Advances in Applied Microbiology, Volume 6

ADVANCES IN

Applied Microbiology VOLUME 6

CONTRIBUTORS TO THIS VOLUME Aris P. Bayan Henry

L.

Ehrlich

Nancy A. Giuffre Herbert S. Goldberg Carl-Goran Hdden

8. W. Koft

P. Margalith

D. Perlman Melvin P. Silverman Irwin W. Sizer Mortimer P. Starr Wayne W. Umbreit

K.

Wuhrmann

ADVANCES I N

Applied Microbidogy Edited by WAYNE W. UMBREIT Department of Bacteriology Rutgers, The State University New Brunswick, N e w Jersey

VOLUME 6

@

1964

ACADEMIC PRESS, New York and London

COPYRIGHT @ 1984,

BY

ACADEMICPRESSINC

ALL RIGHTS RESERVED NO PART OF THIS BOOK MAY BE REPRODUCED IN ANY F O R M BY PHOTOSTAT, MICROFILM, OR ANY OTHER MEANS, WITHOUT WRITTEN PERMISSION FROM THE PUBLISHERS

ACADEMIC PRESS INC. 111 FIFTHAVENUE NEW YORK, NEW YORK 10003

United Kingdom Edition

Published by ACADEMIC PRESS INC. (LONDON)LTD.

BERKELEYSQUARE HOUSE,LONDONW. 1

Library of Congress Catalog Card Number 59-13823

PRINTED I N THE UNITED STATES OF AMERICA

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

AEUSP. BAYAN,Squibb Znstitute for Medical Research, New Brunswick, New Jersey (27) HENRYL. EHRLICH, Rensselaer Polytechnic Institute, Troy, New York (153) NANCYA. GIUFFRE,Squibb Znstitute for Medical Research, New Brunswick, New Jersey (27) HERBERT S. GOLDBERG, Department of Microbiology, School of Medicine, University of hfissouri, Columbia, Missouri (91) CARL-GORAN HEDEN,Department of Bacteriology, Karolinska Znstitutet, Stockholm, Sweden ( 1 ) B. W. KOFT, Department of Bacteriology, Nelson Biological Laboratories, Rutgers, The State University, New Brunswick, New Jersey (227) P. MARGALITH,Laboratory of Microbiology, Department of Food and Biotechnology, Technion-Zsrael Znstitute of Technology, Haifa, Israel (69) D. PERLMAN, Squibb Znstitute for Medical Research, New Brunswick, New Jersey (27) MELVINP. SILVERMAN, Pittsburgh Coal Research Center, U . S. Bureau of Mines, Pittsburgh, Pennsylvania (153) IRWINW. SIZER,Department of Biology, Massachusetts Znstitute of Technology, Cambridge, Massachusetts ( 207) MORTIMERP. STARR,Department of Bacteriology, University of California, Davis, California ( l ) WAYNE W. UMBREIT,Deparfment of Bacteriology, Rutgers, The State University, New Brunswick, New Jersey (227)

K. WUHRMANN, Znstitute of Water Supply, Sewage Purification and Water Pollution Control at the Swiss Federal Institute of Technology, Zurich, Switzerland ( 119) V

This Page Intentionally Left Blank

PREFACE The present volume, the sixth in this series, represents an expansion into areas of applied microbiology other than those solely related to the laboratory. The applied microbiologist has an impact upon and is responsive to changing conditions in the world. The “‘globalimpact of microbiology” is discussed as well as some opinions on what kind of training an applied microbiologist ought to have. The range of the other articles remains broad and considers the microbial action on minerals, the nonmedical uses of antibiotics, water pollution control, some aspects of industrial enzymes, the preparation of radioactive substances by microbial processes, and the influence of some factors, normally considered minor, upon fermentation processes. One difficulty for an Editor, but of course one advantage to the science, is that the area of knowledge covered by applied microbiology is so broad that it is most difficult to cover adequately. Various individuals working in it have quite different views of what ought to be considered in a publication such as “Advances.” Should you find that an area of particular interest to you is not being adequately covered, please let the Editor know and perhaps something can be done about it. It is the object of “Advances” to be as useful as possible and to take advantage of the virtues of the eighteenth century essay as a medium of communication and exchange of useful information.

W. W. UMBREIT

Rutgers University June, 1964

vii

This Page Intentionally Left Blank

CONTENTS CONTRIBUTORS .................................................

V

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

vii

PREFACE

Global Impacts of Applied Microbiology: An Appraisal CARL-GORAN HED~N AND MORTIMER P. STARR I. I1. I11. IV . V. VI . VII . VIII . IX . X. XI . XI1. XI11. XIV. XV . XVI.

Human Ecology ........................................ The Social Responsibility of Scientists ...................... Applied Microbiology and Medicine ....................... General Aspects of the Exploitation of Biological Resources . . . . Microbiological Aspects of Water Resources ................. Applied Soil Microbiology ................................ Nitrogen Fixation ....................................... Microbiological Aspects of Crop Protection . . . . . . . . . . . . . . . . . Biological Control of Insects .............................. Protection of Animal Resources ............................ Prevention of Deterioration ............................... Carbohydrate Waste ..................................... Photosynthesis .......................................... Improved Utilization of Human Resources . . . . . . . . . . . . . . . . . . Microbiology Applied to Biological Research in General ....... International Efforts .....................................

2 5 6 9 10 12 13

14 15 16 17

18 20 20 22 23

Microbial Processes for Preparation of Radioactive Compounds D . PERLMAN. ARIS P. BAYAN.AND NANCYA . GIUFFRE

. . . .

I Introduction ........................................... I1 Methods Used in Preparing Radioactive Compounds by Microbial Processes .............................................. I11 Radioactive Compounds Prepared by Microbial Processes ..... IV Summary .............................................. References .............................................

27 29 36 60 61

Secondary Factors in Fermentation Processes P . MARGALITH

I. I1. I11. IV . V.

Introduction ........................................... Inorganics .............................................. Organics .............................................. Various ................................................ Conclusion ............................................ References ............................................. ix

69 71 81 85

88 88

X

CONTENTS

Nonmedical Uses of Antibiotics HERBERTS . GOLDBERG

I. I1 I11. IV . V

Introduction ........................................... Antibiotics in Animal Nutrition . . . . . . . . . . . . . . . . . . . . . . . . . . . Antibiotics in Plant Disease Control ........................ Antibiotics in Food Preservation ........................... . Antibiotics as Adjuncts in Microbiological Techniques and Procedures ................................................ VI . Public Health Aspects of Nonmedical Uses of Antibiotics . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

.

91

93 98 100 106

109 114

Microbial Aspects of Water Pollution Control K . WUHRMANN

. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Aspects of Microbiological Waste Treatment . . . . . . . . . . . . . . . . . The Problem of Slowly Decomposable Substances in Wastes . .

119 120

The Removal of Nitrogen from Wastes-A Special Contribution of Microbes to Pollution Control .......................... References .............................................

141 150

I I1. I11 IV.

133

Microbial Formation and Degradation of Minerals MELVINP . SILVERMAN AND HENRYL . EHRLICH I. I1. I11. IV . V. VI . VII .

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Geological Concepts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mechanisms of Microbial-Mineral Interactions . . . . . . . . . . . . . . Survey of Microbial Interactions with Inorganic Substances . . . . Sulfur Mineral Deposits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Iron and Manganese Deposits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

153 154 157 161 169 187 198 198

Enzymes and Their Applications IRWINW . SUER

. . . .

I I1 I11 IV V.

Introduction ........................................... Molecular Structure of Enzymes .......................... Enzyme Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Microbial Enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

207 208 214 224 225 226

CONTENTS

xi

A Discussion of the Training of Applied Microbiologists B . W . KOFT

.

I I1. I11. IV V

. .

AND

WAYNE W . UMBREIT

Introduction ........................................... Undergraduate Training ................................. Bachelor Degree with Orientation toward Graduate School . . . . Graduate School Training ................................ Other Problems ........................................

................................................ SUBJECT INDEX. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

AUTHORINDEX

227 231 235 235 238 241 253

This Page Intentionally Left Blank

Global Impacts of Applied Microbiology: An Appraisal CARL-GRAN H E D ~AND N MORTIMERP. STARR Department of Bacteriology, Karolinska Institutet, Stockholm, Sweden; and Department of Bacteriology, University of California, Davis, California

I. 11. 111. IV. V. VI. VII. VIII. IX. X. XI. XII. XIII. XIV. XV. XVI.

Human Ecology ........................................ The Social Responsibility of Scientists ..................... Applied Microbiology and Medicine ....................... General Aspects of the Expbitation of Biological Resources . . . Microbiological Aspects of Water Resources ................ Applied Soil Microbiology ............................... Nitrogen Fixation ...................................... Microbiological Aspects of Crop Protection ................. Biological Control of Insects .............................. Protection of Animal Resources ........................... Prevention of Deterioration .............................. Carbohydrate Waste .................................... Photosynthesis ......................................... Improved Utilization of Human Resources .................. Microbiology Applied to Biological Research in General ...... International Efforts ....................................

2 5 8

9 10 12 13

14 15 16 17 18 20 20 22 23

A conference, bearing the imposing title “Global Impacts of Applied Microbiology” (GIAM), was held in Stockholm from July 29 to August 3,1963. This conference bore the subtitle “A Projection of the Microbiological Research of Today to the Needs of TomorTOW.’’Since the future needs of the world will certainly be most pressing in the developing areas, it was natural for this conference to focus on microbiology as applied to the solution of problems indigenous to such regions. Actually, the Stockholm conference might be regarded as a continuation-within one particular fieldof the United Nations Conference on the Application of Science and Technology For the Benefit of the Less Developed Areas held in Geneva in February, 1963. The Stockholm GIAM conference, which was attended by some 350 representatives of 30 countries, was arranged by the Section for Economic and Applied Microbiology of the International Association of Microbiological Societies (IAMS) in collaboration with the Royal Swedish Academy of Engineering Sciences. It was cosponsored by the World Academy of Art and Science, a relatively new 1

2

CARL-GORAN H E D ~ NAND MORTIMER P. STARR

organization devoted to questions concerned with the interplay of science and society, Professor A. Tiselius and Dr. M. Tveit served as President of the Conference and Secretary-General, respectively. This conference was an interesting and quite unique experiment in that it brought together specialists from many unrelated disciplines: soil microbiology, medicine, fermentation, phytopathology, immunology, food technology, economics, and science administration-to name but a few. It was structured to provide a forum for various viewpoints, and to generate programs for action which could be presented to different governmental and international agencies. Many of the deliberations should, however, be of considerable interest to the individual scientist working in the field of applied microbiology; hence, we have summarized here some of the high points of the GIAM conference. We have neither the ambition nor the competence to present a comprehensive and substantive review of the multidisciplinary field covered by the conference. Rather, we shall prepare a concentrate of some salient ingredients, an “instant” and, we hope, thought-provoking brew, which the reader should dilute with his own experience and imagination. We may have spiced the preparation with a few personal views and prejudices; therefore, we recommend that a factual and unbiased view of the Stockholm conference be obtained by consulting the compIete proceedings which are now in press under the title “Global Impacts of Applied Microbiology” (edited by M. P. Starr, published jointly by Almquist & Wiksell, Uppsala, and Wiley ( Interscience), New York). Since that book comprises the main reference source, we have omitted here citations to the literature which are annotated in that volume.

1.

Human Ecology

The prevailing concern among scientists for the population crisis was evident in the introductory remarks made by S. Brohult who referred to the latest United Nations report on population increase, with its disturbing news that earlier demographic prognoses had been too low. Actually, the 1960 population census estimate surpassed the 1958 prognosis by 75 million. There has been an increase in the food production of our globe, certainly, but, during the 1950’~~ the productivity per inhabitant had remained essentially constant. The Food and Agricultural Organization estimated in 1962 that continued growth in population, in accordance

GLOBAL IMPACTS OF APPLIED MICROBIOLOGY

3

with the United Nations forecast, would necessitate doubling the world supply of food by 1980 and trebling it by the turn of the century, in order to provide a reasonably adequate level of nutrition for all peoples. The GIAM speakers and confreres were gravely concerned that half of the peoples of the world are presently suffering from hunger and disease. G. Borgstrom discussed the biological and chemical limitations in the continued growth of the human biosphere, and threw serious doubt on the possibilities of providing sufficient food to banish the specter of starvation. Agriculturists have been confident that they can continue to raise yields with the aid of modern science and technology. Borgstrom calls this a tragic fallacy and points out that the law of diminishing returns is already operating with regard to minerals, water, etc., in several highly advanced countries. Notwithstanding the potentialities of synthetic chemistry, we must accept the fact that our main source of food, now and in the remote future, must continue to depend on sunlight and carbon dioxide. Borgstrom formulates a new popuIation density concept in which the total biomass within man’s action sphere is related to soil resources, and he shows that the total human biomass has now acquired dimensions which far overshadow those of any other higher mammal. It is, for instance, interesting to consider that the total weight of livestock of all major countries is 860 million metric tons compared with the total calculated weight of humans (187 million metric tons). The livestock is under the direct control of man within his biosphere, and if the biosphere is recalculated in terms of Borgstrom’s population equivalents, it becomes startlingly clear that green plants must carry a feeding burden which is far in excess of the 3 billion humans we normally consider to be the beneficiaries of photosynthesis. A closer estimate would be the equivalent of 17.5 billion human consumers! Horses still require a protein intake that corresponds to that of 653 million people; in other words, the equine consumptive force itself equals that of the humans in the most populous country of the world, China. It is unfortunate that man has thus far been almost completely unable to relate himself properly to his environment. In a situation in which man’s attention is concentrated on the struggle with other human beings, he may tend to forget that he may be in an even more serious struggle with other living organisms, be it insects or

4

CARL-GORAN

HEDEN AND MORTIMER P. STARR

bacteria, It has been estimated that insects eat, steal, or destroy one-third of everything which man grows and stores for the future. Moreover, the World Health Organization has estimated that insects cause one-half of all human deaths, disease, and deformity. A single insect-borne disease, malaria, still infects one-sixth of the human race and claims a human life every 10 seconds! In discussing human ecology and infectious diseases, S. Mudd reminded his audience that an infectious disease is a struggle between two biological systems, each adapted by long evolution to survive under conditions inimical to the other. In earlier days, the birth and death rate and the natural resources necessary to support human populations approached a steady-state condition. Now, a reduction of mortality has brought about unbalanced increases in population out of proportion to our augmented resources. Consequently, it is no longer possible to provide a minimum adequate diet for each member of the human family, and a net increase in its collective misery is the result. This is, of course, particularly true in the newly independent nations in which, as pointed out so eloquently by the Deputy Prime Minister of Israel, A. Eban, there is the sad realization that a nation can be free in every institutional sense of the word and yet lose the essence of freedom in the throes of starvation and want. As mentioned before, much of the discussion at the Stockholm meeting was actually focused on the need for helping the underdeveloped countries and the problems encountered in the limitations of their capacity to accept and adopt a science and technology based on presuppositions and value systems different from their own. This problem is undeniably complex as may be illustrated by a quotation made by E. M. Mrak from a work by G. Hardin: “If we include freedom to breed as one of man’s inalienable freedoms, and if we accept the obligation to share excess food with those who are starving, then how can any nation, class, or religious group that responsibly controls its numbers survive competition with any nation, class, or religious group that refuses to act responsibly?” Our main consolation is that there has been a reduction not only in death rates but also in birth rates in every country that has changed from a predominantly agrarian society to one whose predominantly industrial urban pattern has extended its public education.

GLOBAL IMPACTS OF APPLIED MICROBIOLOGY

5

II. The Social Responsibility of Scientists The fact that the Stockholm conference was initiated by scientists speaks against the popular notion that scientists are indifferent to the social consequences of their work. The great number of world authorities participating also illustrates the widespread feeling of responsibility and concern for the present trends in science administration and sociological development. These factors are much too dynamic to be steered unerringly by means of shortsighted local planning. Furthermore, the potentialities of science and technology are now so enormous that the consequences may b e disastrous if decisions are made by power-seeking and parochially oriented individuals. No doubt the freedom of action, “the political latitude,” is shrinking in an environment in which the general training level is high and whose complex social structure requires specialists at the highest levels, but the application of science and technology in the developing countries certainly involves dangers. Eban elaborated on the problems of controlling the great powers inherent in scientific investigation, which must be enormous when 80 or 90% of all scientists who have ever lived are living now. He stressed that there is no room today either for a scientist without social conscience or for a politician who lacks a basic understanding of the impact of science upon the life of nations and the world community. Beyond the great problems created by nuclear weapon technology, the most urgent human issue now concerns the awakening continents. Eban projected the population increase and the more modest growth of resources into the next century and pointed out the tensions which will ensue. H e found monstrous the suggestion that science is somehow responsible for the perils of nuclear war, for the prospect of world famine, or for the gap between the greater and lesser advanced countries. “The responsibility lies not in science, but in our failure to determine the social direction of scientific progress.” A former United Nations adviser in economic affairs, G. Myrdal, was of the opinion that the world is headed for an economic and political cataclysm if radical changes are not made. In his opinion, the current and now foreseeable economic and social trends in most of the underdeveloped countries have more serious, not to say sinister, directions than is commonly recognized. Particularly dis-

6

CARL-GORAN HEDEN AND MORTIMER P. STARR

turbing is the accelerated rate at which the gap in income between the underdeveloped and industrially advanced countries is widening. It was stimulating to hear the bacteriologist, J. Birkeland, discuss the psychological and sociological climate for scientific investigation, coining terms such as “ecology of science.” He reminded his audience of the Middle Ages when man was preoccupied with problems of salvation and sin, and of the plagues and pestilences that were explained on the assumption that people had sinned. TOO great a preoccupation with life in the hereafter does not provide a favorable intellectual climate for developing the knowledge that will furnish the food and shelter and eliminate the disease of our present world. As perceptively noted by E. C. Stakman, the degree to which man’s intelligence and enterprise enable him to master his environment-instead of being a mere servant of it-is a measure of civilization. In this connection, the relatively young science of microbiology is essential because it is directly concerned with two of the most elemental of all human needs-health and food. The microbe is a prime natural resource of all mankind, regardless of national boundaries. If man makes an effort to understand, control and utilize this resource it may affect his future most profoundly, Science has taught us to live longer and more abundantly by combating dangerous microbes and by utilizing beneficial ones, and applied microbiology now holds a major key to this needful future. As expressed by Stakman, “its potential role is tremendous and it is tremendously important.” The scientists who, together with the international agencies of health and welfare, philanthropic foundations, and pharmaceutical industries, share the responsibility for the present imbalance between mortality and fertility, must continually stress this point. It is a biological certainty that the steady state will be restored but, as rational and compassionate human beings, we cannot, as pointed out by Mudd, leave the restoration to the Horsemen of the Apocalypse, War, Famine, and Pestilence.

111. Applied Microbiology and Medicine Man has many enemies in the microbiological world and some, such as the etiological agents of smallpox, yellow fever, and malaria, are of undoubted ecological importance. In the Western world, control measures have been developed gradually, but in the new

GLOBAL IMPACTS OF APPLIED MICROBIOLOGY

7

nations the development phase has been bypassed and low-cost measures of controlling disease have been imported. As a consequence, the approximate steady-state equilibrium between the available nutrition and actual number of human beings has been disturbed, and individuals, in larger numbers than ever before in the history of the world, are now living in a state of hunger and malnutrition. W. C. Cockburn, from the World Health Organization, discussed the implications of large-scale programs for the control of infectious disease. Apart from smallpox, whose eradication is now a possibility, such programs are relatively recent, but present community campaigns include vaccination programs aimed at tuberculosis, poliomyelitis, pertussis, diphtheria, tetanus, and trachoma. Measles vaccine will follow, and perhaps even preparations active against infectious hepatitis. Immunization methods against tetanus, diphtheria, smallpox, and poliomyelitis have been remarkably effective; their reduced incidence may have its repercussion in the population increase. However, Cockburn emphasizes that not only do immunization procedures save lives, they also ensure a state of health which improves productivity; certainly, they reduce the cost of the health services, a case well illustrated by diphtheria. Such positive contributions to the population balance were also emphasized by J. Ungar, who mentioned the importance of the recently developed trachoma vaccine which may control a disease afflicting approximately one-sixth of the world population. In the population balance, we should not forget that parasitic worms, which infect the people of certain tropical countries, are said to metabolize more of the produce of those countries than do the inhabitants. Obviously, the development of immunization programs against helminthic infections and protozoal diseases may be of the greatest importance in the tropics, but a forecast cannot be attempted in the present state of our knowledge. Much research must go into the development of vaccines having greater safety, potency, and stability than are possessed by present preparations. The economic advantages of large-scale production of vaccines should be exploited, but much remains to be learned about techniques. P. van Hemert emphasized that specialized study of unit processes may offer a way to a better understanding of production, and thus also lead to improvements. Among the possible immunization procedures of the future,

8

CARL-GORAN

H E D ~AND MORTIMER P. STARR

aerosol vaccination was mentioned as a means of reducing the cost and administrative problems of major campaigns. That this is a realistic concept is obvious when one considers the advances in aerobiology which are by-products of the research in biological warfare. It is now known that it is possible to cover large areas with viable microbial aerosols. However, L. D. Fothergill, who discussed the potential ecological consequences of air contaminated with infectious agents, pointed out that the situation is very complicated. Major immunization schemes must always be planned and executed against a background of sound ecological knowledge. For instance, nature’s own immunization activities must never be forgotten, otherwise immunization as well as improvements in the environmental and social conditions may bring about unexpected consequences. The pasteurization of milk had its repercussions in the loss of “natural” immunity to tuberculosis, and no one will deny that an increase in the age of infectian with poliomyelitis has been an undesirable consequence of incomplete community immunization in combination with improved hygiene. Specific responses as well as nonspecific microbiological stimulators are both important in immunology. Pyrogenic lipopolysaccharides might be mentioned, as should also the deoxyribonucleic acid (DNA) digest factor noted by W. Braun, as a stimulator of antibody response. Immunity is the sum of a complex interplay of many known and possibly more unknown factors. Some of those may certainly be manipulated by microbiological techniques, and J. G. Harrar, touching on the importance of the normal bacterial flora of the human intestinal tract, even speculated on the possibility that microorganisms might one day be found that could synthesize compounds which could protect against diseases such as cancer, or that others might slow dawn the process of aging. A. Wettstein and his collaborators made a general survey of microbiological syntheses of pharmacologically active substances. This served as a very striking illustration of the utilization of the biosynthetic capacities of bacteria and molds in modern pharmaceutical research and industry. The long list of useful substances ranges from macromolecules, such as enzymes used as digestive aids or for tissue removal, polysaccharides used as plasma volume expanders and iron carriers, through a number of vitamins, to the ergot alkaloids used by both the obstetrician and the psychiatrist,

GLOBAL IMPACTS OF APPLIED MICROBIOLOGY

9

and the siderochromes which are useful in the treatment of ironstorage diseases. Last, but not least, come the antibacterial and antimycotic antibiotics, some of which are even being used in tumor chemotherapy. Perhaps the most impressive career during the last 20 years is shown by the antibiotics, a history which is astonishingly short considering the fact that antibiosis has been observed almost since man learned to grow microorganisms on artificial media. E. B. Chain reviewed briefly the history of modern antibiotics, and his comparison of death rates from selected causes during the years 1920 and 1960 served as an impressive illustration of the success of modern therapy. He regards the screening of microorganisms for the production of new antibiotics as having been so thorough that it is unlikely that entirely novel structures will be discovered. Against this background, the development of semisynthetic antibiotics is particularly relevant, for example, preparations based on 6-aminopenicillanic acid and the tetracycline derivatives. Even if the danger of creating resistant strains by the continued use of antibiotics has been exaggerated, the development of a wide spectrum of semisynthetic preparations should be a relief to many members of the medical profession. The semisynthetic antibiotics constitute an example of the utility of microorganisms in providing modifications of complex molecules. An even more striking example in the pharmacological field is found in the steroids, the transformation of which was discussed from various points of view by A. Wettstein, D. H. Peterson, and 0. HanE. The future of microorganisms in the pharmaceutical industry is, of course, difficult to predict, but one has reason to be optimistic, considering the advantages that Wettstein and colleagues list for them as compared to higher organisms: 1. great physiological similarity between the individual cells in the population; 2. the possibility of using artificial media; 3. great versatility; 4. relative ease of the preparation of genetic modifications; 5. as compared to chemical synthesis, the microorganism products have the advantage of stereospecificity.

