 Smith, C. E., 98,101,116 Stavely, H.E., 28,47 Smith, E.L., 25,47,88,89,90,92,93,94, Stawicki, S.,89,90,95,96,97,116,117 95,98,99,102,103,104,118, 113, 114, Stearns, T.W., 270,278 116,118, 120, 121 Stebbins, M.E., 89,93,94, 119,120 Smith, K. M., 190,200 Stedman, R. L., 128,132,1.99,140 Smith, L.A., 239(136), 240(136), 669 Stedra, H., 105,119 Smith, G., 4, 20 Stefaniak, J. J., 269,278 Smith, G. N.,137,f4O Steiner, G., 194,200 Smith, H., 238(63), 267 Steinhaus, E. A., 178,180, 181,182,192, Smith, J., 93,116 193,200 Smith, J. H. C . , 239(29), 266 Stephens, R. L., 270, 978 Smith, N.,107,122 Stephenson, E. L., 90,97,118 Smith, M.B., 99,180 Stern, K. G., 105,f24

296

AUTHOR INDEX

Stessel, G. J., 76, 86 Stevens, C. M., 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 41, 47 Stevens, J. M., 89, 118 Stevens, W. F., 89, 105, 121 Stevenson, E. C., 9, 20 Stice, E., 244(143), 269 Stipek, R. W., 90, 99, 111, 119 Stjernholm, R., 37, 38, @ Stodola, F. H., 76, 86, 205, 213 Stokes, A., 78, 86 Stokstad, E. L. R., 87, 88, 89,90, 93, 97, 116, 117, 119, 121, 155, 174 Stone, L., 8, 19, 99, 120 Stone, R. W., 26, 37, 38, 46, 47 Strandskov, F. B., 9, 20 Strassman, M., 38, 47 Stratmann, H., 238(58), 267 Stratton, J. R., 8, 19 Stuckey, R. E., 93, llY Sturgeon, B., 88, 11.2 SribbaRow, Y. P.,88, 121 Sudarsky, J. M., 90, 98, 102, 121 Sugihara, T . F., 272, 278 Sure, B., 270, 278 Sririkova, E. I., 107, 121 Suter, C. M., 123, 140 Sutherland, L., 88, 112, 116 Suthon, M. D., 76, 84 Sutton, T. S., 97, 98, 116 Sueuki, Y., 132, 140 Svachulova, J., 239(144), 269 Swallow, A. J., 59, 72 Sylvester, J. C., 25,33, 46, 98, 100, 120 Seebiotko, K., 89, 90, 116 Szeremi, K., 124, 140 Szilard, L., 215, 218(114), 220(114, 115, 116), 225(115), 231(115), 247(47), 266, 268

Szkolnik, M., 80, 84 Szuecn, J., 270, 273, 278 Szybalski, W., 16, 18

T Tabenkin, B., 25, 47 Taggttrt, M. S., Jr., 253(146), 269 Taira, T., 4, 20 Tajima, M., 145, 147, 158, 173 Takahashi, H., 203, 212, 213

Takahashi, J., 89, 120 Takahashi, M., 212, 214 Takamine, J., 269, 278 Takashi, U., 133, l 4 O Takata, R., 90, 97, 104, 105, 117, 121 Tamiya, H., 238(103), 239(103), 968 Tanada, Y., 184, 190, 193, 200 Tanaka, K., 96,97,105,117,205,206,209, 213 Tanner, F. W., J r. , 76, 86, 212 Tappan, L). V., 90, 117 Tappel, A. L., 58, 63, 71,72 Tarr, H. L. A., 93, 96, 98, 108, 109, 110, 121 Taylor, E. S., 132, 138 Taylor, R. J., 95, 120 Telling, R. C., 219(66), 220(66), 225(66), 233 (66), 234 (66), 248(66), 267 Tempel, A. E., 3, 6, 8, 20 Terada, O., 94,120 Ternberg, J. L., 105, 121 Terni, S. A , , 63, 78 Terry, R. J., 165, 171 Terui, G., 136, 140 Tezuka, E., 131, 132, 140 Thaysen, A. C., 270, 278 Thierfelder, H., 142, 145, 146, 147, 154, 179 Thirumalacher, M. J., 76, 86 Thomas, A. J., 38, 47 Thomas, W. H., 238(84), 267 Thomas, W. J., 240(160), 260 Thompson, C. G., 191, 192, 193,199,200 Thompson, J. V., 191, 195, 199 Thompson, K. W., 105, 122 Thomson, H. M., 184, 200 Thorbecke, G.J., 150, 151,157,159,174 Thornburn, L. A., 89, 115 Thorn, J. A., 25, 47, 99, 116 Thornberry, H. H., 102, 119 Thorne, C. B., 204, 212 Thornley, M. J., 66, 72 Thornton, H. G., 134, 139 Thorp, F., 89, 116 Tice, L. F., 128, 138 Tieman, J. W., 90, 97, 112 Tiffany, L. H., 238(15), 266 Tilley, F. W.,125, 128, 129, 132, 140 Tindall, J. B., 25, 26, 46