IV. General Aspects of the Exploitation of Biological Resources

Recent calculations indicate that the total mass of microbial life on earth is approximately 25 times the total mass of animal life.

10

CARL-GORAN H E D ~ NAND MORTIMER P. STARR

However, the intelligent exploitation of the former has only just begun. Man learned to combat the dangerous microorganisms, and is now considering how best to use the good ones. Next, questions of economics enter the picture, the answers to which may determine whether or not an industrial waste such as suEte liquor and sewage should be treated. Such economic considerations obviously may become of the greatest importance and, against this background, it was interesting to follow the deliberations of the working group on taxonomy and the legal aspects of industrially important microorganisms. L. G. Silvestri regards patents as one of the most powerful incentives for scientific progress. In considering the legal protection of microbiological processes, one enters an area that is full of unsolved problems. Part of the difficulty stems from the fact that the species is the taxon usually accepted for microbiological process patents. The practice has exerted considerable pressure to proliferate the number of species-perhaps beyond the bounds of proper systematics-and the working group expressed the opinion that accepting subspecific taxa for patent purposes would be an improvement over the present rules. One cannot help but reflect that it is unfortunate, from the point of view of the developing countries, that so many microbiological patents and useful industrial practices lie unused because the biological industries in the highly industrialized countries have to compete with industrial chemistry. Even if this is a losing battle for some products in the Western world, the same products might be manufactured microbiologically quite economically in the underdeveloped areas of the world. Perhaps it is an idealistic and unrealistic thought, but could not unused patents and production details be handed over to an international clearinghouse for use in the developing areas? Such an approach might be very helpful, even if the licensing procedures involved a stipulation that the products would have to be used in situ and not for export.

V. Microbiological Aspects of Water Resources Man’s need of water is tremendous, and microorganisms are indispensable agents for pollution abatement and for water recovery. However, for mineralization they need oxygen, which may become a limiting factor. Borgstrom suggests, therefore, that microbial

GLOBAL IMPACTS OF APPLIED MICROBIOLOGY

11

activity be combined with oxygen delivery via algae, and he regards the sewage plants as an integral part of man’s future food-producing centers in which algae, fungi, and bacteria would be the chief livestock, possibly supplemented by insects, fish, ducks, and other animals which are easily controlled. Such plants would offer methods for short-circuiting the carbon, nitrogen, and sulfur cycles and for breaking the tyranny of the calendar year. The losses of silt and sewage not only involve losses of valuable materials, they may also have a profound effect on local marine environments. M. D. Appleman and J. M. Shewan, who discussed microbiology in the mobilization of marine foods, rightly called man a “disturber and a modifier” and then considered not only the factors just mentioned but also projects such as the building of dams which may deny access of fish to their usual spawning grounds. Intelligent planning must take all such effects into consideration. There is a widespread hope that the systematic use of the microflora and fauna of the oceans by deliberate marine farming may hold the key to our future food problems, but Borgstrom emphasized the fact that the productive chains of the seas are more lengthy than those of the land, and that the potentialities of the sea are grossly overrated. Considering all factors, the use of plankton as a primary source of food wouId probably be unrealistic for many years to come. The emphasis must be placed on a better exploitation of the traditional foods now taken from the sea. However, the productivity of the seas and oceans is certainly limited, and much basic and developmenta1 research is needed on this subject. On the basis of such knowledge, it may be possible, as suggested by Appleman, to increase the fertility by such means as intermixing surface and subsurface waters, or by breeding microorganisms adapted to unfertile waters. F. D. Sisler, in discussing the generation of electric energy by microbiological means, regards the seas as a gigantic microbiological fuel cell. Unfortunately, we are far from possessing feasible techniques for tapping the energy; one of the problems is the difficulty of large-scale storage of weak electrical currents. In confined units, however, bioengineering has overcome many of the problems involved in making biological fuel cells, thus exploiting the redox potential generated by bacterial growth,

12

CARL-GORAN H E D ~ NAND MORTIMER P. STARR

VI. Applied Soil Microbiology When we consider soil microorganisms in terms of quantity alone (300-3000 kilos live weight/hectare on well-cultivated land), it is understandable that they have attracted, and will continue to attract, much attention. Some exert dramatic effects, such as those parasitic on crop plants, while others have less striking effects, but the complex interplay of antagonism and symbiosis becomes a major role in determining agricultural productivity. Actually, the decomposition of plant residues and their incorporation into the soil comprise a most fundamental process which was discussed at length in various contexts; for example, M. P. Starr outlined the eliminative split of pectic substances, which is the newly found degradative pathway used by some phytopathogenic bacteria. Such microorganisms obviously exist in a state of delicate balance with other living organisms in the soil, and H. Katznelson drew attention to the possibilities of effecting a biological control by adding organic matter to the soil, thereby stimulating “normal” microorganisms at the expense of the pathogens. One limitation to the intelligent management of composts, stable manures, home wastes, and plant residues stems from our decidedly incomplete knowledge of soil microorganisms. Pertinent in this connection, is M. P. Starr’s report of the recent study, in collaboration with H. Stolp, on a hitherto unrecognized group of predatory, bacteriolytic, and parasitic vibrios. Until they were uncovered recently by H. Stolp, these ubiquitous and remarkable Bdellouibrio, which physically attack and lyse many soil and plant bacteria, were unknown-despite their undoubted significance in soil ecology. The immediate environment of plant roots is of particular importance for the plant. H. Katznelson discussed the complex interaction of root excretion products and microorganisms, and considered how this relation could be manipulated with the greatest benefit to man. The microorganisms in this situation produce amino acids, vitamins, and growth factors as well as lytic and antibiotic substances which may protect the roots against pathogens. However, they may also immobilize nutrients, produce toxic substances, attract pathogens, and repel favorable bacteria. It is obvious that this multifactorial equation is a very difficult one to solve, and that cautious planning should precede change of even one factor, for instance, in the application of agricultural chemicals to the foliar parts of the plant.

GLOBAL IMPACTS OF APPLIED MICROBIOLOGY

13

One of the most interesting aspects of the root microflora is the soIubiIization of minerals, effected by carbon dioxide and organic acids. It should perhaps be mentioned that microbiological leaching has been applied outside the agricultural field. Abandoned mine workings and dumps of waste rock from copper mines contain small amounts of valuable metal which can be recovered by percolation leaching; the rate of such leaching can be significantly increased in the presence of certain microorganisms. In the soil, particularly, the leaching of phosphorus is important and this element, which is, of course, also removed by crops, must be replenished continually. Presently known resources would, according to Borgstrom, last for some 100 years, but the drain is continually accelerating. Microbiological means might be found to release some of the bound resources but, on a long-range and large-scale basis, expensive deep sea mining would seem to be a necessity. As a means of rendering greater amounts of nutritionally important elements available to the plants, H. Katznelson discussed aeration and increasing the organic content of the soil as the most important factors in stimulating microbial activity. However, other possibilities should also be considered, for instance, inoculation with organisms that fix atmospheric nitrogen, produce auxins, gibberellins, antibiotics, lytic substances, vitamins, and amino acids, or solubilize soil minerals. The selection of specific organisms and the exploitation of modern microbial genetics may be very important in this connection.

VII. Nitrogen Fixation In recent years, the increase in world food production has been most notable in calorie-rich crops, and nitrogen consumption has shown a downward trend. Against this background, it is natural that much attention was given to nitrogen fixation at the Stockholm conference, at which the inoculation of legume seed was touched on by several participants. H. Katznelson mentioned that some 50-200lb of atmospheric nitrogen can be added to an acre of soil in 1 year as a result of symbiotic fixation by well-nodulated plants. E. N. Mishoustin stressed the great importance of this practice as a means of economizing in the use of mineral fertilizers. However, symbiotic nitrogen fixation is a complex phenomenon in which antibiotic-producing actinomycetes, ineffective rhizobia, or phages may interfere, and in which even some insecticides may have an

14

CARL-GORAN H E D ~ NAND MORTIMER P. STARR

adverse effect. The area is a challenging one, and J. G. Harrar actually designated the rhizosphere as the most important biological phenomenon associated with the practice of agriculture. He strongly emphasized the importance of studies involving nitrogen fixation, and both he and E. C. Stakman speculated about the possibility that the microbiologist might one day develop nitrogen-fixing microorganisms that will grow on wheat, oats, barley, and other kinds of agricultural plants. However, even short-range studies may have great significance; for instance, the selection of more effective, phage-resistant rhizobia with a wider host range. This approach was also emphasized by a group of specialists on nitrogen fixation who convened to formulate a plan on this subject. They also emphasized the value of research in nonleguminous plants bearing nitrogen-fixing nodules, and in nonsymbiotic fixation of nitrogen. One might perhaps think that chemical nitrogen fertilizers might be the answer to the growing needs, but it should be remembered that the chemical binding of nitrogen is an energy-consuming process, a factor of crucial importance in energy-poor parts of the world. According to Borgstrom, approximately one-sixth of the world’s population, namely 500 million, already depends on artificial nitrogen for its survival. With the present increase in world population, no less than 500 million tons of commercial fertilizers would be needed in the year 2000. This means an annual transport load exceeding the total present maritime trade1 Borgstrom points to biological nitrogen fixation, such as the mass cultivation of nitrogenfixing, blue-green algae, as necessary solutions to this oncoming crisis.

VIII. Microbiological Aspects of Crop Protection It has already been mentioned that protection of crops against pathogenic agents may be achieved by the application of ecological knowledge. In its most extreme form, this may involve the establishment of symbiotic relationships between microorganisms and plants in order to supply the latter with a continuing protection against the attack of pathogens. As pointed out by J. G. Harrar, there are actually many examples of such permanent associations between higher plants and viruses, and, in some cases at least, there is evidence of benefit. Protecting the host plant with certain mild virus pathogens opens up interesting possibilities for its protection against aggressive viruses. However, much remains to be learned

GLOBAL IMPACTS OF APPLIED MICROBIOLOGY

15

about the microbes that menace our food supplies, and about the long-term effects of agents which persist in the soil for long periods. There is much to be gained in such a specific research, particularly in the developing countries in which, according to estimates made by E. C. Stakman, food production could be increased by something like 25-50% merely by the straight application of known methods of controlling pests and pathogens of food and feed crops before and after harvest. The list of plant pathogens is a long one, and many of them have had grave consequences for mankind. Harrar mentioned the “late blight” of potatoes which created havoc and famine in Western Europe, and especially in Ireland, in the last century, the South American leaf disease of the rubber tree which wiped out an entire industry, and the bacterial canker which has destroyed millions of citrus fruit trees. A special working group under R. N. Goodman considered present trends in plant protection against microorganisms. The prophylactic use of microbiological products such as streptomycin may be feasible, but the treatment, particularly of deep-seated vascular infections, presents very difficult problems. However, some amino acids have been used with success, and much attention is now being focused on the possibilities of affecting the host itself rather than the pathogens by chemical substances, including the growth-regulating hormones. The probability of developing a broad spectrum phage for practical plant disease control was regarded as remote.

IX. Biological Control of Insects Chemical control of insects has previously been mentioned briefly, but the use of bacteria and viruses as control agents may become even more important in the future. Actually, J. M. Franz could give a long list of excellent arguments why biological control measures of insects should be regarded as superior to chemical insecticides. Perhaps this type of control may require more supervision, but the system has a high degree of safety to man and domestic animals, and it is self-regulating. Application is simple and may be modified in cases where dusts and sprays show their limitations. The possibility was mentioned of inoculating female insects so that infected larvae act as disseminating agents. The high degree of group specificity in biological control has been mentioned as a disadvantage in comparison with insecticides, but if one considers the important part played by insects as pollinat-

16

CARL-G~RAN HEDEN AND MORTIMER P. STARR

ing agents for at least 50 agricultural crops-including many important fruits and vegetables-and that insects serve as food for many birds and fish which we eat, then group specificity appears to be a very useful attribute. Also, the number of potentially useful agents is very large, including pathogenic viruses, rickettsiae, bacteria, fungi, and protozoa. About 20 different viruses have already been used successfully, and outstanding results have been noted with Bacillus popilliae, which attacks the Japanese beetle, and with Bacillus thuringiensis, which attacks many larvae. Unfortunately, however, only the latter microorganism can be grown in artificial media, a fact illustrating the need for research in the production technology of biological control agents. However, in this, as in many other fields concerned with agricultural production, the great success of farming has led to decreasing interest in supporting scientific studies. E. M. Mrak found that crisis research, i.e., the combating of a specific disease, may well be stimulated, but that the administrators are perfectly happy to wait until a new crisis arises after the first has subsided. He emphasized the importance of accumulating a “savings account” of basic scientific information that eventually will be useful to the evolving countries.

X. Protection of Animal Resources Biological methods of the type mentioned may also become useful in the control of insects carrying infectious agents which may attack livestock, but other measures will dominate the picture. D. T. Berman, who surveyed the veterinary side of recent improvements in applied immunology, emphasized that a harvest of livestock for meat production is low in the underdeveloped countries as compared to the United States where an intensive husbandry and control of epizootic disease has given a fine return. Elsewhere, diseases such as brucellosis, hog cholera, foot-and-mouth disease, and rinderpest still produce a sizeable loss in the total meat production, even if many examples of success can now be found. Such control is noted with certain viral vaccines, and antibiotics have also led to an increase of our food resources. In discussing the effects of antibiotics on biological equilibria, E. B. Chain noted that an improvement is achieved by two routes. One method is by preventing disease in animals, and the other involves the use of antibiotics as food preservatives. One good example of increased food supply as a consequence of penicillin treatment is given by cow mastitis

GLOBAL IMPACTS OF APPLZED MICXOBIOLOGY

17

which, if untreated, may interfere with the supply of milk. Chain also mentioned the use of tetracyclines as preservatives, particularly for fish, and that surface spoilage in meat can be considerably delayed by the use of antibiotics in places where adequate refrigeration is not available.

XI. Prevention of Deterioration The example just given is actually concerned with one of the most important contributions which modern microbiology can make to relieve the worlds food shortage. Organic decay caused by microorganisms certainly is indispensable in nature, but unfortunately it is nonselective. M. P. Starr remarked that spoilage is a rather subjective expression, and that the phenomenon must be considered in a cultural perspective. The same microbiological processes which would be regarded as spoilage in one area would yield highly desirable products in another, fermented herring and sauerkraut being examples. Obviously, the microbiological conversion of one food into another may be regarded either as a prevention of deterioration or as sheer spoilage. In the case of animal fodder, the question of acceptability does not assume the same importance as with human food. Fermentation as a means of preserving organic matter was actually the subject of discussion in a working group in which R. Nilsson reported on the possibilities of making first-class ensilage from organic matter of vegetable as well as animal origin by hydrolyzing polysaccharides to sugars with mold enzymes, thereby creating a favorable environment for the development of lactic acid bacteria. B. E. Malin demonstrated that the development of clostridia in silage could be controlled by certain antibiotics. In the developing countries, certain technical aspects of the preservation of food are of great importance. One example is that of the development of sterilized and dried foods. Such preservation represents a potentially useful means of distributing food from one community in which it is surplus, to another in which there is great need for it. J. K. Scott, who reviewed the effects of technical advancement of the preservation of food, cited “recombined milk” as an example of bulk transfer from one area to another. He discussed the technically important features of heat sterilization and pointed out that hard-and-fast rules can hardly be given for this process because the most advantageous type of heat treatment for

18

CARL-GORAN HEDEN AND MORTIMER

P. STARR

a given commodity will depend on the chemical and physical nature of that commodity. However, the possibilities for modern short-time heat processes are indeed impressive.

XII. Carbohydrate Waste One of the great problems in food processing is concerned with the handling of waste materials, many of which are carbohydrate in nature. G. Borgstrom mentioned whey, a by-product of the cheese industry, as an example. In the United States, whey is produced at a rate of more than 5.4 million metric tons annually, and threefourths of this quantity is obviously disposed of as waste. Borgstrom estimates that this total represents a reservoir of approximately 204,000 tons of sugar and 36,000 tons of protein. This might well be converted microbiologically in the same manner as sulfite waste liquor, molasses, distillery slops, pulp fluid, etc. W. W. Umbreit, L. L. Ingraham, Z. VanBk, and I. MAlek gave impressive surveys of the synthetic powers possessed by microorganisms, and one could not help comparing those powers to the amounts of transformable material available. One of the most important targets would seem to be the mobilization of yeasts, molds, and bacteria to remove the excess carbon and to produce nitrogensupplement materials. Borgstrom reminded his audience that only one-tenth of the caloric intake of the world's household consists of animal products, and that the trend is toward an even more vegetarian diet in which shortages of lysine, threonine, and tryptophan will assume great importance. From man's point of view, the ruminants, of course, constitute the most important carbohydrate-to-protein converters. They are of particular importance because they use cellulose, hemicellulose, and related products which otherwise would never enter into the human biosphere. Against this background, the microbiology of the rumen is worthy of greatly expanded research toward such directions as the balance between bacteria and protozoa, or the importance of sulfate-reducing bacteria. So far, the versatile catalysts available in the large numbers and varieties of microorganisms have not been fully exploited, and much remains to be done in the fermentative upgrading of the cheapest kinds of natural resources. The production of vitamins, amino acids, and other essential nutrient material as well as hormones, organic acids, and alcohols is already established practice. K. Arima dis-

GLOBAL IMPACTS OF APPLIED MICROBIOLOGY

19

cussed the techniques for making microbial enzymes and demonstrated convincingly that rapid production, low costs, and a possibility for improvement by genetic means has initiated a gradual replacement of enzymes from higher animals or plants by products from microorganisms. The use of such enzymes, as well as the microbiologically produced amino acids and flavor substances such as inosinic acid and guanylic acid, may, in the future, profoundly affect both the acceptability and the nutritional value of so-called microbiological foods. These may be deficient in certain amino acids, such as methionine, which is already low in many diets, and here one must consider either supplementation or selection of special kinds of organisms. In this connection, it is noteworthy that several mushrooms seem to be rich sources of sulfur-containing amino acids. J. H. Lichtfield described the mass cultivation of Morchella species in submerged culture. The technique is based on a simple medium containing calcium carbonate as a neutralizing agent and inert support for the mycelium. In this case, the methionine-pluscystine values were low, but the lysine, leucine, and tryptophan contents were satisfactory, and the potentialities of growing the mycelium in waste water substrates, particularly from the food canning and dairy industries, seem promising. H. J. Bunker reviewed the whole subject of microbial foods and emphasized the usefulness of Rhodotomla gracilis in the synthesis of fat and proteins. He regarded the direct conversion of cellulose into protein by bacteria and fungi as unrealistic at the present time. However, G. Borgstrom considered that fermented sawdust might be a valuable concentrated food for pigs and dairy cows, and H. Katznelson thought that the same material might be of tremendous value in agriculture if prepared in a form suitable as a manurial substitute. A. Imshenetski reminded his audience that the cultivation of industrial microbes depends completely and exclusively on human will, whereas agriculture depends on uncontrollable climatic and other unpredictable conditions. Furthermore, the speed with which microorganisms can perform their synthetic tasks makes them attractive for food production. Bunker, for instance, compared a bullock weighing 500 kg and synthesizing about 0.4 kg of protein per 24 hours with a yeast culture of the same weight which was calculated to produce 50 tons of protein in 24 hours. However, there seem to be limitations to the amount of yeast which can be incor-

20

CARL-GORAN HEDEN AND MORTIMER P. STARR

porated into the diet, and undoubtedly new microbial foods must undergo rigorous testing for toxicity, including possible carcinogens, before they can be recommended for human consumption. Of the new foods, yeast grown on hydrocarbon, a microbial nutrient of which the resources are almost limitless, was of particular interest. A. Champagnat briefly reviewed the British Petroleum project, and a working group under the chairmanship of J. C. Senez discussed microbial transformations of hydrocarbons.

XIII. Photosynthesis One of the most interesting of the potential microbial foods is the green algae ChZoreZZa, which is a remarkably efficient producer of protein. They actually utilize the sun’s energy more efficiently than do ordinary land plants, and E. M. Mrak mentioned an estimated 6200 square feet of culture space as being sufficient to produce enough Chlorella to fill the protein needs of a family of five or six persons. Experience has shown, however, that acceptability is a factor of the first magnitude in the consumption of food, and even a weakly unpleasant odor or taste may be enough to prevent the utilization of any constituent as human food. Where customs and taboos are important factors in limiting acceptability, as is true of yeast, the novelty of ChZoreZZa foods makes large-scale use very difficult. However, one should not regard the case as lost because, as E. Heegaard pointed out, the quality and palatability of many food products can be improved by the application of microbial enzymes. This fact was reiterated by K. Arima, who cited several instances in which the enzymes have been used to remove undesirable trace substances and to change food properties. The removal of bitter glucosides from citrus juices is one example.

XIV. Improved Utilization of Human Resources Just as education may be a necessary factor in securing acceptance of microbial foods, so is it an equally important one in making immunization programs acceptable to developing countries, as well as for the future of applied microbiology in general in such areas. Undoubtedly, the practical results will come first to those societies that provide for adequate research and have the wisdom to use the results, and this involves training and education, not only of scientists but of administrators on all levels as well. Since the economy

GLOBAL IMPACTS OF APPLIED MICROBIOLOGY

21

of the developing countries is founded on agriculture and microbiology is an intrinsic part of that agricultural economy, and since these countries have few or no agricultural microbiologists, Mishoustin stated with emphasis that first priority should be given to training designed to apply microbiology to the potential needs of agriculture. Discussions of the working group concerned with trends in applied water microbiology formulated the conclusion that developing areas are not alone in having training problems. K. Baalsrud commented on the small number of individuals throughout the world who are presently dealing with this subject, in spite of the significance of the field. Furthermore, most of the persons involved find themselves forced to attack the problems first through administrative and advisory activities, which necessarily delay, if not actively hamper, arriving at solutions. Training in bioengineering was the subject of a special working group whose report emphasized that this kind of education should ordinarily be offered only to graduate students with a good background in the basic sciences. However, the education of those responsible for operational bioengineering must not be sacrificed to research training. J. W. Foster found that training in the technological art of modern industrial fermentation is given only casually, and nowhere is it conducted on the scale or in the manner needed. There is a need for “hybrids” whose education, training, and interests embrace the breadth of current industrial and applied chemistry as well as microbiology. The scientists who work in applied microbiology can function optimally only insofar as their contact with the moving frontier of science is effective. This contact involves problems of documentation, classification, and retrieval of information, a subject which was discussed by a special working group. This group considered essential the establishment of new research units that are devoted exclusively to the process of information transfer. P. R. Brygoo asserted that the whole of biology, as well as each of its specialities, offers sources of interest for modern microbiology, and that its applications are closely related to almost all branches of human activity. Many abstracting journals exist, but not one of these services covers the field of microbiology in its entirety, which means, categorically, that there is no one reliable information source for the whole area, This lack is particularly true in the interdisciplinary fields in which the problem is quite acute.

22

CARL-GORAN H E D ~ NAND MORTIMER P. STARR

XV. Microbiology Applied to Biological Research in General

As pointed out by E. C. Stakman, microbes can be used to satisfy man’s curiosity as well as his wants, and basic investigations will not only help solve the basic problems of his existence, but will also satisfy his curiosity about the nature of life itself. When one talks about applied microbiology, its applications to the fields of general biochemistry, genetics, etc., are generally not considered, However, it can be claimed that those are the areas wherein the applications of microbiological techniques have yielded the most remarkable results in recent years. Actually, it is doubtful that we would have arrived at our present knowledge of genetically determined inheritance and how it is effected and controlled, if it were not for the use of microorganisms as living research tools. Molecular biology and microbial genetics were actually touched on by several of the participants, and W. Braun gave a fascinating survey of perspectives and horizons in microbial genetics. He discussed the numerous techniques presently available for the creation of useful new strains. For instance, it seems obvious that the relatively promiscuous transfer of information by means of F-factors and colicinogens will provide applied microbiology with a new tool for the creation of useful combinations of characters. Braun even speculated about a future in which new enzymes and other biologically active proteins may be composed artificially in vitro by the provision of novel nucleotide sequences. This may appear to be wishful thinking, but knowledge of the genetic code and its transcription mechanism is accumulating at a pace which certainly opens up new vistas. The bulk of this knowledge comes from basic studies on microorganisms, the study of which has yielded this remarkable “by-product.” Investigations of antibiotics, for instance, required the development of new methods for amino acid sequence determinations, and the biochemists have obtained a number of valuable new specific inhibitors for the study of metabolic reactions. In so doing, as was noted by L. L. Ingraham, they have been exposed to a biological demonstration material which will help in the development of better chemical catalysts. With regard to medical applications, W. Braun showed how short the step is between microbial genetics and the areas of immunology and oncology in higher organisms.

GLOBAL IMPACTS OF APPLIED MICROBIOLOGY

23

XVI. International Efforts The need for international efforts in microbiology on all levels was emphasized, and such efforts are being made by the International Association of Microbiological Societies ( IAMS ) with its numerous specialized sections. This organization originally concentrated its efforts on organizing international congresses and symposia, on matters of taxonomy and nomenclature, and on the coordination of culture collections, but many more functions have now been added. The newer functions are related to the growing importance in general of international and regional efforts in science. No doubt, one reason that collaboration becomes a vital necessity is the complexity and cost of modern research. The success of the regional efforts in Europe concerned with high-energy physics and space research will certainly be followed by the organization on molecular biology; the latter subject, which has great significance for applied microbiology, is developing almost explosively in many other parts of the world. An international effort is also being made in the biological sciences-The International Biological Programwhich is a project sponsored by the International Council of Scientific Unions aimed at a worldwide study of human adaptability and biological resources. From the point of view of the microbiologists, it is interesting to note that photosynthesis and nitrogen fixation form the backbone studies within the section on physiology of terrestrial communities. The greatest impact on microbiology will, however, come from UNESCO, which is now planning a “Microorganisms Decade” at the request of the Japanese delegation. This project was considered during the GIAM conference, which summarized its main considerations in the form of a resolution aimed at UNESCO. It was emphasized that existing organizations should be utilized to effect the plans for this cooperative venture. The IAMS and its sections were requested to support the efforts through its Committee on International Programs. The importance of specialized international conferences was emphasized, and the very small number of participants from the developing countries at the GIAM meeting was deplored. Every effort should be made to ensure participation from such regions at future conferences. IAMS and its sections should support the training of microbiologists in the developing countries and explore every means to stimulate and organize training and research therein. A number of concrete

2.4

CARL-C~~RAN HEDBN AND MORTIMER P. STARR

projects, which were given as an appendix, are now being completed by direct contract proposals from the different IAMS sections. A strong emphasis on the specific problems of the developing countries is a common feature. These problems of the developing areas must be approached from two angles: one is the application of available knowledge, the other is concerned with the development of new knowledge. G. Myrdal emphasized very strongly ,the need for focusing research activities in the advanced countries on solving the problems of the new nations. This would actually demand an enlargement of research and, within the scope of this enlargement, its redirection. The problems involved in balancing population growth and birth control are so enormous that, however successful our endeavours may be, one must predict a considerable increase in the number of individuals in the developing countries. As a consequence, immediate attention must be given to increasing the supply of food, and this indeed calls for international cooperation. Myrdal suggested that the rich countries might set up a number of specialized institutes for basic as well as applied research, which should direct their programs toward solving problems for the peoples living under unfavorable conditions. Such institutes might, for example, participate in an international effort to forecast the results of our scientific progress and, in particular, the effects which substitutes for natural products might have on the economies of underdeveloped countries, Several other types of new international research organizations were suggested. For instance, Silvestri suggested units qualified to make official tests of deposited microbial strains which would provide patent offices with needed information. With the aim of furthering fermentation research and other fields of applied microbiology of importance to the developing countries, C.-G. H e d h suggested the establishment of an international organization for bioengineering and biotechnology, and J. W. Foster advocated international centers for research in systematic mutation and screening. Under present world conditions and with the understandable disinclination of private industry to disclose trade practices, such institutes might also offer a logical and practical way of disseminating technological “know how” in the developing countries. It is quite possible that additional work and inventiveness are required before the same principles that are now in vogue in the industrial nations can become general among the new nations. E. Heegaard

GLOBAL IMPACTS OF APPLIED MICROBIOLOGY

25

noted that anaerobic processes, unsterile operations, unrefined products, simplicity, and adaptation of the fermentative processes to local conditions all assume particular importance in such areas. E. L. Gaden discussed the economic considerations involved in plant design for underdeveloped areas and, for instance, mentioned wooden and concrete vessels as an alternative to stainless steel fermentors. He also discussed the cooling problems, and the fact that labor costs are so low in many developing countries that automation may not be economically sound. J. W. Foster also dwelt upon special conditions to be found in the developing countries and focused attention on the possibilities of cultivating microorganisms on solid or particulate substrates such as cellular grains, brans, or chopped vegetable matter. The advancing of industrial microbiology in the developing countries certainly needs talent, and this will hardly be available if applied microbiology does not attain a certain degree of international prestige. At the present time, the word “applied is regarded by many as a label for an area populated with second-rate scientists. This derogation is unfortunate and might be avoided by the application of what E. M. Mrak calls the “full-spectrum approach,” which has proved to be remarkably successful in agricultural research in the United States. It means simply that applied and highly theoretical research are pursued side by side, a method by which even the purest molecular biologist might be persuaded to think of his studies in terms of their practical implications. A. Eban considered the need for a re-evaluation of priorities in the allocation of the worlds resources, both of money and of scientific man power, and he, together with Birkeland, Borgstrom, and others expressed concern over the effects of the space race. Borgstrom mirrored the feeling of many colleagues when he said that the priority list must be radically revised before world science and technology become further engulfed in the moon illusion. A sense of unity was provided by the unquestioned assumption that microorganisms are a very important natural resource, potentially available to all nations, and that methods now exist and are continually being improved for controlling microbiological processes so that they can be exploited to meet human requirements and to help buy the time needed for attaining a balance between population increase and food supply. We feel that we can hardly end this discursive summary in a more

26

CARL-GORAN HEDEN AND MORTIMER P. STARR

fitting way than by citing a few of E. C. Stakman’s words spoken at the conference banquet in the Stockholm City Hall: “Man has acquired intellectual ability to learn vastly more about the world of microorganisms than he now knows. Is he wise enough to utilize his intellectual powers in researches of the scope commensurate with the needs and opportunities of harnessing the full powers of microorganisms? And is he wise enough to develop the effective code of ethics and the moral purpose to utilize these powers for the benefit of all people and not for the benefit of some and for the destruction of others?”

Microbial Processes for Preparation of Radioactive Compounds D. PERLMAN, ARISP. BAYAN, AND NANCY A. GIUFFRE Squibb Institute for Medical Research, New Brunswick, New Jersey I. Introduction ........................................... 11. Methods Used in Preparing Radioactive Compounds by Microbial Processes .......................................... A. Methods of Addition of Precursor to Microbial Systems . . . B. Recovery of Labeled Compounds ..................... C. Determination of the Purity of Labeled Compounds ...... D. Stability of Labeled Compounds ...................... 111. Radioactive Compounds Prepared by Microbial Processes . . . . . A. Antibiotics ........................................ B. Vitamins .......................................... C. Amino Acids ...................................... D. Carbohydrates and Related Compounds . . . . . . . . . . . . . . . . E. Sterols and Related Compounds ...................... F. Organic Acids and Alcohols .......................... G. Nucleic Acids and Purines ........................... H. Miscellaneous ...................................... IV. Summary ............................................. References ............................................

1.

27 29 29 33 33 34 36 37 47 50 52 55 55 55 55 60 61

Introduction

Organic compounds labeled with radioactive isotopes have found widespread use in research in medicine, chemistry, and biology. In many laboratories they have long ago lost their novelty and have become conventional research tools. Their preparation has become a new branch of practical organic chemistry, and, in some instances, the organic chemist and the biochemist have made use of microbial systems to prepare the desired labeled compounds. The usefulness of microbial systems in preparing radioactive labeled compounds was apparent from the pioneering work of Barker et al. (1940a, b; Carson and Ruben, 1940; Foster et al., 1941)) who were interested in the metabolic pathways in bacteria and fungi. In the course of their studies they grew microorganisms or added other Cl1-compounds to the in atmospheres of fermentation and isolated the labeled metabolites. These experiments, besides elucidating the metabolic pathways present in these 27

28

D. PERLMAN, ARIS P. BAYAN, AND NANCY A. GIUFFRE

microorganisms, also showed the “use of bacteria for the preparation of organic compounds containing radioactive carbon” ( Barker et al., 1940b). Some years later when C1402became available, algae and Thiobacillus thiooxidans were grown in an atmosphere containing CI4O2and the cells were recovered. Since the only carbon available to these cells was C14, all of the carbon in the cell constituents including that in the amino acids of the cell protein, the lipids, and the nucleic acids was labeled. Some laboratories still use processes based on these experiments for preparing labeled amino acids for commercial distribution. The modern bacteriologist, biochemist, pharmacologist, and toxicologist rely heavily on experiments using labeled compounds to show metabolic pathways in various biological systems. Without these labeled compounds these investigations would be carried out at a much slower pace, and to many fewer successful conclusions. Even so, the conclusions of Wood and Perkinson (1952) that the “possibility of synthesis of isotope labeled compounds by microorganisms is often dismissed by the organic chemist for lack of special equipment and because of ‘complexity’ of the mixture of products obtained are still valid 12 years later. A number of reviews and summaries surveying the literature have appeared during the past 15 years. Noteworthy among these are those by Arnstein and Bentley (1950), Thomas and Turner (1953), and Catch (1954a). Techniques useful in the preparation of a number of labeled compounds using microbial systems were mentioned by Catch (1957), Evans and Catch (1956), and, in his recent book “Carbon-14 Compounds,” Catch (1961) devotes a chapter to “Biological Methods of Labelling.” Unfortunately, most of these reviews and essays were intended for the organic chemist, and relatively little attention was paid to the interests of the microbiologist. The standard manual, “Organic Syntheses with Isotopes” by Murray and Williams (1958), provides instructions for preparation of a large variety of labeled compounds but does not mention any microbiological processes. The purpose of the present review is to organize some of the literature concerned with microbial processes found useful in preparing labeled compounds. The variety of labeled compounds prepared by means of microbial processes (or chemical processes using several microbial steps) will be featured. Emphasis will be placed on the microbiological aspects of these processes, and

PREPARATION OF RADIOACTIVE COMPOUNDS

29

technical problems likely to be encountered will be mentioned. However, no detailed procedures for preparing the compounds will be presented, and the reader is advised to consult the original literature. Only radioactive isotopes will be included, and studies with the stable nonradioactive isotopes N15, C13, 01*, and deuterium, often used to prepare labeled compounds, will not be stressed.

II. Methods Used in Preparing Radioactive Compounds by Microbial Processes

Although a wide variety of experimental equipment and procedures have been described in the literature as being useful in preparing labeled compounds by microbial processes, there seems to have been little recognition of the advantages, requirements, and limitations of these processes and techniques. We have reviewed a number of the papers discussing the preparation of various labeled compounds and determined the similarities and differences in the processes described. If allowances are made for problems peculiar to preparation of certain types of chemically unstable compounds, it can be seen that only a few methods have found wide acceptance. Since the advantages and disadvantages of these methods are of concern to the laboratory investigator preparing labeled compounds by microbial processes, these will be stressed in the following discussion. A. METHODS OF ADDITIONOF PRECURSOR TO MICROBIAL SYSTEMS Two methods have been used in the preparation of most labeled compounds by microbial processes : 1. Growth of the microorganism in a medium containing “normal’’ medium constituents (and the labeled material) under conditions where appreciable amounts of the labeled substrate are absorbed by the culture and incorporated into the desired metabolite. 2. Growth of the microorganism in a “normal” medium, collection of the microbial cells, and resuspension of the cells in a solution containing the labeled precursor. Incubation is continued until a “high yield of the desired metabolite is obtained. In some instances cell-free enzymes have been used in converting labeled compounds to desired products. While cell-free enzymes

30

D. PERLMAN, ARIS P. BAYAN, AND NANCY A. GNFFRE

may have application on a preparative scale, e.g., conversion of thymine-C14 to thymidine, there are very few reports of successful “scale-up” of these processes from a microscale to a preparative level. The advantages of using growing cultures in preparing labeled compounds are derived in part from the ease of operation. The selected microorganism is grown in conventional media, under conditions where useful concentrations of the desired metabolite are usually obtained. The radioactive precursor is added to the growing culture and incubation is continued until maximum incorporation is achieved, or some other end point is reached. The fermentation is harvested and the desired labeled metabolite is recovered by conventional procedures. All of these operations can be carried out and standardized without using the labeled materials, and the addition of the labeled precursor does not make an appreciable difference in either the fermentation operations or the type of equipment used. When C14-labeled precursors are used and appreciable amounts of metabolic C1402 may be produced, precautions against contamination of the incubation chamber atmosphere are usually taken. Hunter et al. (1954)) Craig (1960)) Snell (1960)) and Rinehart (1964) are a few of those who have described modified shaken flask-size equipment designed to take care of these problems. They have arranged their flasks in a “closed system so that air is swept through the flask and the metabolic Conis trapped in alkali. The flow rate of the air through the flask must be carefully controlled. If not, the rate of metabolism of the organism is often affected. There may be a need to be concerned about the efficiency of conversion of the precursor to desired metabolite, especially if a costly precursor is used, and in these situations addition of the precursor may be delayed until the growth phase of the fermentation has been finished, or the precursor may be added repeatedly to the growing culture during the incubation period. Karow et al. (1952) obtained about a 1%conversion of glucose C to streptomycin when the g1uc0se-C~~ was added to the fermentation at time of inoculation, while Hunter and Hockenhull (1955) found a 5% conversion when addition was delayed until 60 hours after inoculation. The main requirements of this method of preparing labeled metabolites are listed below.

PREPARATION OF RADIOACTIVE COMPOUNDS

31

1. Use of a Microbial Process Which Gives a Useful Yield of Metabolite This requires exercise of some judgement in selection of cultures, media, fermentation conditions, and precursor. While useful yields of an antibiotic may be obtained when a specially selected strain is grown in a complex medium in a medium size shaken flask, carrying out the fermentation in a test tube or very small flask may be difficult, if not impossible. Considerable effort may be expended in standardizing operations on a “microfermentation scale,” and this should not be discounted in planning a program. 2. Selection of the “Best” Precursor This requires some knowledge of the intermediary metabolism of the fermentation process. If the culture converts glucose to acetate, and acetate is then converted to the desired metabolite, it may be more economical to add labeled acetate to the fermentation rather than g1u~ose-C~~. Since most radioactive compounds useful as precursors are quite costly, some economic considerations must be noted in choosing them. When g1uc0se-C~~ (or other CI4-labeled hexoses) are added to growing cells, much of the carbon is converted to cell substances and other materials usually unrelated to the desired product. A conversion of 10% of g 1 ~ c o s e - Cto ~ ~the desired product is considered a high yield. When S3504 is the precursor for an amino acid, a conversion of 30% is quite satisfactory. On the other hand, when Co5T12is used to prepare vitamin B12-Co57a conversion of 80% of the added precursor is usually expected, and when HC13Bis the precursor for producing 7-~hlortetracycline-C1~~, a conversion of 98% can be expected (Doerschuk et al., 1959).

3. Use of Conditions Which Permit Control of the Microbial Process This is essential for the safety of the laboratory as well as for efficiency of operations. Experiments must be carried out so that the escape of radioactive C 0 2 from fermentations does not result in hazardous conditions, or that radiation from cobalt, sulfur, or phosphorus isotopes does not expose laboratory personnel to undesirable levels of radiation. Problems arising in the use of this method come from difficulties in carrying out of fermentations on a small scale, the selection of

32

D. PERLMAN, ARIS P. BAYAN, AND NANCY A. GIUFFRE

fermentation conditions to give useful rates of incorporation of the labeled precursor, and the multiplicity of labeled compounds often produced from the labeled precursor by the growing microorganism. The products obtained may not be specifically labeled, and obviously this is a limitation where specific labeling is required. The advantages of using “washed or “resting” cells are derived in part from the efficiency of conversion of precursor to desired metabolite: ( 1 ) They contain the needed enzymes required to convert the precursor to the desired metabolite with a minimum of diversion to unwanted side products, and ( 2 ) the conversion is very rapid, and the conversion efficiency is usually very high, with little chance for formation of the unwanted side products. Metabolic inhibitors can be added to prevent formation of these side products, thus simplifying recovery of the desired labeled metabolite. Usually less unlabeled metabolite is added to the “harvested fermentation (to aid in recovery of the labeled metabolite) and thus material of a higher specific activity is isolated. The disadvantages of using washed cells come from difficulties in perfecting laboratory operations. Most fermentation processes are designed to give the highest yield of desired metabolite. Usually, few studies have been carried out to determine the best conditions for production of the desired metabolite by washed cell or resting cell preparations. Thus, experimental programs have to be arranged, and this may take more time than the laboratory investigator wishes to allot to the program. Although most labeled compounds praduced by microbial processes have been formed as a result of adding C’*-labeled precursors to microbial cells, a few compounds have been made by growing the microorganisms in D20 or H20. Ober et al. (1962) grew the paromomycin-producing streptomycete in a complex organic medium containing 10 mc./ml. of tritiated water. The growth of microorganism was not affected by this procedure and the authors (1962b) found the cost of preparing the labeled material less than that of a C14-labeledprecursor. A total of 1.3 gm. of paromomycin was isolated from the fermented medium with a specific activity of 0.75 pc./mg. (as the sulfate salt). The in &TO stability of the tritiated material was checked by exhaustive water washing of the labeled material bound to an ion-exchange column, and by lyophilization of aqueous solutions of the labeled material. Similar studies have been carried out using deuterated water resulting in cell

PREPARATION OF RADIOACTIVE COMPOUNDS

33

components containing deuterium (Chorney et al., 1960; Moses and Calvin, 1959; Marmur and Schildkraut, 1961). Labeling of bacteriophage with P32 has been reported by Kozinski (1961), who first grew Escherichia coli in a P320,-containing medium and then infected the cells with phage +X 174. The phage particles formed contained some of the P32.Marmur and Schildkraut ( 1961) proposed that deuterium-containing phage can be prepared by growing susceptible bacteria in deuterated water (they used up to 95% DzO in their media without markedly affecting growth rates) and the bacteria then can be infected with suitable phages. It is likely that labeled virus can be prepared in an analogous manner using tissue cultures, grown in media containing P3204 or DzO, as host for the virus.

B. RECOVERY OF LABELED COMPOUNDS The procedures used in recovering the labeled material from the fermented medium have in part depended on the type of material involved. Usually labeled antibiotics, vitamins, and steroids have been recovered by normal procedures found useful in isolating these materials from nonlabeled fermentations. Since the efficiency of isolation procedures is usually dependent on the amount present, addition of carrier amounts of the unlabelled product often helps to improve recovery, and this is usually done even though it results in material of lower specific activity. Isolation of labeled cell constituents including amino acids, nucleic acids, and lipids sometimes requires use of specialized techniques. Amino acids may be prepared by acid hydrolysis of the cells and recovery of the amino acids (with or without carrier addition) by ion-exchange and by preparative paper chromatography. Thinlayer chromatographic techniques and paper ionophoresis have also been very helpful. C. DETERMINATION OF THE PVRITY OF LABELED COMPOUNDS Many problems are encountered in determining the purity of labeled materials. Some of these arise from the chemical instability of the compounds, while others are related to the degradation caused by the radioactivity (as will be mentioned in Section 11, D). In most laboratories isotope dilution assays (accurate to about 3%) and radioautography of thin-layer and paper chromatograms are used to determine purity. The latter methods usually permit

34

D. PERLMAN, ARIS P. BAYAN, AND NANCY A. GIUFFRE

detection of radioactive impurities of the order of 0.5% of the material under study. Any chromatographic system which provides high resolution and minimum spreading of the migrating spots is suitable for radioautography ( Schwarz BioResearch, 1963). However, as pointed out by Catch (1961), paper chromatographic systems do not have sufficient resolving power, may give false spots, or may result in loss of material by volatilization. So, care must be used in selection of the analytical method, and, if possible, several methods should be employed, When the material in question has bioactivity, a bioassay combined with a radiochemical analysis will often be more useful than a single method. For example, in examining vitamin BI2 labeled with Co6*,an assay of its growth-promoting activity for Lactobacillus leichmannii and a quantitative chemical assay (based on absorption at 360 mp) together with the radiochemical assay usually give a better estimate of the purity and specific activity of the material than any single method.

D. STABILITYOF LABELED COMPOUNDS

All radioactive substances are unstable. That part of their radiation which is absorbed produces the ions, free radicals, or activated molecules which result in the degradation of the parent compounds. Rosenblum ( 1962) has summarized some available information and pointed out that radiant energy absorbed per unit of time within the body of a radioactive substance may be given in rads by the expression: n X E X 1.6 X 10-l2 Absorbed dose (in rads) = 100 x w where n is the number of disintegrations, E the absorbed energy in electron volts of the particular radiation emitted, 1.6 x 10-l2 is the factor converting electron volts to ergs, 100 is the factor converting ergs to rads, and W is the weight of the sample in grams. Solids and solutions containing 1 mc. and 1 pc. of a 0-emitter such as tritium, C1*,CoBo,and P32 with average energies of 0.055 Mev., 0.050 Mev., 0.093 Mev., and 0.70 Mev., respectively, and of such dimensions as to absorb completely all p-radiation receive large doses of energy as can be seen from the following tabulation of Rosenblum’s calculations.

-

PREPARATION OF RADIOACTIVE COMPOUNDS

35

RADIATIONENERGY ABSORBED ( IN RADS/DAY) BY SELF-IRRADIATION OF P-RAY EMITTERS Isotope H3 c 1 4 coeo p32 Eav.( MeV. ) 0.0055 0,050 0.093 0.70 1 mc./mg. 1pc./mg. 1mc./ml. 1kc./ml.

2.8

x

lo5

280 280 0.28

2.6 x 106 2600 2600 2.6

4.8 x 106 4800 4800 4.8

3.6 x 107 36,000 36,000 36

Information concerning the effect of several types of radiation on aqueous solutions of cyanocobalamin was compiled by Rosenblum (1962). The data collected showed initial vitamin concentration and percentage loss, and he concluded that “at moderate exposures (25,000 y for rads or less), and in concentrations between 10 pg/ml and 5 mg/mI, the number of cyanocobalamin molecules destroyed per 100 ev expended by energetic electrons or by gamma radiation from cobalt-60 was 0.25 to 1.5.”The yield decreased as the exposure dose increased and was less at lower vitamin BlX concentrations. This was true in simple aqueous solutions and in saline. Laufer et al. (1963) observed that while freshly prepared labeled compound may be pure radiochemically as measured by radioautography and isotope dilution, often such compounds deteriorate before use. Degradation may be caused by different circumstances. If the temperature of sterilization of aqueous solutions is too high, or time is prolonged, part of the molecule may hydrolyze. In work with labeled thymidine as much as 5% thymine was formed. Schwarz BioResearch ( 1963) has discussed radiochromatography as a highly sensitive method for determining the purity of radiolabeled compounds. Baker et al. (1958) comment on the deterioration of g l ~ c o s e - 1 - C as ~ ~ follows: “When high specific activity g1uc0se-C~~ is stored in a dry form, it may be subject to destruction by self-irradiation, bacterial degradation, or chemical decomposition due to the presence of a contaminant in the preparation.” They found that the destructive action of this third possibility could be prevented almost completely by storage of the material in frozen solution. They mention in passing that if g l ~ c o s e - 1 - Csolutions ~~ have to be heat sterilized, carrier glucose should be added, and the autoclaving carried out on 0.5 N-HCl solutions. Other types of radiochemical impurity are caused by a variety of radiation effects ( Bayly and Weigel, 1960), particularly with highspecific activity compounds. A primary radiation effect can be

36

D. PERLMAN, ARIS P. BAYAN, AND NANCY A. GIUFFRE

caused by the disintegration of one of the unstable atomic nuclei. This atom, changed to another element, may or may not be eliminated from the molecule. This kind of reaction will result in a radiochemical impurity of the molecule containing another radioactive atom. Secondary radiation effects can cause hydrolysis or decomposition of a molecule or the labeled compounds. Thus, liberation of sulfuric acid from CI4-dextran sulfate can completely decompose the organic moiety of the molecule. The instability of cobalt-labeled cyanocobalamin concerned Mollin and Baker (1955), who noted a gradual fall in potency of solutions containing vitamin BI2 labeled with Co5* or Cooo,and they emphasized the necessity for frequent microbiological assays of solutions of the labeled vitamin. Smith (1959) published more extensive information on this subject, and the observations of Barlow and Sanderson (1960) bear on this matter. The instability of other labeled compounds has also been noted. Smith and Allison ( 1952), working with S35-benzylpenicillin, noted that chromatograms of the material often showed several areas of radioactivity which did not have antibacterial potency. They attributed this to degradation of the penicillin caused in part by the radiation. Similar observations were reported by Perret ( 1953). A number of techniques have been adopted in some laboratories to minimize radiation effects. Storage at as low temperatures as possible usually reduces the rate of chemical degradation. Dispersion over a large area, such as drying radiochemical solutions on paper, reduces adsorption of radiation energy by the radioactive compound. Finally, dilution of the radioactive compound by either adding the unlabeled form or by introducing other substances, e.g., nonpolar solvents, usually has reduced radiolysis. In general, the best practice in working with radioactive compounds is to use them immediately after preparation or receipt from the manufacturer. This will minimize the effects of instability and the complications resulting from impurities caused by radiation.