297

AUTHOR INDEX

Tinker, R. B., 137, 1-40 Tobie, W. C., 129, l 4 O Tochikura, T., 205, dl2 Todd, A. R., 88, 90, 112, 113, 116 Tokue, S., 101, 112 Tolbert, N. E., 38, 47 Tome, J., 26, 37, 47 Tomizawa, K., 211, $14 Tootill, J. P. R., 80,8 4 9 3 , 114 Tozer, H., 11, 20 Traub, F. B., 67, 73 Trebst, A., 103, 104, 122 Trexler, P. C.,142,143,144,145,146,147, 149, 150, 151, 152, 154, 155, 156, 170, 173, 174 Trim, A. R., 129, 140 Tripp, G. E., 65, 72 Troescher, C. B., 94, 112 Triieblood, K. N . , 88, 116 Trump, J. G., 50, 52, 53, 73 Tsunoda, T., 93, 120 Tsutomu, S., 133, l 4 O Tsychiya, H. M., 97, 98, 101, 116 Turinova, T., 98, 120 Turner, D. I., 76, 80, 84 Tuttle, L. W., 59, 72 Tyrrell, E. A., 240(147), 269

U Uei, Y., 167, 173 Udaka, S.,202,205,206,209,211,212,213, ,914 Uemura, S., 209, 212 Ueno, M., 10, 20 Ueno, S., 125, 140 Ugolini, F., 8, 18, 20 Uhl, V. W., 269, 278 Ulrich, W., 124, 139 Underkofler, L. A., 4,8,20,242(149), 249 (148, 149), 251(148), 269 Unekata, H., 5, 20 Unger, E. D., 237(150), 239(150), 243 (151), 869 Urbain, W. M., 66, 73 Ushikoshi, I., 99, 118

V Vago, C., 179, 199 ValentovL, M., 8, 80

Valley, G., 126, 128, 131, 140 Van Abeele, F. R., 25, 33, 46 Van Antwerpen, F. J., 220(123), 268 Van de Graaff, H. J., 50, 52, 53, 73 Van der Sluis, J., 8, 18 Van Lanen, J. M., 99, 120 Van Tieghem, P. E. L., 269, 278 Vaughn, R. E., 239(152), 242(152), 249 (152), 269 Vavra, J., 239(153), 269 Veber, J., 185, 200 Veer, W. L. C., 88, 89, 121, 122 Verbina, N. M., 8, 20 Verkhovtseva, T. P., 102, 118 Vintika, J., 10, 20, 21 Vintikov4, H., 10, 21 Virgona, A., 25, 26, 46 Voros, J., 81,86 Vogel, H. J., 35, 46 Vohra, P., 28,29, 31, 32, 36, 37, 41,47 Vojnovich, C.,4,20,90,102,119,243(124), 249(125), 251(125), 268, 269 Vond&ek, M., 3, 7, 8, 19 von Euler, H., 77, 86, 204, 212 Von Hofsten, A., 239(154), 260 Von Hofsten, B., 239(154), 260 von Lorch, L., 93, 119 von Wowern, J., 130, 139 Vorhes, F. A., 64, 73 Vyas, S. R., 98, 181