111. Radioactive Compounds Prepared by Microbial Processes

Information on a wide variety of radioactive compounds prepared by microbial processes has been summarized in Tables I to VII. We have attempted to stress in these tables the variety of products

PREPARATION OF RADIOACTIVE COMPOUNDS

37

produced and the types of precursors used, as well as some information on the microbiological aspects of the processes. The processes mentioned in these tables were selected from the hundreds of those reported, and no claim is made for complete coverage. Although data on specific activities of products reported before 1956 are usually lacking, these processes are mentioned since useful labeled compounds were produced.

A. ANTIBIOTICS Information on the preparation of 32 labeled antibiotics is summarized in Table I. Although more than 1500 antibiotics have been mentioned in the scientific literature, relatively few have been found useful in treatment of various human, animal, and plant diseases. Some of these have been prepared in labeled form for use in pharmacological and microbiological studies. The recent report by Djerassi et al. (1962)) on the usefulness of biogenetic studies using labeled precursors in structure elucidation, will undoubtedly lead to more laboratories using this approach. Benzylpenicillin (penicillin G-S36) was the first antibiotic to be prepared in radioactive form. Howell et al. (1948) found that addition of S3504did not affect growth and penicillin production by Penicillium notatum (strain 1249B21). Somewhat later, Rowley et al. (1948) carried out experiments using test tube size submerged cultures, and others (Maass and Johnson, 1949a, b; Smith and Allison, 1952) used shaken flask size fermentation. In these experiments sterilized solutions of S3504 were added to media “deficient” in S so that the S35 was a major source of S in the medium. The media were inoculated with Penicillium chrysogenum and the flasks incubated for 4 to 7 days. The chemical instability of benzylpenicillin, together with the instability due to the decay of the S35, resulted in purification problems. Determinations of purity using chromatographic procedures often showed radioactive contaminants in “high” potency preparations (Smith and Allison, 1952; Perret, 1953; Schepartz and Johnson, 1956), and these problems are still encountered today. Specific activities of the order of 1.2 mc./mg. have been repeatedly obtained. A number of laboratories have reported preparation of C-labeled penicillins. Labeling of the penicillin with C13 was successfully carried out by Craig et aE. (1951; Craig, 1960). This stable product

E;:

TABLE I MICROBLAL PROCESSES FOR PREPARATION OF RADIOACTIVE ANTIEXOTICS Product Actinomycin-C14

Altemariol-C14

Microorganism Streptomyces antibioticus

Altemaria tenuis

Method Precursor of addition ~-Methionine-C14H~ ~-Proline-l-C14 To 2-day culture Hydroxy-~-proline-C14 To %day culture ~-Valine-U-C14 To 2-day culture To 2-day culture L-Proline-1-C14 ~-Threonine-U-C14 To 2-day culture Glycine-l-C14 To 2-day culture ~~-Tryptophan-7-C14 To 1-day culture ~-Methionine-C14H~ To 1-day culture Acetate-l-C14

After 19 days

Aspergillic acid-Cl4

Aspergillus f l a w s

~-Isoleucine-C14 ~~-Leucine-C14

Bacitracin-C14

Bacillus licheniformis

~~-Phenylalanine-C~4 Washed cells DL-Aspartic acid-Cl4 Washed cells ~~-Ornithine-Z-C14 Washed cells ~~-Lysine-2-C14 Washed cells

Incorp. ( %)

Ref .a

15

-

11.7 6.2 6.0

At inoculation At inoculation

17 17.1 12.3 15.5

13 pc./gm. (4127 c.p.m. )

75

2.29 pc./mmole 1.19 pc./mmole

44 44

9.5 c.p.m./unit 8.7 c.p.m./unit 2.8 c.p.m./unit 2.3 c.p.m./unit

70 70 70 70

+

Bacitracin-Cl4H3

Bacillus licheniformis

Growing culture D-ISOkUCine-C'4 ~~-Omithine-H3

Cephalosporin-C14

Cephalosporium sp.

Acetate-l-C14

16 to 48 hours after inoculation

U

15 38 38 39 39 39 39 62 62

6 14.6 pc./mmole

76

?

Citrinin-Cl4

Citromycetin-C14 Curvularin-C14 Echinulin-C14

Erythromycin-C14

Erythromycin-Cl4H3

Penicillium citrinum

NaHC140, Propionate-2-Cl4 Acetate-l-C14 Formate-Cl4

After 7 days After 7 days After 7 days After 7 days

-

Aspergillus candidus

Formate-Cl4 Acetate-2-Cl4

After inoculation After inoculation

66 11

Penicillium frequentam

Formate-CI4 Acetate-2-Cl4

To growing culture To growing culture

0.8 2

Curvularia sp.

Acetate-2-Cl4 Acetate-l-Cl4

To 8-day culture To 8-day culture

0.16 3.8

-

13 13

Acetate-2-Cl4 Mevalonic lactone2-c14 Glycine-l-Cl4

To 4-day culture To 4-day culture

0.7 4.25

-

::

$

To 4-day culture

0.8

-

16

Propionate-l-C14 Propionate-2-Cl4 ~-Methionine-C14H,

To washed cells To washed cells To washed cells

j

Aspergillus amstelodumi

Streptomyces erythreus

Acetate-l-Cl4

To washed cells

Propionate-1474

To washed cells

Propionate-l-C14

To 4-day culture

Propionate-l-Cl4H3

To growing culture

-

-

-

-

-

11 11 11 11

20,400 c.p.m./mg. 20

19,400 c.p.m./mg. 1.2 x 109 c.p.m./mmole < 0.01 0.9 x 106 c.p.m./mmole 9 1.8 x 106 c.p.m./mmole 39 -

-

-

59 59 59 59

-

g

F

2

8

E8 +-

20

8

21

5

z

21

rn

??

21 65 28,29

%

TABLE I (Continued) Product

Microorganism

Erythromycin-H3

Precursor Propionate-2,3-H3

Method of addition To washed cells

Incorp. ( %)

-

Sp. activity 2

x 104 c.p.m./mmole

Gliotoxin-Ha

Trichoderma uiride

Gliotoxin-Cl4

Gramicidin-C14

Bacillus brevis

~~-m-Tyrosine-H~ To growing culture 44.3 ~~-Phenylalanine-Ha To growing culture 17.6

2.5 mpc./mg.

-

-

74 78 78

To growing culture ~~-Serine-3-C1* ~~-Methionine-C14H,To growing culture To growing culture Clycine-2-Cl4

2.7 3.4 1.2

~-Proline-C14 At inoculation ~-Leucine-C14 At inoculation ~-Phenylalanine-C14 At inoculation ~-Valine-C14 At inoculation ~-0rnithine-2-Cl4 At inoculation

4.8 3.8 2.2 1.9 3.6

-

77 77 77 77 77

Acetate-l-C14

1

-

8

-

16.6

0.29 pc./mmole

31

-

-

36

At inoculation At inoculation After 6 days incubation

7 2.5 2

-

14 14

Griseofulvin-C14

Penicillium griseofulvum Penicillium patulum

Griseofulvin-2-ethoxy derivative

Penicillium griseofulvum

Choline-C14H3 DL-EthionineCH,C14H3

Methymy cin-C14

Streptomyces venezuelae Penicillium griseof ulvum

Methionine-C14H3 Propionate-l-Cl4 Acetate-l-C14 Propionate-l-C14

M ycelianamide-C14

Ref.a

After 18 days

78 78 78

10

Mycophenolic acid-CI4

Penicillium brevicompatum

Acetate-l-Cl4 Methionine-C14H3

After 27 days incubation

0.4

Neomycin-Cl4

Streptomyces fradiae

Glucose-U-Cl4

After 1day incubation

19.5

Novobiocin-C14

Streptomyces nivew

Methionine-C14H3

Nystatin-Cl4

Streptomyces mursei

Propionate-l-Cl4 Acetate-l-Cl4

Paromomycin-H3

Streptomyces rimosus H2O

At inoculation

Patulin-Cl4

Penicillium patulum

At inoculation At inoculation

Glucose-l-C14 Acetate-2-Cl4

-

10

-

-

61

15

0.75 pc./mg.

-

Cell-free system

6-Methylsalicylate-C14 Cell-free system Acetyl-CoA-C14

284 c.p.m./mg.

9, 10

18,400 c.p.m./mmole 235 c.p.m./pmole 1,130 c.p.m./pmole 3,960 c.p.m./pmole 2,200 c.p.m./pmole

6-Methylsalicylate-C14 At inoculation Glucose-Cl4

-

Cell-free system

6-Methylsalicylate-Cl4 Washed mycelium Penicillic acid-Cl4

Penicillium cyclopium

Acetate-1-04 Malonate-C14H2 Malonate-l-C14

Penicillium urticae

Diethylmalonate-2-C14

After 7 days incubation

-

-

2.3 35

9.5 15

-

-

5

5

l9

rp P

TABLE I (Continued) Product Benzylpenicillin-S35

Microorganism Penicillium notatum 1249B21 Penicillium ch ysogenum 4176 4176 4176

Wis. 48-701 Wis. 51-20 Wis. 51-20 Wis. 48-701 Wis. 48-701

Benz ylpenicillin-Cl4

Abbott 4-25 Astra 1155 Penicillium chysogenum Wis. 49-133 Upjohn BC-65

Precursor

Method of addition

Incorp. ( "/o )

Sp. activity

Ref.a

S3504

At inoculation

3.4

15.56pc./gm.

32

S3504

At inoculation

-

3700 c.p.m./unit

42

S3504 S3504

At inoculation At inoculation At inoculation At inoculation At inoculation At inoculation At inoculation At inoculation

-

8700 c.p.m./unit 0.05 pc./unit 0.095 pc./unit 1.23 mc./mg. 0.6mc./pg.

43 55 56 64 63 54 30 30 71 72 58 50

~ 3 . 5 0 ~

S3504 S3504 S3504 S350, ~-Cystine-S35 S350,; cystine-S35 S3504;valine-Cl4 W04 S3504 Phenylacetate-C14

Phenylacetate-C14

At inoculation At inoculation At inoculation After 1day incubation

4176

Acetate-2-Cl4

-

4176

Formate-C14

-

12 16

-

20 0.8 2.5

-

35

-

58

-

2.5 pc./mmole 0.17 pc./mmole

-

175pc./gm. 12.4 mc./mg. 12 x 106 c.p.m./mmole 10.5 x 105 c.p.m./mole 184 c.p.m./pmole 3820 c.p.m./pmole

27 60 47 47

tY

9

E

v)

w m

9

?

Wis. 48-701 Wis. 48-701 Wis. 48-701 Wis. 48-701

Glycine-Cl4 DL-CyStine-C14 DL-Valine-C14 Cystine-Nlb, C14,

Wis. 48-701

Diphenylacetyl-l-C14 During incubation ~-Cystine-S35 Phenylacetate-l-Cl4 ~-Valine-U-C14 S3504 At inoculation

Wis. 51-20 Synnematin-S35 Cephalosporium ( D-4-amino-carboxysalmosynnematum n-butylpenicillin-S35) Emericillopsis terricola

Prodigiosin-C14 Pyocyanine-CI4

Streptomycin-C14

Serratia marcescens Pseudomonus aeruginosa

Streptomyces griseus

S3504

S35

During incubation During incubation During incubation During incubation

At inoculation

S3504

At inoculation

Glycine-2-Cl4 Glycerol-l-C14

At inoculation At inoculation

~-Alanine-U-C14

At inoculation

DihydroxyacetoneAt inoculation 1,3-C14 Glucose-Cl4 At inoculation Starch preparation-C14 At inoculation Glycine-2-Cl4 ~ 1 4 0 ,

At inoculation Gassed culture

1 3.4 1.7

2.3 pc./mmole 17.2 pc./mmole 4.16~c./mmole 7.5, c14;-

9, s35 C14, 1.5pc./mmole S35, 0.2 pc./mmole 3.7 1140 pc./mmole 0.27 169 pc./mmole 9.2 x 104 c.p.m./pmole

-

I

-

3.3

0.3

-

27.4 x 104 c.p.m./pmole 3.2 x 105 c.p.m./mg.

-

3 3 3 1,2

1 30 30 7

7 49 57

11.9 x 104 c.p.m./mmole 2.2 x 104 c.p.m./mmole

-

5

84 um g

8

* 2 3

35

8

35 17

23

6 v)

0.5 1.3 0.9

0.5

6770 c.p.m./mg. 1.8 x 105 c.p.m./mg. 3600 c.p.m./mg. 2.9 pc./mmole

38 38 51 33

b P

w

TABLE I (Continued) Product

Streptomycin-C14 (Continued)

Microorganism Streptomyces griseus ( Continued)

Streptomyces aureofaciens

Precursor

Method of addition

( %)

Sp. activity

Ref.a

5 15

29 pc./mmole 23.8 x 10-2 pc./mmole 7288 c.p.m./mmole

34

Glucose-U-Cl4 N-methylglucosamine-C14 Glucose-Cl4

After 60 hours After 60 hours After 60 hours

-

L-Arginineguanidine-Cl4 ~-Methionine-C14H~ Fonnate-Cl4 Clycine-2-Cl4 Starch-U-Cl4 Glucose-6-C14 ~-Methionine-CH~l4 Acetate-1-C14 Ha36

After 96 hours

8.5

At inoculation At inoculation After 48 hours After 48 hours After 48 hours After 48 hours After 48 hours At inoculation

7 0.016 52 17 15 48

7-chlortetracycline-CPs Streptomyces aureofaciens 6-demethyl-7Streptomyces chlortetracycline-C14 aureofaciens S-hydroxytetraStreptomyces rimosus Acetate-2-Cl4 cycline-Cl4 Glucose-U-C14 Canna residue-Cl4 Acetate-2-Cl4 Acetate-l-Cl4

A A

Incorp.

-

13

0.46 pc./mg.

After 1day After 1day After 1day

5.0 ca. 6

190 pc./mmole 2.56 pc./mmole

-

-

After 2 days

-

-

-

5

Streptomyces rimosus Ethionine-C14H2CH3

-

46 31a

41 66,69 66,69 67 68

x 105 c.p.m./mmole

5-hydroxy-4-N-ethylmethyltetracycline-Cl4

P

19a 19a 48 48 48 48 48 23,24

98

-

34

-

26 25

k

Valinomycin-C14

Streptomyces fuloissimus

References: 1. Arnstein et al. (1954). 2. Arnstein and Grant ( 1954a). 3. Amstein and Grant (1954b). 4. Bassett and Tanenbaum ( 1960). 5. Bentley and Keil (1961). 6. Bemlohr and Novelli (1960). 7. Bhuyan et al. (1958). 8. Birch et al. (1958a). 9. Birch et al. ( 1958b). 10. Birch et al. ( 1 9 5 8 ~ ) . 11. Birch et al. (1958d). 12. Birch et al. ( 1958e). 13. Birch et al. ( 1959). 14. Birch et al. ( 1960a). 15. Birch et 02. (1960b). 16. Birch et al. ( 1961). 17. Blackwood and Neish ( 1957). 18. Bu’Lock and Ryan (1958) 19. Bu’Lock and Smalley (1961). 19a. Candy et al. ( 1964). 20. Corcoran et al. ( 1960). 21. Corcoran (1961). 22. Djerassi et QZ. (1962). 23. Doerschuk et al. (1956). 24. Doerschuk et al. (1959). 25. Dulaney et al. ( 1962). 26. Gatenbeck (1961).

L-Valine-1-C14

-

At inoculation

4.15 pc./mmole

44

a

27. Gordon et al. (1953). 28. Griesebach et QZ. (1960a). 29. Griesebach et al. (1960b). 30. Halliday and Arnstein ( 1956). 31. Hockenhull and Faulds (1955). 31a. Homer (1964). 32. Howell et al. ( 1948). 33. Hunter et al. ( 1954). 34. Hunter and Hockenhull ( 1955). 35. Ingram and Blackwood (1962). 36. Jackson et al. (1962). 37. Kaneda et al. (1962). 38. Karow et al. (1952). 39. Katz and Weissbach ( 1962). 40. Katz et al. ( 1962). 41. Kelly et al. (1961). 42. Maass and Johnson ( 1959a). 43. Maass and Johnson (1949b). 44. MacDonald ( 1960). 45. MacDonald ( 1961). 46. Majumdar and Kutzner ( 1962). 47. Martin et al. (1963). 48. Miller et al. (1956). 49. Nara and Johnson (1959). 50. Nathorst-Westfelt et al. ( 1963). 51. Numerof et al. (1954). 52. Ober et al. (1962a).

53. 54. 55. 56. 57. 58. 59. 60. 61. 62. 63. 64. 65. 66. 67. 68. 69. 70. 71. 72. 73. 74.

75. 76. 77. 78.

Ober et d.(196213). Perret ( 1953). Rowley et al. ( 1945). Rowley et ~ l ( .1950). Santer and Vogel (1956). Saunders and Sylvester (1957). Schwenk et al. ( 1958). Sebek (1953). Sebek (1955). Sivak et al. (1962). S m i t h and Allison ( 1952) . Smith and Hockenhull (1952). Smith et al. ( 1962). Snell et al. (1955). Snell (1958). Snell et ~ l (.1960). Snell (1960 1. Snoke (1960). Stevens et a2. (1953). Stevens et al. (1956). Tanenbaum and Bassett (1959). Taubman et al. (1963). Thomas (1961). Trown et al. (1962). Winnick et al. ( 1961). Winstead and Suhadolnik ( 1960).

2 %

E

g

Ei

5

5

3 8 5

2a

A

ur

46

D. PERLMAN, ARIS P. BAYAN, AND NANCY A. GIUFFRE

was used in solving analytical problems in their quality control laboratories. Others have prepared C14-labeledpenicillins by adding acetate-Cl4, formate-Cl4, phenylacetate-C14, and cystine derivatives labeled with C14 to growing cultures of P. chrysogenum. When sufficient phenylacetate or the other benzylpenicillin precursor was added to the growing culture, penicillin G predominated in the antibiotic mixture. If the addition of the phenylacetate was not carefully supervised other penicillins containing some C14 were formed (Martin et al., 1953). The specific activities of the C14labeled penicillin has been lower than those of the S35-penicillins due to dilution of the hot precursor by cold precursor from the medium. Sebek (1953) obtained high conversions of added phenylacetate-C14 to benzylpenicillin, with little dilution of the radioactivity. Penicillin G labeled with CI4, S36, and N16 was prepared in the course of studies on the biogenesis of the thiazolidine ring (Arnstein and Grant, 1954a), and penicillin G labeled with tritium has also been prepared (Arnstein and Crawhall, 1957) in similar studies. Penicillin G labeled with S35has been prepared on a commercial scale (Mohberg and Johnson, 1958; Saunders and Sylvester, 1957). This material was of limited usefulness due to the short halflife of S36.More recently commercial availability was announced of C14-labeledpenicillins of high specific activity, prepared by treating 6-aminopenicillanic acid (from fermentation processes) with C14labeled phenacetyl chloride (J. R. Catch, personal communication). Nathorst-Westfelt et d.(1963) reported on the preparation of S35labeled 6-aminopenicillanic acid to be used in a chemical synthesis of “new” penicillins. The microbiological problems encountered in preparing labeled penicillins are typical of those encountered with most of the antibiotics. If a penicillin labeled with S35is desired, it is necessary to reduce the S35 content of the medium to a minimum so the added S36will constitute a major portion of the total sulfur in the medium. This often results in reducing penicillin yields to about one-fourth “normal” (Smith and Hockenhull, 1952; Saunders and Sylvester, 1957), and this in turn makes it necessary to add “cold carrier penicillin during recovery operations. The end result, if adequate precautions are taken, is a quantity of penicillin of useful specific activity, perhaps one-tenth that produced in the fermentation. Some of the SS5 is incorporated into the penicillin, while much of it ends up elsewhere (Gordon et al., 1954; Segel and Johnson, 1961).

PREPARATION OF RADIOACTIVE COMPOUNDS

47

Streptomycin was the second antibiotic to be produced in labeled form, and there are several reports on processes useful for the production of this material (see Table I ) . A variety of precursors have been used, resulting in products of varying specific activity (Hunter, 1956). The highest activity was obtained when Streptomyces griseus cultures were grown in media containing soybean meal and a C14-hexose (Karow et al., 1952; Hunter and Hockenhull, 1955). Apparently, the carbon compounds of the soybean were not incorporated into the streptomycin structure (Hunter, 1956; Donovick, 1956). As mentioned earlier, delayed addition of the C14hexose resulted in a higher incorporation of labeled precursor into the streptomycin. Most of the labeled compounds listed in Table I were prepared as a result of biogenetic studies of the mechanism of formation of these particular antibiotics. A few, notably the penicillins, streptomycin, and bacitracin, have been used in “mechanism of action studies.” 6-Demethyl-7-chlortetracycline-C14 was prepared for pharmacological studies (Kelly et al., 1961), and it is likely that other antibiotics will be prepared in labeled form for this purpose in the future.

B. VITAMINS A few of the vitamins which have been prepared in labeled form are listed in Table 11. The most useful of these has been the cobaltlabeled vitamin BIZ, which has found use in clinical tests for pernicious anemia. Most of the clinical tests have used CoeO-labeled material, and, as pointed out by Smith (1956), this material has several disadvantages. Co6O is readily available at low-specific activities, but not at high-specific activities needed for some investigations. The relatively long half-life (5.3 years) severely limits the safe total dose for any patient. Accordingly, C O (half-life, ~ ~ 72 days) made nearly carrier free by treatment of nickel in an atomic pile, C O (half-life, ~ ~ 72 days) made by cyclotron treatment of iron, and Co5’ (half-life, 270 days), also from the bombardment of iron in the cyclotron, have been used in preparation of labeled BIZ. Specific activity values of 12 mc./mg. have been reported for C O ~ ~ vitamin BI2 (Smith, 1956) and over 100mc./mg. attained in experimental runs. Neutron irradiation of Blz resulted in material with a specific activity of 0.012 pc./mg. from the Co60 and 0.005 pc./mg. from the P3*(Smith, 1952). These products were of too low activity

MICROBIAL PROCESSES FOR Product fl-Carotene-Cl4 fi-Carotene-Cl4

Microorganism

+P

ca

TABLE I1 PREPARATION OF RADIOACTIVE VITAMINS AND GROWTHFACTORS Precursor

Anabaenu variubilis Blukeslea trispora

C1402 Mevalonate-2-Cl4

Blakeska trispora

Dimethylacrylic acid-3-04 Dimethylacrylic acid-3-Cl4 Acetate-l-Cl4

B-Carotene-Cl4

Chlorellu pyrenoidosa

P-Carotene-Cl4

Acetate-Z-Cl'

Pyridoxamine-C14

Phycomyces blakesleeana Phycomyces blukesleeana Candidu utilis

Riboflavin-C14

Ashbya gossypii

Glucose-C14

Riboflavin-Cl4

Ashbya gossypii

6,7-Di-C14H3-8ribityllumazine Adenine-8-Cl4

Serine-3-Cl4

Riboflavin-C14

Ashbya gossypii

Riboflavin-Cl4

Eiemothecium ashbyii Threonine-U-Cl4

Method of addition

Incorp. (%)

Gassed culture 0.5 After 1 day incu- bation -After 1 day incubation After 1 day incubation After 5 days incu- ca. 1 bation After 5 days incu- ca. 1 bation At inoculation and after 1 day incubation After 2 days incu- 0.6 bation To cell-free extracts 15 ;!