W Wacker, A., 93, 122 Wagenaar, R. O., 60, 71 Wagner, M., 144, 145, 146, 148, 149, 150, 151, 152, 153, 155, 156, 157, 158, 159, 162, 170, 172, 173, 174 Wagner, R., 134,140 Wagner, R. B., 26, 37, 47 Wakaki, S., 239(155), 245(155), 249(155), 260 Wakamatsu, H., 204, 212 Wakizaka, Y., 204, 205, 213 Waksman, S. A., 4, 20, 269, 278 Walker, A. D., 37, 46, 95, 121 Walker, N., 136, 140 Walker, T. K., 35, 46 Wall, J. C., 93, 116 Wallace, J. H., 240(156), 260

AUTHOR INDEX

Wallen, V. R., 78, 79, 86 Waller, J. G., 95, 1.21 Wallis, R. C., 192,800 Walters, V.,132, 138 Walton, R.B., 11, 11 Ward, G.E., 90, 116 Wasser, H. B., 189, 800 Watanabe, H.,243(157), 860 Watson, C.J., 103, 180 Watson, J., 90,117 Wayman, A. C . , 25, 47 Weeks, B.M., 56, 71 Wehmer, C.,269, 878 Weinhouse, S.,38, 47 Weinstein, M.J., 16, 80 Weintraub, A., 5,d0 Weiser, H.H., 93, 118, 134, 159 Weiser, J., 182, 184,185,194,800 Weiss, G.J., 51, 70 Weissbach, H.,88, 99, 106, 118 Welch, A. D., 87,97, 181 Welsch, M.,4, 81 Wenner, H.A., 161, 174 Werkman, C.H., 204,818 West, A. S., 194, 199 West, J. M., 244(85), 867 West, R.,88, 188 Westveer, W. M., 126, 140 Weygand, F.,93,103, 104, 118 Wheeler, M.C., 249(158), 860 White, G.F., 183, 184, 189,800 White, J. G., 88, 116 White, R. T., 178, 183, 199, 800 Whitehead, C. W., 25,46, 46, 47 Whitfield, G. B., 89, 90,97, 116 Whitmarsh, J. M., 104, I88 Whittier, E. O., 239(135, 159), 248(135, 159), 249(135, 159), 869, 860 Wiesen, C. F., 98, 101,116 Wijmenga, H.G., 88,89,95,105,181,188 Wild, U. G.,133, 140 Wild, G.M., 202,812 Wiley, A. J., 237(72), 239(72), 867, 270, 878

Wilharm, G., 107, 108, 109, 118 Wilkin, G. D., 37, 46 Wille, H.,185,200 Willeke, L.,78, 86 Willkie, H. F., 243(151), 860

Williams, N. J., 62, 71 Williams, 0.B., 124, 158 Williams, P.L., 100,106,114 Williams, W. L . , 92,93, 117, 155, 178 Wilson, J. L., 129, 140 Wilson, J. N., 239(27), 240(27), 866 Wilson, R.A., 80, 84 Wilson, R. E., 216(43), 220(43), 225(43), 231(43), 239(43),866 Winder, F. G., 37, 46' Windsor, E.,208,814 Winitz, M.,168, 171, 178, 203, 211, 818, 819

Winkler, K. C., 220(31), 866 Winnick, T.,37, 47 Winter, A. G., 78, 86 Winter, A. R., 97, 98, 116 Wintersteiner, O., 28, 47 Wise, G.H., 168,173 Wisthoff, R.T., 11, 19 Witebsky, E.,163, 174 Witkus, E.,98, 181 W i t h , B., 129, 159 Wolf, D.E.,88,89,91,113, 114,117, f88 Wolf, F. J., 101, 104, 188 Wolf, P. A., 126, 140 Wolfe, P.A., 147, 154,175 Wolfrom, M.L., 55, 73 Wolin, E.F., 62, 66, 73 Wollman, E.,147, 154, 171 Wolnak, B.,89, 105, 118, 181, 188 Wood, T. R., 87, 88, 89, 97, 98, 99, 101, 102, 115, 118,119, 191 Woodbury, D. H., 61, 78 Woodbury, D.T., 103,104,115, 110,128 Woodruff, H. B., 4, 8, 81, 25, 46, 93, 95, 104, f f 4 , 118, 188 Woods, D. D., 37, 47 Woodward, E.R., 9, 80 Woolley, D. W., 107, 188 Work, E.,208, 818 Wostman, B. S., 149, 150, 151, 156, 157, 159,160,178, 174 Wright, B. E., 38, 47 Wright, E.S., 131, 139 Wright, J. M., 77, 86 Wright, L. D., 93, 180 Wright, R. D., 104, 198 Wright, W. H., 147, 154, 175