Sp. activity

Ref.a

1 &mg. 435 c.p.m./mg.

6

1020 c.p.m./mg.

1

750 c.p.m./mg.

1

1

9,lO I

1870 c.p.m./ymole 685 c.p.m./pmole

-

9-1 1 12 16-18

14,19

?

After 3 days incu- 13 bation After 2 days incu- 4 bation

9770 c.p.m./pmole

-

13 8

Ftiboflavin-C14

Eremothecium ashbyii Serine-U-Cl4

Riboflavin-Cl4 Riboflavin-Cl4

Eremothecium ashbyii Adenine-U-Cl4 Candida jlareri Adenine-U-C14

Vitamin B12-Co60 Vitamin Bl,-Co60 Vitamin B,,-CoGo Vitamin B1,-Co57 Vitamin Bl,-Co58 Vitamin B,,-Co58 coenzyme Vitamin Bl2-P32

Streptomyces griseus Streptomyces griseus Streptomyces griseus Streptomyces griseus Streptomyces griseus Propionibacterium shermanii Streptomyces griseus

Vitamin B12-C14 Vitamin B1,-C14

Streptomyces sp.

Vitamin B12-C14 a

C060( NO,), Co6O-salt Co60S04 Co57-salt C05R-salt C058c1~ P3204 Methionine-C14H3 8-Aminolevulinic acid-1,4-Cf4 Porphobilinogen-C14

After 2 days incubation

-

6.9

-

After 1 day incu- 18 bation At inoculation ca. 4 At inoculation 17.2 64 At inoculation

-

-

At inoculation

20

After 1 day incubation At inoculation

0.01

-

-

7

23,800 c.p.m./pmole 5750c.p.mJmg.

15 2

67 pc./mg. 120 pc./mg. 0.25 pc./mg. 13.5mc./mg. 12 mc./mg.

0.35 pc./mg.

-

ca. 1

-

-

ca. 1

-

20 23 4

24 24 25 23 3 522

21

References: 1. Anderson et 02. (1960). 2. Audley and Goodwin (1962).

3. Bray and Shemin (1958). 4. Chaiet et al. (1950). 5. Corcoran and Shemin (1957). 6. Glover and Shah ( 1957). 7. Goodwin and Jones (1956). 8. Goodwin and Horton (1960). 9. Lilly et al. ( 1958).

10. 11. 12. 13. 14.

15. 16. 17. 18.

Lilly et d. (1960). Lotspeich et al. (1963). Lunan and West ( 1963). Maley and Plaut ( 1959a). Maley and Plaut (195913). McNutt (1956). Plaut (1954a). Plaut (1954b). Plaut and Broberg (1956).

19. 20. 21. 22. 23. 24. 25.

Plaut ( 1960). Rosenblum and Woodbury ( 1951). Schwartz et al. (1958). Shemin et al. (1956). Smith et al. (1952). Smith (1956). Toohey and Barker (1961).

50

D. PERLMAN, AXIS P.

BAYAN, AND

NANCY A. GIUFFRE

to be useful (Smith, 1952; Rosenblum, 1959) as far as clinical tests were concerned. C14-labeled B12 has been prepared by adding precursors of the corrin ring to various fermentations. The resulting products have been of very low-specific activity, although useful in studies on biogenesis of the corrin ring (Schwartz et al., 1958; Shemin et al., 1956; Bray and Shemin, 1958; Corcoran and Shemin, 1957). p-Carotene-Cl4 prepared from Phycomyces fermentations has been useful in studying the distribution of this vitamin in rats (Lilly et al., 1960) and in showing the biogenesis of this vitamin in this mold (Lotspeich et al., 1963). Much of the C14-labeled acetate added to the Phycomyces fermentation was converted to lipids, ergosterol, and other cellular products, and relatively small quantities were found in the p-carotene. The studies of the biogenesis of riboflavin in yeasts have utilized isotopic tracer techniques, and Plaut and associates have been able to ascertain the origin of all of the carbons of this vitamin. It is likely that when material of high-specific activity is available it will be useful in pharmacological studies. C. AMINOACIDS A number of microbiological processes have been used to prepare C14-labeled amino acids, and several of these are being operated on a commercial basis. Absorption of C1402by growing cultures of Chlorella species (Schieler et al., 1953; Catch, 1954a, b; Wada et al., 1955), Rhodospirillum rubrum (Tarver et al., 1952), Brucelk abortus (Marr and Wilson, 1951), and T h ~ o b a c thiooxidans i~~~ (Frantz et al., 1952) resulted in formation of cell proteins containing C14labeled amino acids. These amino acids were recovered from the cells by acid hydrolysis of the cell protein and separation of the “free” amino acids on ion-exchange columns. An alternate procedure, using Torula utilis grown in media containing C14-carbohydrate, has resulted in labeled amino acids of specific activity equivalent to those obtained from Clorella or R. rubrum (Laufer et al., 1963). Data showing the efficiency of these processes are summarized in Table 111. Between 25 and 40% of the C14 absorbed by Chlorella is fixed in the protein (Schieler et al., 1953; Catch, 1954b, 1961; Erb and Maurer, 1960; Peyser, 1963), and similar observations have been made with R. rubrum (Tarver et al., 1952) and T . thiooxidans

51

PREPARATION OF RADIOACTIVE COMPOUNDS

(Frantz et al., 1952). About half of the labeled hexose is “fixed by the T. utilis cells, and about 30% of that fixed is recovered as protein hydrolyzable to amino acids (Laufer et al., 1963). Acetate is an alternative to the hexose in the yeast process (Gilvarg and c1*-LABELED AMINOACIDs

TABLE 111 MICROBIAL PROTEIN HYDROLYZATES

FROM

Chlorella pyrenoidosa grown on media with c 1 4 0 2 as C source

1ncorp.a (%) 1.5 0.8 1.5

Amino acid Alanine Arginine Aspartic acid Cystine Glutamic acid Glycine Histidine Iso1eucine Leucine Lysine Methionine Phenylalanine Proline Serine Threonine Tyrosine Valine

2.0 0.7 0.2 0.8 1.7 0.8 1.3 0.8 0.6 0.7 0.8 1.4

Sp. activitya (mc./ milli- Sp. activityb atom C ) (c.p.m./mg.) 120 114 240 1658 160 2336 70 200 664 80 98 240 800 240 45 240 88 240 469 360 200 120 160 360 200

-

1491 4 48 1 88 10

RhodoTorulu utilis spirillum on C14rubrum molasses on C1402 1ncorp.C

1ncorp.d

(%)

(%)

1.6 0.9 1.2 0.2 2.0 0.8 0.5 1.2 1.5 2.0 0.3 1.0 0.7 0.9 0.9 0.8 1.2

4.4 -e

5.5 0.2 5.2 4.0 1.o 0.6 1.1 0.6

-e

1

1.4 4.1 2.3 2.3

Peyser (1963). Schieler et al. (1953). Laufer et al. (1963). Tarver et al. (1952). e Phenylalanine arginine = 6.7. a

*

+

Bloch, 1951; Ehrensvard et al., 1951), but is less efficiently converted to amino acids and thus less desirable. labeled methionine and cystine have been prepared by a number of microbial processes. Schliissel (1951) and his associates ( Schlussel and Fenedegen, 1951; Schlussel et al., 1951) used Torula utilis cultures, Wood and Perkinson (1952) and Wood and Mills

52

D.

PERLMAN, A R E

P.

BAYAN, AND

NANCY A. GIUFFRE

(1952) used Saccharomyces, Cowie et al. (1952) found Escherichiu coli of value, and Gordon et al. (1954) proposed the use of Penicillium chrysogenum. In the experiments with the mold about 20% of the S35 added to the culture was recovered as m e t h i ~ n i n e - S ~ ~ , while in the Torula studies 18% was cystine-P5. Laufer and Gutcho (1955) grew a number of yeast species on S35-containing media and isolated g1~tathione-S~~ from the radioactive cells. Between 4 and 9% of the added S35 could be recovered as glutathione-S35. The usefulness of ~elenomethionine-Se~~ in clinical medicine has resulted in a need for this material. Treatment of selenomethionine in a graphite reactor apparently did not yield a material of high enough activity (McConnell et at., 1962), and a method based on a microbiological process using bakers' yeast was found more satisfactory (Blau, 1961). Selen~methionine-Se~~ with a specific activity of 10 to 20 mc./mmole has been obtained (Hansson and Blau, 1963) from protein hydrolyzates of yeast grown in media containing H2Se7503. Some ~elenocystine-Se7~ was also recovered. A process based on E . coli was apparently less efficient for production of selen~methionine-Se~~ ( Tuve and Williams, 1961).

D. CARBOHYDRATES AND RELATED COMPOUNDS A few of the C14-labeled carbohydrates and related compounds produced by microbial processes are listed in Table IV. The glucose-C14 and mannose-C14 produced by Torula utilis are obtained by acid hydrolysis of the cell polysaccharide material, Other polysaccharide products from yeast include yeast mannan-C14, yeast glycogen-C14, and trehalose-Cl4 ( Chen, 1959). Fermentation of glycerol-1, 3-G4with Acetobacter species yielded a nearly quantitative amount of dihydroxyacetone-1, 3-C14 ( Blackwood and Neish, 1957). Segal and Topper (1957) isolated ~-fucose-Cl~ and D-glucose-C14 from slime obtained from Aerobacter aerogenes grown in g1uc0se-C'~ media. The dextran-Cl4 produced by enzyme treatment of sucrose-C14 by a Leuconostoc mesenteroides preparation was used in clinical studies of the usefulness of dextran as a blood plasma expander (Scully et at., 1952). The rnann0~e-C'~ [from yeast and from hydrolysis of cell wall of Chlorella (Catch, personal communication) 1, the galactose-C14, and the g1uc0se-C~~ mentioned in Table IV are all produced by these microbial processes on a commercial basis.

MICROBIAL PROCESSES FOR ~~~~~~~

~

PREPAXATION OF

TABLE IV C14-LABELa, CARBOHYDRATES A N D

RELATED

COMPOUNDS

~

~

~~~

Incorp. Product

Microorganism

Cellulose-C14

Acetobacter xylinum

Dextran-Cl4

Leuconostoc mesenteroides Chlorella pyrenoidosa Penicillium charksii

Erythrose-Cl4phosphate Galactose-C14 Galactose-C14 Glucose-Cl4

“Marine alga” Torula utilis

Glycerol-CI4 Myo-Inositol Mannitol-Cl4

“Marine alga” Chlorella Leuconostoc mesenteroides Torula utilis

Mannose-Cl4 Sucrose-C14

Chlorella Pseudomonas saccharophila

References: 1. Blakeley and Blackwood ( 1957). 2. Calbiochem ( 1963). 3. Gander (1960). 4. Laufer et al. (1963).

Method of addition

(%)

Sp. activity

Glucose-l-Cl4 Mannitol- 1-C14 Sucrose-Cl4

At inoculation At inoculation At inoculation

3.7 28 7.8

0.16 pc./mg. C 0.32 pc./mg. C 107 pc./gm.

6 8

~ 1 4 0 ~

Gassed culture

0.1

-

7

%

Glucose-l-C14 Glucose-Z-Cl4 Acetate-l-Cl4

At inoculation At inoculation At inoculation Gassed culture At inoculation

1570 c.p.m./pmole 1930 c.p.m./pmole

3 3

cl

4800 c.p.m./mole 9.3 mc./mmole 400 Pc./mg.

3

8

2 4

?

2

88

Precursor

c1402

Canna leaf molasses-C14

ca. 8

Gassed culture Gassed culture At inoculation

-

-

9.3 mc./mmole 50 mc./mmole 103 mpc./mmole

Canna leaf molasses-Cl4

At inoculation

ca. 8

400 cl.c./mg.

~ 1 4 0 ,

Gassed culture Cell-free extracts Cell-free extracts

-

c1402 c1402

Fmctose-C14

Fructose-CI4 Glucose-C14-l-PO,

4 mc./mmole

-

-

Ref .a

5

10 1 4

10 9 9

‘d %I

2!

50 zC

i 2

5

5. Minor et al. (1954a). 6. Minor et al. (1954b). 7. Moses and Calvin (1958). 8. Scully et a2. (1952).

9. Wolochow et al. (1949). 10. Nuclear-Chicago Corp. (1964).

w w

TABLE V MICROBIAL PROCESSES

FOR

PREPARATION OF C14-LABELED STEROLS AND RELATED

PRODUCTS

Incorp. Precursor

Method of addition

(%)

Eburicoic acid-Cl4

Polyporus sulfureus

Formate-Cl4 Acetate-2-CI4

At inoculation At inoculation

1.7 5.1

Ergosterol-C14

Saccharomyces cerevisiue

Acetate-l-C14 Methionine-C14H3 Methionine-C14H,

82 68 14

Lanosterol-Cl4

Saccharomyces cerev isiae

Acetate-l-CI4

To washed cells At inoculation To cell homogenates To cell homogenates

Squalene-Cl4

Saccharomyces cerevisiue

Acetate-l-Cl4

To medium after 30 minutes

-

488 c.p.m./mg.

Zymosterol-C14

Saccharomyces cerevisiae

Acetate-l-C14

To medium after 30 minutes

-

352 c.p.m./mg.

Acetate-l-CI4

To cell homogenates

Product

Microorganism

References: 1. Alexander and Schwenk (1957). 2. Alexander et al. ( 1958). 3. Dauben et al. (1957a). 4. Dauben et al. (195%).

0.2

0.3

Sp. activity 4250 c.p.m./mg. C 9200 c.p.m./mg. C

1.4 x lo6 c.p.m./mg. 3 x 105 c.p.m./mg. 4200 c.p.m./mg. 313 c.p.m./mg.

1200 c.p.m./mg.

a

5. Hanahan and al-Wakil ( 1952). 6. Schwenk et al. (1955). 7. Schwenk and Alexander (1958).

Ref .a

4

PREPARATION OF RADIOACTIVE COMPOUNDS

55

E. STEROLSAND RELATED PRODUCTS Some of the data on (?*-labeled sterols and related products of microbial fermentations are summarized in Table V. The specific activities of most of the compounds mentioned were high enough to be useful in determinations of site of incorporation of precursors, but too low for use in other biological studies. Microorganisms have also been used in transforming labeled steroids into useful derivatives. For example, Rhizopus arrhizus was used (in studies on mechanisms of enzymic hydroxylation) to convert androstenedione-6a,7-H; to GfLhydroxyandrostenedione, 6ketoandrostenedione, and lla-hydroxyandrostenedione( all containing the tritium label) (Baba et al., 1963), Rhizopus nigricans converted 3,20-pregnenedione-lla,12a-H:to 3,20-pregnenedione-lla-o1 (Hayano et al., 1958)) and Calonectria decora converted progesterone-llaJ2a-Hi to 12fi,15a-dihydroxyprogesterone (Hayano et al., 1959).

F. ORGANIC ACIDSAND ALCOHOLS Data on a few of the organic acids produced in microbial fermentations of U4-labeled substrates are summarized in Table VI. Of this group, citric, kojic, lactic 1(+) and l(-) isomers, lactobacillic, puberulic, and stipitatic acids are only available through microbiological processes. Acetic, butyric, fumaric, oxalic, and shikimic acids can be produced by chemical processes, and gibberellins have been prepared with a tritium label (Rosenblum, 1959). G. NUCLEICACIDSAND PURINES

A few of the microbial processes for producing labeled nucleic acids and purines are reviewed in Table VII. Several of these have been useful in preparing material of high-specific activity, while others were the results of mechanism studies. These labeled materials have been widely used in biochemical studies on the mechanisms of nucleic acid formation.

H. MISCELLANEOUS A large number of organisms have been grown in media containing radioactive isotopes, and cells containing the isotope recovered for further investigation. For example, Aiba and Yamamoto (1959) grew Serratia rnarcescens in P3*04-containingmedia and used the

MICROBIAL hOCESSES

TABLE VI C1'-LABELED

FOR PREPARATION OF

ALCOHOLS A N D

ORGANIC

ACIDS

Incorp. Product Acetic acid Amy1 alcohol Butyric acid Citric acid

Microorganism Clostriduim luctoacetophilum Saccharomyces cerevisiae Clostriduim iizctoacetophilum Aspergillus niger

Precursor

Method of addition

( %)

At inoculation ( as Na,C1403 ) Resting cells

28

48 88 48

~ 1 4 0 ,

At inoculation At inoculation At inoculation Gassed culture

-

~ 1 4 0 ,

a-Aminobutyric acid-2-04 ~ 1 4 0 ,

Amtate-l-Cl4 Acetate-1-04

7.9

Fumaric acid

Rhizopus nigricans

~tha1101-i-cl4

At inoculation

Kojic acid

Aspergillus

Lactic acid

Lactobacillus delbrueckii Lactobacillus leichmunnii Rhbopus o y m Rhizopus MX Escherichia coli Lactobacillus arabinosus

Acetate-2-04 Glycine-2-Cf4 Glucose-U-04

4 days after inoculation At inoculation

40

Glucose-U-W

At inoculation

40

Glucose-U-Cl4 pyUvate-C14H3 Me&onine-C14H3 Vaccenic acid-1-04

To washed cells At inoculation At inoculation At inoculation

fivus-0 yzoe

Lactobacillic acid

2.3

0.77

Sp. activity 1350 c.p.m./mmole

Ref.a

696 c.p.m./mole

10

1650 c.p.m./mmole 4250 c.p.m./mmole 686 c.p.m./pmole 60 c.p.m./mg. BaCo3 131c.p.m./mg. BaCo, 95 pc./mole 53.7 pc./mole 241 c.p.m./mg.

3 3 4

3

12 8 1 1 5

228 c.p.m./mg.

5

-

-

9

28

14.9 c.p.m./mole

6

-

7 11

42 92

-

Malic acid

AspergiUus niger

~ 1 4 0 ,

Gassed culture

-

Oxalic acid

Aspergillus niger

Acetate-1-U'

At inoculation

-

Oxaloacetic acid

Thwbacillus thiouxidans

c1402

To cell-free extract

Puberulic acid

Penicillium aurantiovirens

GIucosc+~-C~~ Acetate-l-Cl4 Acetate-2-Cl4

At inoculation At inoculation At inoculation

n-Propanol

Saccharmyces cerevisiue

a-Aminobutyric acid-24214

To resting cells

7.9

696 c.p.m./pmole

10

Shikimic acid

Escherichiu coli

Glucose-U-C14

At inoculation

6

0.3 pc./mg.

15

Stipitatic acid

PeniciUium stipitatum

Glucose-l-Cl4 Glucose-l-C14

At inoculation 5.1 After 7 days incu- 20 bation After 7 days incu- 34 bation

Gibberellins a

Fusarium noniliforme

Acetate-1-04 Acetate-2-Cl4

During incubation During incubation

0.01

-

-

0.9 2

63 c.p.m./mg. BaCO, 21 c.p.m./pmole

12 4

-

14

-

13 13 13

-

-

-

2 2

-

2

542 c.p.m./mg. 328 c.p.m./mg.

16 16

References:

1. Arstein and Bentley (1953).

7. Chalk, K. J. I., and Kodicek, E. (1961). 13. Richards and Ferretti (1960). 2. Bentley (1960). 8. Foster et al. (1949). 14. Suzuki and Werkman (1958). 3. Bhat and Barker ( 1948). 9. Gibbs and Gastel (1953). 15. Zaprometov ( 1961). 4. Bomstein and Johnson (1952). 10. Guyman et al. (1961). 16. Zweig and DeVay ( 1959). 5. Brin et al. (1952). 11. Hofmann and Liu (1960). 12. Mosbach (1952). 6. Carson et al. (1951).

TABLE VII MICROBIALPROCESSES FOR PREPARATION OF RAD~OACTIVENUCLEIC ACIDS AND PURINES Product Adenine-U-Cl4

Microorganism

Precursor

Method of addition

Incorp. ( %)

Sp. activity

-

-

Ref .a

1 7

Anabaenu cylindrica Chlorella eUipsoidu

c1402 c1402

Gassed culture Gassed culture

Adenosine-5’po4-c14

Chlorella pyrenoidosa

c1402

Gassed culture

0.2

240 mc./mmole

5

Cytidine-5’-

ChloreUa pyrenoidosa

c1402

Gassed culture

0.2

216 mc./mmole

5

Uracil-2-Cl4

Cell-free extract

28 mc./mmole

5

Anubaena cylindrica Chlorella ellipsoidu

c1402 ~ 1 4 0 ,

Gassed culture Gassed culture

Chlorella pyrenoidosa

c1402

Gassed culture

po4-c14

Deoxyuridine-2-Cl4 Escherichiu coli Guanine-U-CI4 Guanosine-5‘PO,-C~~

-

27

0.1

-

-

1 7

196 mc./mmole

5

Thymidine-2-Cl4

Escherichia coli

Thymine-2-C14

Cell-free extract

25

30 mc./mmole

5

Thymidine-CH33

Escherichia cold

Thymine-CH33

Cell-free extract

32

6700 mc./mmole

5

Uridine-2-Cl4

Escherichia coli

Uracil-2-Cl4

Cell-free extract

20

20 mc./mmole

5

Nucleic acid-Cl4

Escherichiu coli Rhodospirillum rubrum

A’I”-C14 Guanine-8-Cl4

Cell-free extract After 2 days growth

100 50

Torula utilis

Canna leaf molasses-C14

At inoculation

3

10.4 c.p.m./mmole

4 2

100 pc./mg.

3

Ribonucleic acid-Cl4

Towla utilis

Adenine-8-Cl4 Uracil-B-Cl4

Deoxyribonucleic acid-Cl4

Chlorella pyrenoidosa

C1402

Torula utilis

Adenine-8-C14 Uracil-B-Cl4

References : 1. Audley and Goodwin (1962). 2. Goodwin and Passorn (1961). 3. Laufer et al. (1963). 4. Moldave ( 1960).

After 1 day incu- 55+ bation After 1 day incu- 55+ bation Gassed culture After 1 day incubation After 1 day incubation

-

3

-

3

10 w./mg.

"

5

-

3

%

5

-

3

cl

a

5. Peyser (1963). 6. Strange et al. (1962). 7. Wada ( 1957).

E

8 Z

8

P 8

9 8

6

H

60

D. PERLMAN, ARIS P. BAYAN, AND NANCY A. GIUFFRE

labeled cells in studing the characteristics of fibrous air-sterilization filters. Stonier (1956) labeled crown gall bacteria with PSzby growing them in media containing P3’O4, and then studied the properties of the bacteria. For example, he noted that lysis of his labeled cells occurred and attributed this to the presence of an undetected phage (Stonier, 1960). The absorption of isotopes has interested some workers studying fixation of isotopes by animals. Most microorganisms will absorb metallic ions found in synthetic media, and the experiments of Morgan ( 1961) using Rhodmonas, Ochromonas, Navicula, and bacteria to study absorption of Ce137, Fe60, Sr87, Zn”, Ce141, CeIS7,CoB0,C U ~ ~ , FeS9,Re103, SrE9,and UZ38gave about the results expected. Tritium absorption studies were carried out with bacteria (Alpen, 1960) and Chloretla (Weinberger and Porter, 1954), and absorption of the hydrogen isotopes ( from aqueous solutions) varied somewhat to indicate that these may be preferentially excluded from the cells. Tritium gas was not fixed by a number of bacteria (Smith and Marshall, 1952) in a different manner from hydrogen. A few other uses of microbial systems to produce labeled materials include the production of labeled Clostridium perfringens toxin by growing the bacteria in media containing hydrolyzed protein from labeled C . pyrenoidosa cells. The labeled toxin was used in animal studies to localize and trace the toxin in the organs (Ellner, 1961). Labeled ergot alkaloids have been prepared from Claviceps cultures in biosynthetic studies (Vining and Taber, 1963) and these may be useful in pharmacological investigations.

IV. Summary Microbial processes have been used to prepare a wide variety of labeled compounds including amino acids, nucleic acids, antibiotics, organic acids, polysaccharides, vitamins, sterols, pigments, and phage, Most of these have been labeled with C14, and a few prepared with tritium, S35, P3*, or cobalt isotopes. The advantages of using microbial processes lie in the variety of complex organic compounds that can be prepared with “relative simplicity,” the specific activity of the materials prepared (as compared with similar materials prepared by chemical synthesis), the preparation of the “naturally occurring” form of the compound, and the availability of process “know how” for preparation. The disadvantages are

PREPARATION OF RADIOACTIVE COMPOUNDS

61

derived from the fact that products of biosynthetic origin may not be specifically labeled, biosynthetic methods may be difficult to “scale-up” to a preparative scale, and often the final product (when growing cultures are used) is difficult to isolate in pure form. Several types of microbial processes have been successfully used in the preparation of labeled compounds. These include: (1) growing the microorganism in media containing a source (or precursor) of the labeled compound which can be utilized by the culture and transformed into the desired product; ( 2 ) adding the labeled source material to washed microbial cells and isolating the synthesized metabolite containing the labeled atom; ( 3 ) growing the microorganism in tritiated water-containing media to obtain tritium labeled products; and (4)adding the labeled precursor to crude enzyme preparations derived from microorganisms and isolating the labeled conversion product, The first method has been most widely used and among its special requirements are: (1) selection of a strain of the organism producing large amounts of the desired metabolite from the commercially available labeled compounds useful as precursors; ( 2 ) selection of a process and conditions which result in little dilution of the radioactivity of the added precursor, thus insuring a product of high-specific activity; and ( 3 ) selection of a process which results in a formation of the desired product in such a form that it can be easily used in biochemical or other investigations. Labeled compounds synthesized by chemical processes will successfully compete with those from microbial processes when the cost of producing the latter is of the same order as the former, or the complexity of recovering the microbial product is too much of a problem. It is likely that in the future more labeled compounds will be prepared by methods involving a combination of microbial operations and chemical manipulations.

REFERENCES Aiba, S., and Yamamoto, A. (1959). J . Biochem. Microbiol. Technol. Eng. 1, 129-141. Alexander, G. J., and Schwenk, E. (1957). J. Am. Chem. SOC. 79, 45544555. Alexander, G. J., and Schwenk, E. (1958). J. Biol. Chem. 232, 611-616. Alexander, G . J., Gold, A. M., and Schwenk, E. (1958). J . Bwl. Chem. 232, 599-609. Alpen, E. L. (1960). Biochim. Biophys. Actu 43, 317-321.

62

D. PERLMAN, ARIS P. BAYAN, AND NANCY A. GIUFFRE

Anderson, D. G., Norgard, D. W., and Porter, J. W. (1960). Arch. Biochem. Biophys. 88, 68-77. Amstein, H. R. V., and Bentley, R. (1950). Quart. Reu. Chem. SOC. 4, 172194. Amstein, H. R. V., and Bentley, R. (1953). Biochem. J . 54, 517-522. Arnstein, H. R. V., and Crawhill, J. C. (1957). Biochem. J . 67, 180-187. Arnstein, H. R. V., and Grant, P. T. (1954a). Biochem. J . 57, 353-359. Amstein, H. R. V., and Grant, P. T. (1954b). Biochem. J . 57, 360-368. Amstein, H. R. V., Clubb, M. E., and Grant, P. T. (1954). In “Radioisotope Conference” (J. E. Johnston, ed.), pp. 306-312. Butterworths, London. Audley, B. G., and Goodwin, T. W. (1962). Biochem. J . 84, 587-592. Baba, S., Brodie, H. J., Hayano, M., Peterson, D. H., and Sebek, 0. K. (1963). Steroids 1, 151-156. Baker, N., Gibbons, A. P., and Shipley, R. A. (1958). Biochim. Biophys. Acta 28, 579-586. Barker, H. A., Ruben, S., and Kamen, M. D. (1940a). Proc. Natl. Acad. Sci. U.S. 26, 426-430. Barker, H. A., Ruben, S., and Beck, J. V. (1940b). Proc. N d . Acad. Sci. U.S. 26,477-482. Barlow, G. H., and Sanderson, N. D. (1960). Biochim. Biophys. Acta 41, 146-148. Bassett, E. W., and Tanenbaum, S. W. (1960). Biochim. Biophys. A C ~ 40, Q 535-537. Bayly, R. J., and Weigel, H. (1960). Nature 188, 384-387. Bentley, R. (1960). Biochem. Biophys. Res. Commun. 3, 215-219. Bentley, R., and Keil, J. G. (1961). Proc. Chem. SOC. pp. 111-112. Bemlohr, R. W., and Novelli, G . D. (1960). Biochim. Biophys. Acta 41, 541-543, Bhat, J. V., and Barker, H. A. (1948). J . Bacterial. 56, 777-779. Bhuyan, B. K., Mohberg, J., and Johnson, M. J. (1958). J. Bacteriol. 76, 393-399. Birch, A. J., Massy-Westropp, R. A., Rickards, R. W., and Smith, H. (1958a). J . Chem. SOC. pp. 360-365. Birch, A. J., English, R. J., Massy-Westropp, R. A., Slaytor, M., and Smith, H. (1958b). J . Chem. SOC. pp. 365-368. Birch, A. J., English, R. J., Massy-Westropp, R. A., and Smith, H. (1958~). 1. Chem. SOC. pp. 369-375. Birch, A. J., Fitton, P., Pride, E., Ryan, A. J., Smith, H., and Whalley, W. B. (1958d). J . Chem. SOC. pp. 4576-4581. Birch, A. J., Blance, G. E., and Smith, H. (1958e). 1. Chem. SOC. pp. 4582-4583. Birch, A. J., Musgrave, 0. C., Rickards, R. W., and Smith, H. (1959). J. Chem. SOC. pp. 3146-3152. Birch, A. J., Pride, E., Rickards, R. W., Thomson, P. J., Dutcher, J. D., Perlman, D., and Djerassi, C . (1960a). Chem. G Ind. (London) p. 1245. Birch, A. J,, Cameron, D. W., Holloway, P. N., and Rickards, R. W. (1960b). Tetrahedron Letters 25, 26-31.

PREPARATION OF RADIOACTIVE COMPOUNDS

63

Birch, A. J., Blance, G. E., David, S., and Smith, H. ( 1961). J . Chem. SOC. pp. 3128-3131. Blackwood, A. C., and Neish, A. C. (1957). Can. J. Microbiol. 3, 165-169. Blakeley, E. R., and Blackwood, A. C. (1957). Can. J. Microbiol. 3, 741744. Blau, M. (1961). Biochim. Biophys. Actu 49, 389-390. Bomstein, R. A., and Johnson, M. J. (1952). J . Biol. Chem. 198, 143-153. Bray, R., and Shemin, D. (1958). Biochim. Biophys. Acta 30, 647-648. Brin, M., Olson, R. E., and Stare, F. J. (1952). Arch. Biochem. Biophys. 39, 214-218. Bu’Lock, J. D., and Ryan, A. J. (1958). Proc. Chem. SOC. pp. 222-223. Bu’Lock, J. D., and Smalley, H. M. (1961). Proc. Chem. SOC. pp. 209-211. Calbiochem. (1963). Bulletin #34. Candy, D. J., Blumsom, N. L., and Baddiley, J. (1964). Biochem. J . 91, 31-35. Carson, S. F., and Ruben, S . (1940). Proc. Natl. Acad. Sci. U.S. 26, 422426. Carson, S. F., Foster, J. W., Jefferson, W. E., Phares, E. F., and Anthony, D. S. (1951). Arch. Biochem. Biophys. 33, 448-458. Catch, J. R. (1954a). Rept. Progr. Appl. Chem. 39, 353-359. Catch, J. R. (1954b). Proc. 2nd Radioisotope Congr. Oxford, EngE. 1, 258261. Catch, J. R. (1957). Anal. Chem. 29, 1726-1730. Catch, J. R. ( 1961 ) . “Carbon-14 Compounds.” Buttenvorths, London. Chaiet,-L., Rosenblum, C., and Woodbury: D. T. (1950). Science 111, 601602. Chalk, K. J. I., and Kodicek, E. (1961). Biochim. Biophys. Acta 50, 579581. Chen, S. L. (1959). Biochim. Biophys. Actu 32, 480-484. Chorney, W., Scully, N. J., Crespi, H. L., and Katz, J. J. (1960). Biochim. Biophys. Acts 37, 280-287. Corcoran, J. W. (1961). 1. Biol. Chem. 236, PC27. Corcoran, J. W., and Shemin, D. (1957). Biochim. Biophys. Acta 25, 661662. Corcoran, J. W., Kaneda, T., and Butte, J. C. (1960). J . Biol. Chem. 235, PC29. Cowie, D. B., Bolton, E. T., and Sands, M. K. (1952). Arch. Biochem. Biophys. 35, 140-145. Craig, J. T. (1960). In “Radioactivity for Pharmaceutical and Allied Research Laboratories” ( A . Edelman, ed.), pp. 73-88. Academic Press, New York. Craig, J. T., Tindall, J. B., and Senkus, M. (1951). Anal. Chem. 23, 332333. Dauben, W. G., Ban, Y., and Richards, J. H. (1957a). J . Am. Chem. SOC. 79,986-970. Dauben, W. G., Fonken, G. J., and Boswell, G. A. (1957b). J. Am. Chem. SOC. 79, 1000-1001.

64

D. PERLMAN, A R I S P. BAYAN, AND NANCY A. GNFFRE

Djerassi, C., Seidel, P. C., Westley, J., Birch, A. J., Holzapfel, C. W., Rickads, R. W., Dutcher, J. D., and Thomas, R. (1962). Abstr. 142nd Meeting Atlantic City, New Jersey, Am. Chem. SOC. p. 5P. Doerschuk, A. P., McCormick, J. R. D., Goodman, J. J., Szumski, S. A., Growich, J. A., Miller, P. A., Bitler, B. A,, Jensen, E. R., Petty, M. A., and Phelps, A. S. (1956). J. Am. Chem. SOC. 78, 1508-1509. Doerschuk, A. P.,McCormick, J. R. D., Goodman, J. J., Szumski, S. A., Growich, J. A., Miller, P. A., Bitler, B. A., Jensen, E. R., Matrishin, M., Petty, M. A., and Phelps, A. S. (1959). J. Am. Chem. SOC. 81, 30693075. Donovick, R. (1956). Giom. Microbial. 2, 324. Dulaney, E. L., Putter, I., Drescher, D., Chaiet, L., Miller, W. J., Wolf, F. J., and Hendlin, D. ( 1962). Biochim. Bbphys. Acta 60, 447-449. Ehrensvard, G., Reis, L., Saluste, E., and Stjernholm, R. (1951). J. Bid. Chem. 189,93-108. Ellner, P. D. (1961). 1. Bacteriol. 82,275-283. Erb, W., and Maurer, W. (1960). Bwchem. 2. 332, 388-395. Evans, C. C., and Catch, J. R. (1956). Proc. Intern. Conf. Peaceful Uses of Atomic Energy, Geneva, 1955 14, 33-40. Foster, J. W., Carson, S. F., Ruben, S., and Kamen, M. D. ( 1941). Proc. Natl. Acad. Sci. U.S . 27,590-596. Foster, J. W., Carson, S . F., Anthony, D. S., Davis, J. B., Jefferson, W. E., and Long, M.V. (1949). Proc. Natl. Acad. Sci. U.S . 35, 663-672. Frantz, I. D., Feigelman, H., Werner, A. S., and Smythe, M. P. (1952). J. Biol. Chem. 195,432-438. Gander, J. E. (1960). Arch. Biochem. Biophys. 91,307-309. Gatenbeck, S. ( 1961). Blochem. Biophys. Res. Commun. 6, 422-426. Gibbs, M.,and Gastel, R. ( 1953). Arch. Biochem. Biophys. 43, 33-38. Gilvarg, C., and Bloch, K. ( 1951). J. Biol. Chem. 193, 339-346. Glover, J., and Shah, P. P. (1957). Biochem. J. 67,15P. Goodwin, T.W., and Horton, A. A. (1960). Biochem. J. 75, 53-57. Goodwin, T. W., and Jones, W. T. G. (1956). Biochem. J . 64, 9-13. Goodwin, T. W., and Passorn, P. N. A. (1961). Experientia 17, 221-222. Gordon, M.,Pan, S. C., Virgona, A,, and Numerof, P. (1953). Science 118, 43. Gordon, M., Numerof, P., and Pan, S. C. (1954). J . Am. Chem. SOC. 76, 4037-4038. Griesebach, H., Achenbach, H., and Griesebach, U. C. ( 1960a). Naturwissenschaften 47, 206. Griesebach, H., Achenbach, H., and Hofheinz, W. (1960b). 2. Nuturforsch. I%, 560-568. Guyman, J. F., Ingraham, J. L., and Crowell, E. A, (1961). Arch. Biochem. Biophys. 95,163-168. Halliday, W. J., and Amstein, H. R. V. (1956). Blochem. J. 64, 380-384. Hanahan, D. J., and al-WakiI, S. J. (1952). Arch. Blochem. Biophys. 37, 167-171. Hansson, E., and Blau, M. (1963). Biochem. Biophys. Res. Common. 13, 71-74.

PREPARATION OF RADIOACTIVE COMPOUNDS

65

Hayano, M., Gut, M., Dorfman, R. I., Sebek, 0. K., and Peterson, D. H. (1958). J . Am. Chem. SOC. 80, 2386-2387. Hayano, M., Gut, M., Dorfman, R. I., Schubert, A,, and Siebert, R. (1959). Biochim. Bwphys. Acta 32, 269-270. Hockenhull, D. J. D., and Faulds, W. F. (1955). Chem. &. Ind. (London) p. 1390. Hofmann, K., and Liu, T. Y. (1960). Biochim. Biophys. Acta 37, 364-365. Homer, W. H. (1964). J. Biol. Chem. 239, 578-581. Howell, J. F., Thayer, J. D., and Labaw, L. B. (1948). Science 107, 299300. Hunter, G. D. (1956). Giorn. Microbiol. 2, 312-324. Hunter, G. D., and Hockenhull, D. J. D. (1955). Biochem. 1. 59, 268-272. Hunter, G. D., Herbert, M., and Hockenhull, D. J. D. (1954). Biochem. J. 58, 249-254. Ingram, J. M., and Blackwood, A. C. (1962). Can. J . Microbiol. 8, 49-56. Jackson, M., Dulaney, E. L., Putter, I., Shafer, H. M., Wolf, F. J.. and Woodruff, H. B. (1962). Biochim. Biophys. Acta 62, 616-619. Kaneda, T., Butte, J. C., Taubman, S. B., and Corcoran, J. W. (1962). J . Biol. Chem. 237,322-328. Karow, E. O., Peck, R. L., Rosenblum, C., and Woodbury, D. T. (1952). J . Am. Chem. SOC.74, 3057-3059. Katz, E., and Weissbach, H. (1962). Biochem. Biophys. Res. Commun. 8, 186-190. Katz, E., Prockop, D. J., and Udenfriend, S. (1962). J . Biol. Chem. 237, 1585-1588. Kelly, C. G., Kanegas, L. A,, and Buyske, D. A. (1961). J . Pharm. Expt2. Therap. 134, 320-324. Kozinski, A. W. ( 1961). Virology 13, 377-378. Laufer, L., and Gutcho, S. (1955). U. S. Patent 2,711,989. Laufer, L., Gutcho, S., Castro, T., and Grenner, R. (1963). 145th Meeting New York, New York, Am. Chem. SOC.p. 1P. Lilly, V. G., Barnett, H. L., Krause, R. F., and Lotspeich, F. J. (1958). Mycologia 50, 862-873. Lilly, V. G., Barnett, H. L., and Krause, R. F. (1960). West Virginia Agr. Expt. Sta. Bull. 441T, p. 61-76. Lotspeich, F. J., Krause, R. F., Lilly, V. G., and Barnett, H. L. (1963). Proc. SOC. Exptl. Biol. Med. 114, 444-447. Lunan, K. D., and West, C. A. (1963). Arch. Biochem. Biophys. 101, 261268. Maass, E. A., and Johnson, M. J. (1949a). 3. Bacteriol. 57, 415-422. Maass, E. A,, and Johnson, M. J. (19498). J . Bacterdol. 58, 361-306. McConnell, K. P., Mautner, H. G., and Leddicotte, G. W. (1962). Biochim. Biophys. Acta 59, 217-218. MacDonald, J. C. (1960). Can. J . Microbiol. 6, 27-34. MacDonald, J. C. (1961). J . Bwl. Chem. 236, 512-514. McNutt, W. S. (1956). J . Biol. Chem. 219, 365-373. Majumdar, S. K., and Kutzner, H. J. (1962). Appl. Microbiol. 10, 157-168. Maley, G. F., and Plaut, G. W. E. (1959a). J . Biol. Chem. 234, 614-647.

66

D. PERLMAN, ARIS P. BAYAN, AND NANCY A. GIUFFRE

Maley, G. F., and Plaut, G. W. E. (1959b). J. Am. Chem. Soc. 81, 2025. Marmur, J., and Schildkraut, C. L. (1961). Nature 189, 636-638. Marr, A. G., and Wilson, J. B. (1951). Arch. Biochem. Biophys. 34, 442448. Martin, E., Berky, J., Godzesky, C., Miller, P., Tome, J., and Stone, R. W. (1953). J . Biol. Chem. 203, 239-250. Miller, P. A,, McCormick, J. R. D., an 1 Doerschuk, A. p. ( 1956). Science 123, 1030. Minor, F. W., Greathouse, G. A., Shirk, H. G., Schwartz, A. M., and Harris, M. (1954a). J . Am. Chem. SOC. 76, 1658-1661. Minor, F. W., Greathouse, G. A., Shirk, H. G., Schwartz, A. M., and Harris, M. (1954b). J. Am. Chem. SOC. 76, 5052-5054. Mohberg, J., and Johnson, M. J. (1958). J. Bacteriol. 76, 385-392. Moldave, K. (1960). Biochim. Biophys. A d a 43, 188-196. Mollin, D. L., and Baker, S. J. (1955). Biochem. SOC. Symp. (Cambridge, End.) 13, 52-58. Morgan, G. B. (1961). Quart. J . Florida Acad. Sci. 24, 94-100. Mosbach, E. H. (1952). Arch. Biochem. Biophys. 35, 435-441. Moses, V., and Calvin, M. (1958). Arch. Biochem. Biophys. 78, 598-600. Moses, V., and Calvin, M. (1959). Biochim. Biophys. Acta 33, 297-312. Murray, A., and Williams, D. L. (1958). “Organic Syntheses with Isotopes.” Wiley (Interscience), New York. Nara, T., and Johnson, M. J. (1959). J. Bacteriol. 77, 217-226. Nathorst-Westfelt, L., Schatz, B.-A., and Thelin, H. (1963). Acta Chem. Scand. 17, 1164-1165. Nuclear-Chicago Corp. ( 1964). Radiochemical Bulletin No. 9. Numerof, P., Gordon, M., Virgona, A., and O’Brien, E. (1954). J. Am. Chem. SOC. 76, 1341-1344. Ober, R. E., Fusari, S. A., Coffey, G. L., Gwynn, G. W., and Glazko, A. J. (1962a). Atom Light 22 (New England Nuclear Corp., Boston). Ober, R. E., Fusari, S. A., Coffey, G. L., Gwynn, G. W., and Glazko, A. J. ( 1962b). Nature 193, 1289-1290. Perret, C. J. (1953). J. Gen. Microbial. 8, 195-196. Peyser, P. ( 1963). Personal communication. Plaut, G. W. E. (1954a). J. Biol. Chem. 208, 513-519. Plaut, G. W. E. (1954b). J. B i d . Chem. 211, 111-116. Plaut, G. W. E. (1960). J. Biol. Chem. 235, PC41-PC42. Plaut, G. W. E., and Broberg, P. L. (1956). J . Biol. Chem. 219, 131-138. Richards, J. H., and Ferretti, L. D. (1960). Biochem. Biophys. Res. Commun. 2, 107-110. Rinehart, K. L. (1964). “Chemistry of the Neomycins and Related Antibiotics.” Wiley, New York. Rosenblum, C. ( 1959). Nucleonics 17, 80-83. Rosenblum, C. H. (1962). In “Vitamin B,, und Intrinsic Faktor,” 2. Europaisches Symposion Hamburg ( H. C. Heinrich, ed. ), pp. 294-305. Enke, Stuttgart. Rosenblum, C., and Woodbury, D. T. (1951). Science 113, 215.

PREPARATION OF RADIOACTIVE COMPOUNDS

67