299

AUTHOR INDEX Wuest, H. M., 90, 18.2 Wyss, O., 124, 133, 158

X

Yonehara, Y., 76,84 Yuji, M., 133, 140 Yvoire, M., 272, 8Y8

Z

Xeros, N., 190, 800

Y Yacowitz, H., 93, 112, 118 Yakobson, L. M., 204, 215 Yall, I., 38, 46 Yamaguchi, A., 76, 84 Yamaguchi, Y . , 239(155), 245(155), 249 (155), 250 Yamamoto, T., 96, 181 Yamatodani, S., 4, 80 Yazaki, H., 205, 212, 815, $14 Yanagita, T., 132, 133, 140 Yokoi, K . , 143, 158, 175

Zajicek, J., 8, 80 Zakrzewsky, K., 239(70), 867 Zaumeyer, W. J., 76, 80,84,86 Zelinka, J., 7, 13, 17, 18 Zelle, M. R., 60, 71 Zender, R., 60, 79, 73 Ziegler, D. W., 240(160), 960 Zinn, R. E., 89, 106, 181, 189 ZoBell, C. E., 249(161), 253(161), 860 Zodrow, K., 89,90,95,96,97,98,108,109, 110, 116, 117, 118 Zook, H. D., 26, 37, 47 Zweifach, B. W., 157, 174

SUBJECT INDEX A Absorption of antibiotics by plants, 77-81 Acetate in penicillin biosynthesis, 37 Acetic acid production by continuous fermentation, 242 Acetone production, 249-251 Agaricus, 262-5, 268, 27G3 Alcohol production by continuous fermentation, 216, 241-7, 249-251 Amino acids, production by fermentation processes, 201-214 Animals, germfree, 141-174 Antianemia factor, 88 (see also “Cobamides”) Antibiotics fermentations, as source of cobamides, 89 growth promotion effect in animals, 155 in plant disease control, absorption and translocation, 77-81 mode of action, 81-2 selective toxicity, 76-7 status of, 75-85 production by continuous fermentations, 243-8 protection of fermentation processes by use of, 9-1 1 use with phenol, 136-7 Antibodies in germfree animals, 149-151 Antimicrobial activity of phenol, 123-140 Antimicrobial agents (see also “Phenol ”), protection of fermentation processes by use of, 8-12 Auxotrophs of E . coli, 93 of M . glutarnicus, 207, 209, 211

B Bacillus popilliae, 177 Bacillus thuringiensis, pathogenicity for Lepidoptera, 178 Bacteria

effects of ionizing radiation on, 60-3 factors affecting activity of phenols on, 123-140 insect diseases, 176-9 Beaveria bassiana in insect disease, 180 Benzylpenicillin, 23 (see also “penicillin”) Bioassay for cobamides, 9 2 4 use in antibiotic translocation and absorption studies, 77-8 Biosynthesis of chlortetracycline without aseptic conditions, 12-3 of penicillin, mechanism, 2347 Biotin effects on L-glutamate formation, 207

C Carpocupsa pornonella, 195 Cesium-137, 51 Chickens, germfree rearing, 145-9 Chloramphenicol absorption and translocation in plants, 78-80 mode of action in plant disease, 81-2 production by continuous fermentation, 245 Chlorobiurn, 254 Chlortetracycline, 78 biosynthesis without maintenance of aspetic conditions, 12-3 Chromatiurn, 254 Chromatography, separation of cobamides, 95-6 Clostridium acetobutylicurn, 249-51 Cobalt, 91-102 Cobalt-60, 51, 103 Cobamides analytical methods for the determination, 92-7 microbial synthesis, 87-122 of “natural”, 97 nomenclature, 90-2