Rowley, D., Miller, J., Rowlands, S., and Smith, E. L. (1948). Nature 161, 1009-1010. Rowley, D., Cooper, P. D., Roberts, R. W., and Smith, E. L. (1950). Biochem. 1. 46, 157-161. Santer, U. V., and Vogel, H. J. (1956). Biochim. Biophys. Actu 19, 578-579. Saunders, A. P., and Sylvester, J, C . (1957). Appl. Microbid. 5, 149-151. Schepartz, S. A., and Johnson, M. J. (1956). 1. Bucteriol. 71, 84-90. Schieler, L., McClure, L. E., and Dunn, M. S. (1953). J . Biol. Chem. 203, 1039-1044. Schliissel, H. (1951). Biochem. 2. 321, 421-425. Schliissel, H., and Fenendegen, L. ( 1951). Biochem. Z . 321, 533-537. Schliissel, H., Maurer, W., Hock, A., and Hummel, 0. (1951). Biochem. 2. 322, 226-229. Schwartz, S., Ikeda, K., Miller, I. A., and Watso, C. J. (1958). Science 129, 40-41. Schwarz BioResearch, Inc. (1963). SBR Tech. Brochure 64D1. Schwenk, E., Alexander, G. J., Stoudt, T. H., and Fish, C. A. (1955). Arch. Biochem. Biophys. 55, 274-285. Schwenk, E., Alexander, G. J., Gold, A. M., and Stevens, D. F. (1958). J . B i d . Chem. 233, 1211-1213, Scully, N. J., Stavely, H. E., Skon, J., Stanley, A. R., Dale, J. F., Craig, J. T., Hodge, E. B., Chomey, W., Watanbe, R., and Baldwin, R. (1952). Science 116, 87-89. Sebek, 0. K. (1953). Proc. SOC. Exptl. Biol. Med. 84, 170-172. Sebek, 0. K. (1955). Arch. Biochem. Biophys. 57, 71-79. Segal, S., and Topper, Y. J. (1957). Biochim. Biophys. Acta 25, 419-420. Segel, I. H., and Johnson, M. J. (1961). J . Bacteriol. 81, 91-98. Shemin, D., Corcoran, J. W., Rosenblum, C., and Miller, I. M. (1956). Science 124, 272. Sivak, A., Meloni, M. L., Nobili, F., and Katz, E. (1962). Biochim. Biophys. Actu 57, 283-295. Smith, E. L. (1952). Brit. Med. Bull. 8, 203-205. Smith, E. L. (1956). Brit. Med. Bull. 12, 52-56. Smith, E. L., and Allison, D. (1952). Analyst 77, 29-33. Smith, E. L., and Hockenhull, D. J. D. (1952). J . Appl. Chem. 2, 287-288. Smith, E. L., Hockenhull, D. J. D., and Quilter, A. K. J. (1952). Biochem. 1. 52, 387-388. Smith, E. L. (1959). Lancet I, 387. Smith, G. N., and Marshall, R. 0. (1952). Arch. Biochem. Biophys. 39, 395-405. Smith, R. L., Bungay, H. R., and Pittenger, R. C. (1962). Appl. Microbid. 10, 293-296. Snell, J. F. (1958). U. S. Patent 2,843,526. Snell, J. F. (1960). In “Radioactivity for Pharmaceutical and Allied Research Laboratories” (A. Edelman, ed.), pp. 113-135. Academic Press, New York. Snell, J. F., Wagner, R. L., and Hochstein, F. A. (1955). Proc. Intern. Conf. Peaceful Uses of Atomic Energy, Geneva, 1955 12, 431-434.

68

D. PERLMAN, A R I S P. BAYAN, AND NANCY A. GIUFFRE

Snell, J. F., Birch, A. J., and Thomson, P. L. ( 1 9 0 ) . J. Am. Chem. SOC. 82, 2402. Snoke, J. E. (1960). J. Bacteriol. 80, 552-557. Stevens, C. M., Vohra, P., Inamine, E., and Roholt, 0. A. (1953). J. B i d . Chem. 205, 1001-1006. Stevens, C. M., Inamine, E.,and DeLong, C. W. (1956). J. Bid. Chem. 219, 405-409. Stonier, T. (1956). J . Bacterwl. 72, 259-268. Stonier, T. (1960). J. Bacteriol. 79, 880-888. Strange, L., Kirk, M., Bennett, E. L., and Calvin, M. (1962). Biochim. Biophys. A C ~ 61, U 881-695. Suzuki, I., and Werkman, C. H. (1958). Arch. Biochem. Biophys. 77, 112123. Tanenbaum, S . W,. and Bassett, E. W. (1959). J. B i d . Chem. 234, 18611866. Tarver, H., Tabachnick, M., Canellakis, E. S., Fraser, D., and Barker, H. A. (1952). Arch. Biochem. Biophys. 41, 1-8. Taubman, S. B., Young, F. E., and Corcoran, J. W. (1963). Proc. N,utl. Acad. Sci. U.S . 50, 955-967. Thomas, R. (1961). Biochem. J. 78, 748-758. Thomas, S. L., and Turner, H. S . (1953). Quart. Reu. Chem. SOC. 7, 407443. Toohey, J. I., and Barker, H. A. (1961). J. Biol. Chem. 236, 560-563. Trown, P. W., Abraham, E. P., Newton, G. G. F., Hale, C. W., and Miller, G. A. (1962). Bwchem. J. 84, 157-166. Tuve, T., and Williams, H. H. (1961). J . B i d . Chem. 236, 597-601. Vining, L. C., and Taber, W. A. (1963). Can. J. Microbwl. 9, 291-302. Wada, T. (1957). J. Cen. Appl. Mbrobiol. 3, 137-145. Wada, T., Nomura, N., Mitsui, H., Marvo, B., Tamiya, H., and Akabori, S . (1955). J. Cen. Appl. Microblol. 1, 142-150. Weinberger, P., and Porter, J. W. (1954). Arch. Biochem. Biophys. 50, 160-165. Winnick, R. E., Lis, H., and Winnick, T. (1961). Biochim. Biophys. Actu 49, 451-462. Winstead, J. A., and Suhadolnik, R. J. (1960). J . Am. Chem. SOC. 82, 16441647. Wolochow, H., Putnam, E. W., Duodoroff, M., Hassid, W. Z., and Barker, H. A. (1949). J. B i d . Chem. 180, 1237-1242. Wood, J. L., and Mills, G. S. (1952). J. Am. Chem. SOC. 74, 2445. Wood, J. L., and Perkinson, J. D. (1952). J . Am. Chem. SOC.74, 2444-2445. Zaprometov, M. N. (1961). Biokhimiyu 26, 597-602. Zweig, G., and DeVay, J. E. (1959). Mycologia 51, 877-886. Note added in proof: After this manuscript was completed, a conference sponsored by EURATOM was held (Nov. 13, 1963) on “Methods of Preparing and Storing Marked Molecules” (Presses AcadBmiques EuropBennes, 1964). Among the products prepared by microbiological processes are: fl-hydroxybutyrate, yeast ribonucleic acids, actinomycins, gibberellic acid, galactose, and glycerol.

Secondary Factors in Fermentation Processes P. MARGALITH Laboratory of Microbiology, Department of Food and Biotechnology, Technion-Israel Institute of Technology, Haifa, Israel I. Introduction ........................................... 11. Inorganics ............................................. A. pH .............................................. B. Sulfites ........................................... C. Halogens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Cations ........................................... E. Cyanides .......................................... 111. Organics .............................................. A. Ionones ........................................... B. Barbiturates ....................................... IV. Various ............................................... V. Conclusion ............................................ References ............................................

69 71 71 72 74 78 79 81 81 84 85 88 88

1. Introduction The successful outcome of a fermentation process is in most cases conditioned primarily by factors that influence the growth of the organism concerned with the particular process. The propagation of yeast for food or fodder is a typical case in which optimal conditions for the reproduction of the yeast cell are obligatory for the process at large (White, 1954). In other cases where the industrial interest lies more in the production of a certain metabolite, it is rather the suboptimal conditions for growth and reproduction, and not the optimal conditions as expressed by dry-weight determination, that have to be provided in order to secure the maximum yield of the desired metabolite. Of the many examples available it is enough to mention the citric acid fermentation or the production of penicillin. These processes are discussed by various authors (e.g., Johnson, 1954; Sylvester and Coghill, 1954). However, all these factors may be classified as primury, since they are chiefly concerned with the propagation of the organism. Nutritional requirements such as carbon sources, both for energy and assimilation, nitrogen sources for protein metabolism, minerals, and, if necessary, vitamins are essential for the growth and reproduction of the organism. The intensive propagation of microbial 69

70

P. MARGALITH

cells for industrial purposes has created the necessity of also supplying air in large amounts for the majority of fermentation processes, making oxygen a primary factor with all its engineering and economic implications (Finn, 1954; Gaden, 1960; Richards, 1961). NO doubt, a microbial process is chiefly governed by these primary factors, and when a new product is evaluated it is these that are the first to be controlled, with a view of both the scientific and economic aspects. The category of primary factors may, however, be further extended. Raw materials for microbial processes in the food industry, such as milk, cucumbers, or olives for their respective lactic fermentation, should be looked upon in a similar way (Judkins and Keener, 1960; Cruess, 1958). On the other hand in the fermentative production of pharmaceuticals, precursors for a specific product such as phenylacetic acid for the formation of the pencillin G molecule by Penicillium chrysogenum ( Singh and Johnson, 1948), or various steroid compounds to be transformed by microbial processes (for a recent review see Stoudt, 1960) should be also considered primary factors. All these substrates for microbial activity have been dealt with in many reviews and will not be considered here further. Moreover, even minor amounts of a nutrient such as halides, not necessary for the reproduction and growth of microorganisms but essential for the biosynthesis of halogen-containing metabolites, e.g., chloramphenicol, chlortetracycline, or griseofulvin, should be included in the category of primary factors since their presence in the nutrient medium is obviously necessary. In their absence dehalogenated metabolites will accumulate. For a very thorough review on halogenation in microbial systems see Petty (1961). However, there are quite a number of factors which affect the outcome of many fermentation processes, that cannot be included in this category because their exact identity is not known sufficiently or cannot be considered nutrients or precursors in the strict sense. It is this group of secondary factors we are chiefly concerned with in this review. Thus, secondary factors in microbial processes may be defined as those chemical or physical parameters that change completely or only in part the outcome of a fermentation process, not by increasing this or that nutrient or trace element in the medium, but by introducing a factor not essential for the growth of the organism itself nor as a precursor for the respective metabolite. Such a factor

SECONDARY FACTORS IN FERMENTATION PROCESSES

71

would influence the process by steering the microbial activity toward the desired product.

II. lnorganics One of the early cases in which the shift of a metabolic process by an external factor has been recorded is undoubtedly the historical observation by Pasteur (1861) in which the presence of an excess of oxygen together with a limited concentration of sugar in the medium would avert the alcoholic fermentation of yeast. Under these conditions the assimilation of carbohydrates was promoted with a concomitant reduction of the alcoholic fermentation to a minimum. This principle, today widely known as the Pasteur effect, has had a pronounced impetus on the development of the yeast industry. Almost all modern plants employ the “Zulaufverfahren” or “exponential feeding” process for the commercial production of compressed yeast. The reader is referred to the many textbooks dealing with this problem (e.g., Walter, 1953; White, 1954). It is to be pointed out, however, that the abundant oxygen supply, although suppressing the fermenting capacity of Sacchuromgces cerevisiae, actually restores the normal reproductive power of the organism to that of a nonfermenting ( i n sen= stricto) yeast or hyphal fungus, where oxygen as a normal growth promoter would be considered a primary factor.

A. pH Hydrogen ion concentration may be considered a secondary factor only in those cases where the biosynthesis of the specific metabolite is promoted at certain pH levels, but not at others at which the propagation of the microorganism as such will still be optimal. In contrast to bacteria and actinomycetes, hyphal fungi and yeasts are relatively more able to invade acid environment. In a compilation by Cochrane (1958) of pH optima of various fungi, in many cases, values as low as 4.0 and even less have been recorded. On the other hand the production of penicillin by Penicillium chrysogenum has been shown to take place primarily at pH levels between 6.8 and 7.8, although optimal mycelial growth could be obtained at pH values as low as 3.5 when controlled by means of an automatic device (Johnson, 1952). Hydrogen ion concentration in industrial operations is usually controlled by the supply of less utilizable

72

P. MARGALITH

carbon sources, e.g., dextrin or lactose, suitable nitrogen sources, e.g., corn steep liquor, and calcium carbonate, leading to a rise in pH values after about 24 hours when good mycelial growth has already taken place. The determination of the pathway leading toward the optimal biogenesis of penicillin through the control of pH is therefore a typical case where a secondary factor governs, among others, the result of the process.

B. SULFITES Studies on the mechanism of the alcoholic fermentation have yielded many basic contributions to microbial chemistry with farreaching implications in the field of general biochemistry. The understanding of the major steps of the glycolycis of sugars made early workers aware of the potentialities involved in the artificial diversion of the normal alcoholic fermentation with a view to obtaining intermediates usually not present, or only in minor amounts, in the medium of a fermenting yeast. As early as 1911 Neuberger and co-workers discovered the fact that, by the addition of sulfites to an alcoholic fermentation by yeast, acetaldehyde, the normal intermediate, is fixed by the bisulfite ion, thus interrupting the enzymic reactions leading to the formation of ethanol. The enzyme system that would normally reduce acetaldehyde to ethyl alcohol would now reduce dihydroxyacetone to glycerol, yielding in addition to glycerol, acetaldehyde, carbon dioxide, and minor amounts of ethanol (Neuberg and Reinfurth, 1918). The interest in a biotechnological process for the production of glycerol along these lines (Neuberg’s second form of fermentation) increased enormously on the eve of World War I when the demand for large supplies of glycerol for the war effort became critical. The first realization of Neuberg’s findings as a commercial process was attempted by Connstein and Ludecke (1919). In spite of the production of over 1000 tons of glycerol per month in a plant set up under wartime conditions, both the biotechnological as well as the engineering aspects of the process were rather poor and production could not compete with peacetime glycerol from other sources. During World War I1 and thereafter there was again considerable interest in the microbial process. This was also because of the impact of the synthetic detergent industry upon soap production. However, the production of glycerol was probably never realized in a modern well-designed unit. The actuality of the

SECONDARY FACTORS IN FERMENTATION PROCESSES

73

biotechnological process is apparently still there, because of great technological improvements to be expected when the production can be run on a continuous line, overcoming some of the major difficulties in purification of the product, employing an ion-exchange technique, or rather because of strategical reasons (Harris and Hajny, 1960). The reader is referred to the basic article by Underkoffler (1954) and, more recently, to the article by Freeman and Donald ( 1957a) for further information concerning various developments of the fundamental process, raw materials, and yields. The reaction between the bisulfite ion and acetaldehyde of the fermentation process has been studied rather intensively. It is now generally assumed that a complex is formed between the bisulfite ion and acetaldehyde to yield a-hydroxysulfonic acid ( Sheppard and Bourns, 1954): OH CH,*GH

+ HS0,-

--*

I

CH,*CSO,-

I H It is clear that this reaction will be most effective under pH conditions that will allow the maximum dissociation of the bisulfite ion. This takes place at pH values around 6.7-7.0. Under these conditions the undissociated part of the sulfite molecule is very small, causing very little inhibition of yeast activity [see Hirsch (1956) for an appraisal of dissociation values of antimicrobial agents], Interestingly, according to a recent study by Freeman and Donald ( 1957b), minimum toxicity of SOz (0.374, in peptone-glucose broth) was between 8.0-8.5, when expressed as percentage of survivals. It should be further pointed out that the diversion of the alcoholic fermentation is carried out by the stoichiometric fixation of the acetaldehyde intermediate, hence large amounts of sulfite have to be employed for a successful run. Maximum yields of glycerol were obtained when about twice as much sodium sulfite as sugar were employed. Working with a 20% solution of fermentable sugar at a sulfite dosage of 40%, a concentration of 80,000 p.p,m. of both free and bound SO2 would be present in the medium. This is probably a unique case, where such a high concentration of an antiseptic is employed in a biotechnological process (Freeman and Donald, 1957a).

74

P. MARGALITH

Whether the microbial process of glycerol production will again become of commercial interest, or will only retain its historical importance, cannot be decided at present. However, this is undoubtedly the first case in which a non-nutritive chemical was employed for the diversion of a metabolic pathway into an abnormal yet industrially desirable process. The production of glycerol through the chemical activities of yeast under conditions not suitable for alcoholic fermentation has also been attempted by other methods. Among others, maintaining very high p H levels by means of several additions of sodium carbonate to the fermenting mash led to fair yields of glycerol, acetic acid, and ethanol (according to Neuberg’s third form of fermentation) (see Eoff, 1918). The alkaline process for the production of glycerol from sugar may, therefore, be considered another characteristic case for the regulation of a fermentative process through p H adjustment, completely inadequate for the normal alcoholic fermentation by yeast (see Section, 11, A).

C. HALOGENS The effect of chloride ions on the metabolism of industrially important organisms has already been referred to in the introduction. In the greater majority of cases the halogen would be included in the category of primary factors when incorporated into the halogencontaining metabolite. However, chlorine appears to play no nutritional role in the metabolism of fungi (Foster, 1949) and probably not in that of the bacteria, although this has still to be established. In the absence of chloride ions, the biosynthesis of chlorine-containing metabolites can no longer take place. Usually this results in the production of a dechlorinated analog. The deliberate denial of chlorides should not be considered critical unless precautions have been taken to eliminate all chloride ions in the media supplied by tap water, corn steep liquor, or other ingredients, a point correctly emphasized by the reviewer on halogenation in microbial systems (Petty, 1961). In a chloride-free medium Streptomyces aureofaciens will produce tetracycline, the dehalo analog of chlortetracycline (Minieri et al., 1956).

SECONDARY FACTORS I N FERMENTATION PROCESSES

Chlortetracycline

75

Tetracycline

A similar course of events would take place in the fermentation of chloramphenicol by Streptomyces venezuelae, where dehalo analogs accumulate in the absence of chloride ions in the medium. m C H?H I CH- CH,OH I

p C ~ ?H- ~ ~ - ~ ~ , ~ ~ 2 OzN'

I CHC1,

Chloramphenicol

+

I

I

NH C=O I CH.9

N - Ac etyl-p- ni t rophenyl se r inol

Smith ( 1958) showed that by growing a chloramphenicol-producing strain in a medium devoid of chloride, in addition to the dehalo analog of chloramphenicol (N-acetyl-p-nitrophenylserinol ), other derivatives of p-nitrophenylserinol, such as N-propionyl and N butyryl, would accumulate. It was further found that in the absence of chloride ions, the culture was capable of producing acyl derivatives of p-nitrophenylserinol in amounts equivalent to 400 yg./ml. chloramphenicol as assayed chemically. Upon the addition of chloride to the medium, the production of N-acylated derivatives was blocked and only ,the halogen antibiotic was synthesized in yields of 150 yg./ml. These observations indicate that chloroacetylation of p-nitrophenylserinol led to the production of a compound which inhibited maximum synthesis of other p-nitrophenyl compounds. If not, one would expect the same amount of p-nitrophenyl compounds to be synthesized both in the presence and in the absence of chlorides, with a shift in ratio of chlorinated to simple acylated derivatives. However, one actually observes a sharp depression in the amount of total p-nitrophenylserinol groups synthesized and a shift from several acylated compounds to chloramphenicol.

76

P. MARGALITH

These studies throw some light upon the mode of action of the halogen steering factor upon the biosynthesis of chloramphenicol. However, it would be interesting to learn more about the reason for chlorination not proceeding until all the antibiotic precursor is exhausted, without suppressing the synthetic capacity of the organism. Further studies with higher yielding strains may supply the required answer. Another typical case in which the secondary effect of chloride ions could be demonstrated was the fermentation of griseofulvin. When Penicillium brefeldianum was grown on a chloride-poor medium (Raulin and Thom) no dehalo analog of griseofulvin was produced, but instead fulvic acid was synthesized. OCHa 0

OCH,

CH, 0+kc;=c;=o0/ HC-CH, \I CI

CH,

Griseofulvin

HO HOOC

0

Fulvic acid

According to Dean et al. (1957), fulvic acid cannot be readily related to the biochemical steps involved in the production of griseofulvin. It seems, therefore, that in this case not only is the chloride ion incorporated into the antibiotic molecule, but also its presence in the nutrient medium shifts the metabolic pathway toward a completely different goal. Moreover, it was pointed out that variations in the environment of the fungus had a more profound effect on its metabolism than have variations in the species. In this case, therefore, the halogen ion assumes the role both of a primary as well as a secondary factor. In connection with halogen metabolism the secondary effect of certain compounds influencing the halogenation of the tetracycline molecule should also be mentioned. Because of economic consideration, many attempts have been made to suppress halogenation without inhibiting the biosynthesis of the dehalogen analog. The most interesting results were obtained by Lein et al. (1959),who showed that in the presence of some thio organics good yields of tetracycline were obtained employing chlorinating strains of S . aureofaciens. In the case of 2-benzoxazo1ethio1,

SECONDARY FACTORS IN FERMENTATION PROCESSES

77

-

2 Benzoxazole thiol

at a level of less than 10 pg./ml., over 90% tetracycline at a total yield of over 3000pg./ml. was obtained. It is not known whether this discovery is being actually used for industrial purposes since nonchlorinating strains obtained by genetic manipulations are also available (Kollir and Jarhi, 1960). However, the antihalogenating activity of these compounds should be considered in view of the general approach of secondary factors in fermentation processes. Halogenation is by no means limited to the incorporation of chloride ions. Although iodo and fluoro metabolites are usually not found in the plant kingdom, attempts to brominate acylated p-nitrophenylserinol (Smith, 1958) or tetracycline (e.g., see Sensi et al., 1955) via microbial halogenation have been successful. It is felt, however, that this is no longer within the scope of this review; the reader is again referred to the excellent survey by Petty (1961). Incidentally, little has been done so far on the mode of action of the halogenating enzymes. There are only few data (Goodman et al., 1959) pointing toward the possibility that copper-containing oxidases are necessary for the halogenating system. An interesting case in which the secondary effect of chlorides upon the nature of a fermentation process not concerned with the production of a halogen-containing metabolite has been described by Oxford and co-workers (1949). These workers showed that in the presence of relatively high concentrations (about 0.5 N ) of chlorides, such as sodium, magnesium, or potassium chloride, the formation of oxalic acid in the fermentation of citric acid by a strain of Aspergillus niger, in a medium containing cane molasses, could be almost entirely inhibited. This effect was more pronounced when working with media at a relatively low hydrogen ion concentration. Since there is no evidence for the requirement of the chloride ion for the growth of the mold, it may be concluded that some enzyme system concerned with the formation of oxalic acid may be specifically inhibited. No doubt the observation by early workers (e.g., Currie, 1917) that the initial pH adjustment with hydrochloric acid had a favorable effect on citric acid production may be related to the same effect.

78

P. MARGALITH

D. CATIONS Very interesting data on the influence of a secondary factor in a fermentation process have been submitted by Brack (1947). Working on the metabolites of the fungus Penicillium patulum it was found that the presence of certain cations affects not only quantitativeIy but also qualitatively the accumulation of fermentation products. In a simple chemically defined medium containing a source of carbon, nitrogen, and minerals, in the absence, or with only traces of iron, both gentisyl alcohol and patulin are synthesized. When the amount of iron was increased, the synthesis of gentisyl alcohol was suppressed with a simultaneous promotion of patulin formation. A complete reversal of this ratio was effected by the addition of zinc ions, when gentisyl alcohol was synthesized predominantly. With manganese the formation of gentisyl alcohol was again depressed in favor of patulin, although the total yield was somewhat lower (see Table I, compiled from Brack, 1947).

OH Gentisyl alcohol

Patulin

In spite of the fluctuations of the dry material obtained in the different media, it is obvious that no correlation exists between the extent of growth and the metabolites formed. Neither of the two products contain iron or zinc so that the explanation of this shunt metabolism must be confined to enzymic grounds. It is possible that the presence of some of the cations is necessary for the oxidative ring opening prior to patulin formation, which may be inhibited by other ions without interfering with the general pathways leading to the synthesis of this group of metabolites (Bu’Lock, 1961). An alternative explanation would be the inhibition of the enzyme transforming 6-methylsalicylic acid to 6-formylsalicylic acid, which leads to the accumulation of gentisaldehyde or gentisic acid to be later reduced to the alcohol (Bassett and Tanebaum, 1958).

79

SECONDARY FACTORS IN FERMENTATION PROCESSES

TABLE I BIOSYNTHESIS OF GENTISYL ALCOHOL AND PATULIN BY P . patulum Gentisyl alcohol Dry weight (mg./ (mg./100 ml.) liter)

Cation added6 Fe+ f ( 10-4 M ) Fe++ (10-3 M ) Fe++ ( 2 x lO-3M) F e + + (10-4 M ) Zn++ ( 10-7-10-5 M ) F e + + (10-4 M ) Mn++ ( 10-7-10-5 M )

+ +

250

Patulin (mgJ liter)

-

1280 420 90

200 700 1140

300-600

2000

50

190-230

20

800

c

G/P 6.4 0.6 0.08 40.0 0.025

a The basal medium contained (gm./liter): glucose, 40; NaNO,, KH2P04, 1; KCI, 0.5; and MgSO4.7H,O, 0.5 in doubly distilled water.

3;

E. CYANIDES Although the production of citric acid by fermentation processes has been known since the eve of the current century (Wehmer, 1893), much work has been done in recent years to develop more economic procedures, such as the application of the now wellestablished technique of submerged culture, for the industrial fermentation of citric acid (Schweiger and SnelI, 1949). Great efforts have also been devoted to improvements in the balance of cationic constituents in the medium for the minimal formation of mycelial felts and maximum synthesis of the desired product. It has now been well established that the accumulation of citric acid occurs as a result of deficiencies in one or more of the essential nutritive elements, above all ferric and zinc ions. For a critical review on the conflicting statements concerning the optimal levels of certain cations see Underkoffler (1954). Considerable work has been directed toward the use of lowprice carbohydrate sources for the production of citric acid. Among the cheapest sources of sugar on the world market are the various types of molasses such as blackstrap and beet molasses. However, in working with this type of raw material very low yields of citric acid have been encountered. Attempts to purify these molasses by a ferrocyanide treatment were reported by various workers (e.g., Gerhardt et aZ., 1946; Bernhauer et al., 1949) immediately after World War I1 when cystalline sugar was still considered too expen-

80

P. MARGALITH