300

30 1

SUBJECT INDEX

nutritional value, 110-2 peptides, 105 precursors, 102-3 radioactive, preparation of, 103-4 unnatural, 106-9 Compost pasteurization, 267 synthetic, in mushroom production, 266 Contamination in continuous fermentations, 209,229230,246-7 protection of fermentations, 1-21 Continuous fermentation cell production, 237-241 chemical products studied, 249 theory, 219-236 general equation development, 221-5 general equation extension, 225-9 mathematical symbols, standardization, 219-221 of oscillation phenomenon, 231-2 of prediction from batch data, 233-4 of product formation, 234-5 of stability, 223-5 of undesirable organism effects, 229231 summary, 2356 Continuous industrial fermentations, 215-260 classification of operat,ions and processes, 217-9 mixed culture, 252-5 products, 241-252 acetic acid, 242-3 antibiotics, 243-8 ethyl alcohol, 241-2 Coxiella popilliae, see “Rickettsiella popilliae ” L-Cysteine in penicillin biosynthesis, 2831

D 5,6-Dimethyl-alpha-benzimidazolylcobamide cyanide, 101,104 Diphosphopyridine nucleotide in Lglutamic acid formation, 204 Disease of insects, 17b200 of plants, control by antibiotics, 75-85

Dosimetry in radiation preservation, 54 DPN, 204 (see “Diphosphopyridine nucleotide”) Drug preservation by ionizing radiation, 49-73

E ’

Enzymes, effect of radiation, 59 Escherichia coli, 61,93,124-8,132, 136, 204,208, 209,211,251

F Fermentation classification by importance of contamination, 5-7 contamination sources in aseptic processes, 7-8 continuous industrial, 215-260 deep-vat method for higher fungi, 269 protected, 1-21 protection by antimicrobial agents, 8-12 sterility testing, 2-5 Food preservation by ionizing radiation, 49-73,169 Fungi in insect diseases, 179-181 large scale growth of higher, 261-8

G Germfree animals additional research needs, 167-171 applications, 153-167 characteristics, 148-153 methods and equipment, 142-148 techniques, 141-174 Glutamic acid fermentation, 203-8

H Higher fungi definition, 262 large-scale growth, 261 beta-Hydroxyvaline in penicillin biosynthesis, 35-6

I Immunology, study of, in germfree animals, 1624

302

SUBJECT INDEX

Induced radioactivity, threshold energy levels for the production of, 64 Inorganic salts, effect on antimicrobial activity of phenol, 126-8 Insect control by microorganisms, 175, 176-8, 179, 182, 187, 191-8 by radiation, 65 Insect diseases, 175-200 Insect microbiology, 17.5200 Intestinal flora, growth-depressing action, 160 Ionizing radiation action on peptides and proteins, 56-8 applications, 65-67 economic considerations in food and drug preservation, 68-69 effect on microorganisms, 60-3 on wholesomeness, 63-5 physical and chemical change produced, 64-60 preservation of foods and drugs, 49-73 Ionophoresis, separation of cobamides, 96 Isotopes in the study of penicillin biosynthesis, 26,28-39,41-2

lysine production, 209 pathways of glucose oxidation, 208 Microorganisms ionizing radiation effects, 60-63 genera grown in continuous culture, 239 Microsporidia causing insect disease, 183-5 Mixed culture fermentation, 252 Morchella, 270, 272-6 production in submerged culture, 272-6 Mushrooms history of culture, 263 industrial research, 265-9 submerged culture, 270-5 Mutants bioassay of cobamides, 93 continuous fermentations, 230-1,247 Mycelium, dewatering, 275

N Nematode in insect diseases, 193-8 Nematode-bacterial. disease complex, 195-8

0 J Japanese beetle blue disease, 185-8 microsporidian infection, 183 type A milky disease, 177-9, 194

1 beta-Lactam-thiazolidine nucleus of penicillin, precursors of, 28-36 Lactobacillus lactis, 9 2 4 Lactobacillus leichmannii, 9 3 4 Lysine biosynthetic pathway, 209 fermentation, 208-210

M Mammals, germfree rearing, 146-8 Metarrhizium anisopitae in insect diseases, 179 Micrococcus glutamicus L-glutamic acid, fermentation by, 205-8

Ochromonas malhamensis, 94 L-Ornithine biosynthetic pathway in M . glutamicus, 211 fermentation, 210-1 Oscillation phenomenon in continuous fermentations, 231 Oxygen tension, effect of antimicrobial activity of phenol, 125 Oxytet#racycline,78

P Pasteurization by radiation, 66 Penicillin mechanism of biosynthesis, 2347 plant disease control, 78 precursors, beta-lactan thiazolidine ring nucleus, 26-36 condensation, 38-43 of side chain, 25-26 production by continuous fermentation, 244-6 protected fermentations, 9-11, 16

303

SUBJECT INDEX

Penicillium chrysogenum, 23 Penicillium notatum, 23 PH causing oscillation in continuous fermentation, 231 effect on antimicrobial activity of phenol, 126 Phenols factors affecting the antimicrobial activity, 123-140 oxidation by bacteria, 134-6 use with other inhibitors, 136-7 Phenylacetic acid as side chain precursor of benzylpenicillin, 25-6, 38-41 Photosynthetic bacteria, see “Chromatium” and “Chlorobium” Phytotoxicity of antibiotics, 77 Plant disease status of antibiotics in control of, 7585 Protected fermentation, 1-21 prospectives in, 13-17 Protozoa in insect disease, 181-5

R Radiation (see also “Ionizing radietion”) high energy, nature of, 50-7 sources of, 51-4 injury, use of germfree animals in study, 154 preservation, 49-73 Rat, germfree, 151 Reductive amination in biosynthesis of L-glutamic acid, 204 Resistance to phenols, 133 Rickettsia in insect disease, 185-8 Rickettsia melolonthae, 185-8 Rickettsiella popilliae, 185-8

s Salmonella typhosa, 124-6, 130-132 Serology, germfree animals in study, 162-4 Sewage mixed-culture continuous fermentation, 252 vitamin BIZand cobamides in, 104-5

Soaps effect on antimicrobial activity of phenol, 128-130 Spawn preparation, method, 265 Staphylococcus aureus, 124-7, 129-133 Sterility testing, problems in fermentation processes, 2-6 Sterilization cold (see “Radiation preservation”) for germfree animal techniques, 145 by radiation, 66 Streptomycetes chlortetracycline production, 12-3 cobamide production, 89, 98, 101, 107 phage contamination, 4, 11 streptomycin production, 14 Strep tomyc in absorption and translocation in plants, 78-81 mode of action in plant disease, 81-2 phytotoxicity, 77 production by single-stage continuous fermentation, 243 Submerged culture food yeasts, 209-270 molds, 270 mushrooms, 270-5 production problems of higher fungi, 275 Sulfur formation by natural continuous fermentation, 253

T Temperature effect on antimicrobial activity of phenol, 124-5 on viral infections of insects, 192 TPN, see “Triphosphopyridine nucleotide,” 204 Transamination in biosynthesis of Lglutamic acid, 204 Translocation of antibiotics by plants, 77-81 Triphosphopyridine nucleotide in Lglutamic acid formation, %4 Trogoderma inclusum fungus infections, 182

304

SUBJECT INDEX

V L-Valine in penicillin biosynthesis, 31-5 Virology potential of germfree animals, 161 use of germfree animals in study of, 158 Viruses diseases of insects, 188-193 effects of ionizing radiation, 63 insect-parasite relationship, 190-1

Vitamins effect8 of radiation on, 63 metabolism in germfree animals, 156 in Morchella mycelium, 273 Vitamin Blz , see “Cobamides”

Y Yeast production by continuous fermentation, 216,237-8