sive for the production of citric acid. These studies resulted in remarkable improvements in the yields of citric acid, when media containing molasses were treated after sterilization with ferrocyanide at a level of 0.4-0.8 gm./liter. Martin (1955) devoted considerable efforts to the elucidation of the nature of the ferrocyanide effect. Working with a simple chemically defined medium containing minute amounts of ferric and zinc ions (0.018 mM), considered suboptimal for the propagation of A. niger and citric acid production, it was found that the growth process of the mold was extremely sensitive to the presence of ferrocyanide, whereas the sequence of reactions leading to acid synthesis was relatively insensitive. The breakdown of ferrocyanide to cyanide could not be the cause of the inhibition since 100 times as much cyanide was required to obtain a similar effect using NaCN. On the other hand, ferrocyanide at extremely low levels caused both a stimulation of growth and acid production. Since the medium contained suboptimal concentrations of trace elements, the effect of ferrocyanide could not be explained on the basis of metal removal. Optimal concentrations of ferric and zinc ions for growth and production with the strain and medium employed in this research were considered 0.09 mM. It was therefore postulated that the stimulatory effect of ferrocyanide in this case was due to the inhibition of isocitric acid dehydrogenase, thus promoting the accumulation of citric acid in the medium [see Ramarkrishan and Martin (1955) for the biochemistry of citric acid formation]. This enzyme was found to be sensitive to ferrocyanide but not to cyanide. However, in the case of media containing molasses much higher concentrations of ferrocyanide had to be used in order to obtain a similar stimulating effect. It was, therefore, suggested that ferrocyanide operated through the following mechanisms: ( 1) removing excess of cations that favor growth but not production ( Shu and Johnson, 1948) and ( 2 ) exerting the inhibitory action upon the isocitric acid dehydrogenase enzyme. High levels of residual ferrocyanide in the treated molasses medium will not suppress growth as long as the pH is maintained around neutrality where ferrocyanide toxicity is reduced to a minimum. With the production of acidity and fall of pH, ferrocyanide resumes its toxicity and inhibits further mycelial growth. Thus, ferrocyanide controls the fermentation process via its changing toxicity as a function of hydrogen ion concentration. This observation is very much reminis-

SECONDARY FACTORS IN FERMENTATION PROCESSES

81

cent of the antimicrobial activity of many compounds such as sulfite, benzoic acid, and propionic acid ( Hirsch, 1956). However, so far no data are available in literature on the effect of these antimicrobial agents on the production of citric acid. Although it has been shown that the effect of ferrocyanide upon the citric acid-producing aspergillus was not due to the liberation of cyanide, other studies (Chughtai and Walker, 1954) have indicated that under certain conditions the addition of cyanide also has a stimulatory effect upon this fermentation. However, it was suggested that this effect was due more to a general stimulation of physiological functions of the mold, as manifested in considerable increases in the consumption of sugars, production of mycelial felt, and formation of organic acids, and less to the activation of any specific process. It was postulated that in glucose cultures prepared with preformed felts of A. niger, cyanide promotes the tricarboxylic acid cycle by restricting more highly oxidative side reactions. It is not known whether the addition of cyanide to the fermentation of citric acid has been adopted in commercial usage.

Ill. Organics A. IONONES The production of carotenoids by microbial processes has recently received considerable attention by a group of workers of the Northern Utilization Research and Development Division, Peoria. In a number of papers published during the last 6 years, numerous data have been made available to the public concerning the production of fl-carotene by various organisms, members of the Choanopheraceae. It has been shown that by the propagation of plus and minus mating types of the same species, and in some cases also by interspecific mating, increased yields of 0-carotene were obtained (Hesseltine and Anderson, 1957; Anderson et al., 1958). Crosses such as Blakesteea trispow NRRL 2566( +) X NRRL 2561(-) gave particularly high yields. Incidentally, this is one of the few cases known in literature where the concept of heterothallism has been applied successfully to a production process. So far, however, little is known of the nature and mode of action of this stimulating phenomenon. It was further shown (Anderson et ul., 1958) that by adding p-ionone (O.l%, addition after 2 days) to the fermenting medium,

82

P. MARGALITH

which contained, in addition to the basal nutrilites, vegetable oil and a surface active agent, the yield of 0-carotene in the mycelium of a mated culture of B. trispma was raised from 6,5mg./lOOml. in the control medium to 12.9 mg./100 ml. These results were further improved in more recent work by the incorporation of kerosene into the ionone-containing medium, when such high yields as 86 mg./100 ml. or 17 mg./gm. dry fermentation solids were obtained (Ciegler et al., 1962,). The effect of p-ionone on carotenogenesis has been described by MacKinney et al. (1952). This group of workers was chiefly concerned with the metabolic pathways leading to the biosynthesis of polyenes. Employing the stationary culture of Phycomyces bhkesleeunus, three- to fourfold increases in carotene were obtained. On the other hand, in the presence of methyl heptenone, an open-ring analog of 0-ionone, at a similar concentration, a completely different spectrum of carotenoid biogenesis was formed. Whereas in the presence of 0-ionone almost 100% of the carotenoids were 0-carotene, when substituted with methyl heptenone, phytofluene, neurosporene, 5- and p-carotene accumulated in the mycelium, the prevalent fraction being, this time, phytofluene (MacKinney et al., 1954)) or phytoene (Nakayama et al., 1957). In another communication (MacKinney et ul., 1953), an attractive hypothesis was put forward regarding the part played by 0-ionone in the biosynthesis of 0-carotene. Since 0-ionone was effective in the stimulation of p-carotene production, while structurally similar compounds such as a-ionone, methyl heptenone, and citral were not, it was suggested that 0-ionone, or a greater part of its product. molecule, should be regarded as precursor to the This hypothesis was tested by Engel and co-workers (1953), employing p-ionone labeled at position 6 and 9 with carbon-14. Incubating P. blukeskeanus under similar conditions to those employed by MacKinney et a2. (1952) a fivefold stimulation of carotenogenesis was observed, but no radioactivity was detected in the 0-carotene isolated from the mold.

7% CH=CH-C=CH7

8

D

10

7% CH=CH-C=CH-CH=

11

CH,

p- Carotene

(%)

12

IS

14

15

83

SECONDARY FACTORS IN FERMENTATION PROCESSES

p-Ionone

Methyl heptenone

Citral

Coming back now to the fermentation work carried out by the Peoria group, it is very likely that in the case of B. trispora also, grown under submerged conditions, p-ionone does not promote the production of p-carotene via the incorporation of the preformed ring structure, although this still awaits confirmation. One might expect some differences since in the latter case (Ciegler et al., 1959) a-ionone was also effective, contrary to the observation of MacKinney et al. (1952) and Engel et d.(1953) with Phycmgces of the related Mucoraceae. @-Iononein the fermentation of (J-carotene should, therefore, be regarded as a steering factor concerned with some metabolic succession of enzymic reactions leading to the synthesis of p-carotene. In their earlier works, Hesseltine and Anderson (19571, and Anderson and co-workers (1958) state that 75 to 80% of the total carotenoids were p-carotene. However, in increasing the production because of the improvement of fermentation techniques by the incorporation of various oils, detergents, and (J-ionone, it was pointed out (Ciegler et al., 1959) that approximately 95% of the total carotenoids were all trans-p-carotene. Unfortunately, no data are given regarding the composition of the carotenoid mixture under the improved conditions of fermentation when p-ionone was omitted from the medium. Recalling the observation by MacKinney et a2. (1954) that, in the presence of methyl heptenone, p-carotene constituted only 32% of the total carotenoid mixture, it is possible that p-ionone antagonizes the effect of methyl heptenone. It should be pointed out that Birkinshaw and Morgan (1950) described the occurrence of methyl heptenone in several fungi such as Endoconidiophora coerulens and Endoconidiophora virescens. Sprecher (1961) demonstrated high concentrations of the ketone in the ether extracts of these ascomycetes. The possibility that this compound may appear also in other fungi should, therefore, not be excluded. In this case the effect of p-ionone could be explained by a general

84

P. MARGALITH

promotion in the biosynthetic activity of the polyene-synthesizing microorganism and a concomitant suppression of non-p-carotene carotenogenesis.

B. BARBITURATES

A new group of secondary factors that influence critically the outcome of an industrial fermentation process has been suggested by Margalith and Pagani (1961b) who worked on the production of rifomycin. The antibiotic principles produced by Streptomyces mediterranei in a conventional nutrient medium have been shown to be of a complex nature, differing both in antibiotic potency and toxicity. The production of these antibiotics was seriously handicapped since no reliable results on the antibiotic potencies of the mixture could be obtained, Control of the fermentation as well as extraction procedures were considered unsatisfactory. Of the five different antibiotic principles contained in the fermentation broth, fraction B seemed to be the most interesting (Sensi et al., 1960). However, the production of this fraction, which was only a minor constituent of the active mixture, had to be carried out by column fractional extraction or similar procedures. Obviously, such methods could not be suggested for production purposes. Several attempts were made in order to change the ratio between the different fractions. Genetic manipulations of the organism resulted in no improvement, Of over 3000 isolates tested, only in one case was there a change in the normal biosynthesis, yielding larger amounts of the less desirable fractions (Margalith and Pagani, 1961a). It was noted, however, that working with a Verona1 buffer to keep the pH from falling due to excessive acid production, the color of the broth assumed a rather characteristic pinkish red character. This was found to be due to the predominant production of fraction B. By maintaining the pH at a similar level employing other buffering agents, it could be established that the prevalent biosynthesis of the desired fraction was not due to the maintenance of the hydrogen ion concentration. Since no evidence could be put forward that the stimulative action was due to the incorporation of the added compound, the action of the barbiturate was considered to influence some metabolic pathways intimately concerned with the production of the antibiotic principles of the rifomycin complex. Further studies gave additional support to the hypothesis that the

SECONDARY FACTORS IN FERMENTATION PROCESSES

85

barbiturate had a selective effect on the formation of fraction B, this being realized by a general increase of the productivity capacity of the organism (see also Ferguson et al., 1957) and the concomitant suppression of most of the other components of the antibiotic complex. By comparing various derivatives of the barbituric nucleus it could be established that compounds of the general formula ( I )

(I)

were the most effective. The order of decreasing activity was: 5,5diethyl-, 5-methyl-, 5-ethyl-, and 5-phenyl-5-ethyl-barbituric acid. The nonsubstituted barbituric acid as well as other analogs, such as 5-amino, 5-nitro, or 5,5-dipropyl or 5,5-diallyl barbiturates, showed no stimulation of the synthesis of fraction B. Interestingly, 5,sdimethyl barbiturate stimulated the synthesis of fractions C-D. Many questions are still open concerning the mode of action of these barbiturates on the enzyme systems involved in the pathways leading to the biogenesis of the rifomycin antibiotics. Whatever their function may be, these compounds should be incorporated into the class of secondary factors in the fermentation of S. mediterranei.

IV. Various Going over the patent literature covering improvements on known fermentation practices, one is struck with the abundance of details published dealing with minor modifications of processes through the incorporation of all kinds of chemical compounds into the medium (e.g., Schweiger and Snell, 1949; Moyer, 1953; Takida and Tawara, 1955). It is very hard to distinguish between modifications dealing with substantial improvements and those that are patented in order to secure priority or other commercial interests. No doubt, many improvements claimed by various authors have given satisfactory results in the hands of the original workers, but, since these have not been confirmed by others, they are rarely

86

P. MARGALITH

employed in industry, although they continue to be cited in literature. Secondary factors, which must be still gathered under this rather unfortunate heading, are those that presumably are contained in various raw materials employed widely in the fermentation industry. The only thing we know about them is that they very favorably affect certain processes as viewed from the industrial yields obtained. In this respect they have much in common with the nonidentifiable growth factors from nutrition studies, e.g., Dam et al. (1959). One of the best examples available is the use of distillers solubles in the production of novobiocin (Hoeksema and Smith, 1961). This raw material, prepared from the stillage of whiskey distillations, has proved an excellent nutrient ingredient for the novobiocin streptomyces (Streptomyces niveus, Streptomyces sphericus), both from the economic and fermentative point of view. A peak yield of over 570 pg./ml. has been obtained when a medium consisting of practically only distillers solubles and Cerelose was used in comparison to the much lower yields obtained from a simple chemically defined medium supplemented with vitamins and amino acids similar to the gross composition of distillers solubles. In spite of extensive fractionation work no evidence for a specific stimulating factor could be demonstrated (Eble and Lager, 1961). I t is possible that increased knowledge concerning the metabolism of this industrially important streptomyces will lead to better understanding of the factors specifically concerned with the biosynthesis of this antibiotic. Incidentally, quite recently there have appeared in the literature some indications concerning a new group of growth factors present in distillers solubles tentatively named BI3 (e.g., Dansi et al., 1962), which may also have some influence on the fermentation of novobiocin. Similar observations have been made with other fermentation processes from the time of the extensive studies on the production of penicillin (Jarvis and Johnson, 1947) up to the more recent research on the biosynthesis of erythromycin ( Stark and Smith, 1961). One usually obtains much higher yields employing a natural “complex” medium in comparison to the chemically defined substrate. It is generally assumed that this difference is due to the fact that complex media support heavier growth. If so the use of chemically

SECONDARY FACTORS IN FERMENTATION PROCESSES

87

defined media, containing all known ingredients of the natural media, should yield similar results. This, indeed, is of little importance to the fermentation technologist as long as he gets good results, but is of considerable interest for the understanding of the biosynthetic machinery. So far, however, data available are not sufficient for the evaluation of natural products commonly employed in fermentation processes. It is also possible that one of the critical factors involved in the question of superiority of complex natural material is not the concentration of nutrilites but rather their availability and, what may count even more, the rate at which they are made available through the attack of microorganisms. The productivity of P. chysogenum in a lactose medium is a good example (Jarvis and Johnson, 1947). The fact that substantial improvements have been obtained in the yields of an antibiotic in a synthetic medium, because of the incorporation of a chemical at low concentrations, has, however, a direct bearing on the present discussion. Ferguson and co-workers (1957) have shown that the addition of certain derivatives of barbituric acid to a synthetic medium has increased the production of streptomycin by a factor of up to 4.5 over the control medium. Since these compounds could not be considered precursors of the antibiotic (see also Margalith and Pagani, 1961b) the increase, approaching yields in a complex medium, was considered to be effected through the intervention with some essential metabolic process which eventually determines the activity of the enzyme system concerned with the synthesis of the antibiotic. Hence, barbiturates would assume the role of some unknown compound( S ) present in the complex medium. An alternative explanation may be related to the observation that during the propagation of Streptomyces griseus (Ferguson et d.,1957) in a barbital medium, a mycelium more resistant to autolysis emerged. These workers suggested that if the enzyme system involved in the formation of the antibiotic is more stable within the cell the emergence of autolysisresistant hyphae might account for the higher results obtained in the chemically defined medium. It would be very desirable to obtain more information on the behavior of mycelium of other antibiotic-producing organisms with a view to comparing the outcome of fermentation processes in chemically defined and complex media.

88

P. MARGALITH

V. Conclusion Several cases in which the presence of a certain chemical factor in the nutrient medium substantially influences the fate of a fermentation process have been reviewed. Both organic and inorganic compounds have been shown to shift the normal metabolism of several biotechnological processes into a pathway leading to an increased or even predominant production of an industrially important metabolite. Interferences with normal metabolic patterns due to genetic manipulations have not been recorded, although it is realized that hereditary characters of a specific strain are of primary importance in fermentation work at large. No attempt has been made to cover all cases of shunt metabolism. Only those that have already shown to be, or potentially are, of practical importance have been recorded. Undoubtedly, future studies will reveal many more cases which have been already described in other disciples of microbiology and which, before long, wil be harnessed for industrial purposes. Except for occasional data (Mann et at., 1955) little is known of secondary factors that may influence processes such as the transformation of steroids or the production of enzymes, although considerable developments are to be expected in the near future.

REFERENCES Anderson, R. F., Arnold, M., Nelson, G. E. N., and Ciegler, A. (1958). J. Agr. Food Chem. 6, 543. Bassett, E., and Tanebaum, S. (1958). Experientin 14, 38. Bernhauer, K., Rauch, J., and Gross, G . (1949). Biochem. 2. 319, 499. Birkinshaw, J. H., and Morgan, E. N. (1950). Biocheh. J. 17, 55. Brack, A. (1947). Helv. Chim. Actu 30, 1. Bu’Lock, J. D. (1961). Adunn. Appl. Microbiol. 3, 283. Chughtai, I. D., and Walker, T. K. (1954). Biochern. J . 56, 484. Ciegler, A., Arnold, M., and Anderson, R. F. (1959). J. AppE. Microbial. 7, 98. Ciegler, A., Nelson, G. E. N., and Hau, H. H. (1962). J . Appl. Microbiol. 10, 132. Cochrane, V. W. (1958). “Physiology of Fungi,” p. 20. Wiley, New York. Connstein, W., and Ludecke, K. (1919). Ber. 52, 1385. Cruess, V. W. (1958). “Commercial Fruit and Vegetable Products,’’ pp. 708-733. McGraw-Hill, New York. Currie, J. N. (1917). J . Biol. Chem. 31, 15. Dam, R., Morrison, A. B., and Norris, L. C. (1959). J. Nutrition 69, 277. Dansi, A,, Dal Pozzo, A., Zanini, C., and Rotta, L. (1962). Chim. Ind. ( M i l a n ) 44, 839.

SECONDARY FACTORS IN FERMENTATION PROCESSES

89

Dean, F. M., Eade, R. A., Moubasher, R. A., and Robertson, A. (1957). Nature 179, 366. Eble and Lager. Cited by Hoeksema, H., and Smith, C. G. (1961). Progr. Ind. Microbiol. 3, 91. Engel, B. G., Wursch, J., and Zimmerman, M. (1953). Helu. Chim. Acta 36, 1771. Eoff, J. R. (1918). U.S. Patent 1,288,398. Ferguson, J. H., Huang, H. T., and Davisson, J. W. (1957). J . Appl. Microbiol. 5, 339. Finn, R. K. (1954). Bucteriol. Revs. 18, 254. Foster, J. W. (1949). “Chemical Activities of Fungi,” p. 537. Academic Press, New York. Freeman, G. G., and Donald, G. M. S. (1957a). J. Appl. Microbiol. 5, 197. Freeman, G. G., and Donald, G. M. S . (195713). J . Appl. Microbiol. 5, 211. Gaden, E. L. (1960). J. Appl. Microbiol. 8, 123. Goodman, J. J., Matrishin, M., Young, R. W., and McCormick, J. R. D. (1959). J . Bucteriol. 78, 492. Gerhardt, P., Dorrell, W., and Baldwin, I. L. (1948). J . Bacteriol. 52, 555. Harris, J. F., and Hajny, G. J. (1960). Biotechnol. Bioeng. 2, 9. Hesseltine, C . W., and Anderson, R. F. (1957). Mycologia 49, 449. Hirsch, P. ( 1956). “Chemische Konservierung von Lebensmitteln,” p. 80. Steinkopf, Dresden. Hoeksema, H., and Smith, C. G. (1961). Progr. Ind. Microbiol. 3, 91. Jarvis, F. G., and Johnson, M. J. (1947). J . Am. Chem. SOC. 69, 3010. Tohnson, M. J. (1952). Bull. World Health Org. 6 , 99. Johnson, M. J. ( 1954). In “Industrial Fermentations” (L. A. Underkoffler and R. J. Hickey, eds.), Vol. 1, p. 410. Chemical Publ. Co., New York. Judkins, H. F., and Keener, H. L. (1960). “Milk Production and Processing,” pp. 334-376. Wiley, New York. KollLr, J., and JQrai, M. (1960). Acta Microbiol. Acad. Sci. Hung. 7 , 5. Lein, J., Sawmiller, L. F., and Cheney, L. C. (1959). J . Appl. Microbiol. 7, 149. MacKinney, G., Nakayama, T., Buss, C. D., and Chichester, C. 0. (1952). J. Am. Chem. Soc. 74, 3456. MacKinney, G., Chichester, C. O., and Wong, P. S. (1953). J . Am. Chem. SOC. 75, 5428. MacKinney, G., Chichester, C. O., and Wong, P. S. ( 1954). Arch. Biochem. Biophys. 53, 479. Mann, K. M., Hanson, F. R., O’Conell, P. W., Anderson, H. V., Brunner, M. P., and Karnemaat, J. N. (1955). J . AppZ. Microbiol. 3, 14. Margalith, P., and Pagani, H. (1961a). J . Appl. Microbiol. 9 , 320. Margalith, P., and Pagani, H. (1961b). J . Appl. Microbiol. 9, 325. Martin, S. M. (1955). Can. J. Microbiol. 1, 644. Minieri, P. P., Sokol, H., and Firman, M. C. (1956). US. Patent 2,734,018. Moyer, A. J. (1953). J. Appl. Microbwl. 1, 1. Nakayama, T.,Chichester, C. O., Lukton, A., and MacKinney, G. (1957). Arch. Biochem. Biophys. 66,310.

90

P. MARGALITH

Neuberg, C., and Reinfurth, E. (1918). Biochem. 2. 89, 365. Oxford, A. E., Sephton, H. H., and Van der Westhuizen, G. C. A. (1949). J . S . African Chem. lnst. 2, 95. Pasteur, L. (1861). Compt. Rend. A c Q ~Sci. . 52, 1260. Petty, M. A. (1961). Bacteriol. Revs. 25, 111. Ramakrishnan, C. V., and Martin, S. M. (1955). Arch. Biochem. Biophys. 55, 403. Richards, J. W. ( 1961). Progr. Ind. Microbiol. 3, 141. Schweiger, L. B., and Snell, R. L. (1949). U.S. Patent 2,476,159. Sensi, P., DeFerrari, G. A., Gallo, G. G., and Rolland, G. (1955). Farmaco (Pavia), Ed. Sci. 10, 337. Sensi, P., Greco, A. M., and Balotta, R. (1960). Antibiotics Ann. 1959/1960 p. 262. Sheppard, W. A,, and Bourns, A. N. (1954). Can. J . Chem. 32, 4. Shu, P. A., and Johnson, M. J. (1948). Ind. Eng. Chem. 40, 1202. Singh, K., and Johnson, M. J. (1948). J . Eacteriol. 56, 339. Smith, C. G. (1958). J . Bacteriol. 75, 577. Sprecher, E. ( 1961 ). Arch. Mikrobiol. 38, 299. Stark, W. M., and Smith, R. L. (1961). P r o p . Ind. Microbid. 3, 211. Stoudt, T. H. (1960). Aduan. Appl. Microbiol. 2 , 183. Sylvester, J. C., and Coghill, R. D. (1954). In “Industrial Fermentations” ( L . A. Underkoffler and R. J. Hickey, eds.), Vol. 2, p. 219. Chemical Publ. Co., New York. Takida, T., and Tawara, K. ( 1955). Japanese Patent 4435 (’55). Underkoffler, L. A. ( 1954). In “Industrial Fermentations” ( L. A. Underkoffler and R. J. Hickey, eds.), Vol. 1, p. 252. Chem. Publ. Co., New York. Walter, F. G. (1953). “The Manufacture of Compressed Yeast.” Chapman & Hall, London. Wehmer, C. (1893). French Patent 228,554. White, J. (1954). “Yeast Technology,” pp. 27-41. Wiley, New York.

Nonmedical Uses of Antibiotics HERBERT S. GOLDBERG Department of Microbiology, School of Medicine, Uniuersity of Missouri, Columbia, Missouri

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . Economic Aspects ...................................... 11. Antibiotics in Animal Nutrition . . , . , . . . . . . . A. Current Status . . . . . . . .. . . . . . . .. .. . . . B. Antibiotic Levels . . . . . .. .. . . .. . . . . . . . . . . . . .. . . . . . . .. C. Mode of Action of Antibiotic Growth Stimulation . . . . . . . . 111. Antibiotics in Plant Disease Control . . . . . . . . . . . . . . . . . . . . . . . Antibiotics of Importance . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Antibiotics in Food Preservation . . . . . . . . . . . . . . . . . . . . . . . . . . A. Canning . . . . . .. .. .. . . . . . , . . . . B. Dairy Products . . . . . . . . . . . . . . . . C. Fresh Meats and Poultry . .. . . . . . . . . . . . . . . . . .. D. Fruits and Vegetables . . . . . . . . . . . ........... E. Fish and Sea Food . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . V. Antibiotics as Adjuncts in Microbiological Techniques and Procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Tissue Culture . . . . . .. . . . . .. .. . . ... B. Microbial Classification . . . . . . . . . . ... VI. Public Health Aspects of Nonmedical Uses of Antibiotics . . . . . A. Tetracyclines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Streptomycin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C . Penicillin . . . . D. Nisin-Subtilin-Bacitracin . .. .. . . . . . . . . . . .. . . .. .. . . . E. Tylosin-Oleandomycin .. . .. . . .. . . . . . . F. Research .................. References . ..................

91 92 93 93 94

97 98 98 100 100 101 102 103 104 106 106 108 109 110 112 112 113 113 114 114

1. Introduction The impact of antibiotics on the treatment of infectious disease and indeed on the practice of medicine is well appreciated by most individuals. However, the multiple uses of antibiotics in areas outside of human and veterinary medicine, although quite extensive, are not so well known. It is the purpose of this review to present the current status of nonmedical uses of antibiotics, revealing its past, present, and potential contribution to food and agriculture. In addition, the microbiologist in particular has found ways in which antibiotics have helped solve problems in the research laboratory, and these will also be discussed. 91

92

HERBERT S . GOLDBERG

ECONOMIC ASPECTS In order to assess the relationship between antibiotics produced for nonmedical and medical (including veterinary) uses, a brief look at some figures is in order (Table I). The three major nonmedical uses of antibiotics are in animal feed supplements, crop protection, and food preservation. The data indicate that in 1961 more than half of the total United States antibiotic production was for nonmedical use. Over a 10-year span, 1951-1961, antibiotic usage, nonmedically, increased to the point where almost two million pounds were allocated for food preservation and feed and crop usage. A $45,000,000 annual industry has become a wellestablished part of the economy. TABLE I ANTIBIOTIC PRODUCTION FOR NONMEDICAL USES ( UNITEDSTATES)1951-1961

Year 1951 1952 1953 1954 1956 1960 1961 1961

Antibiotic use Feed supplement Feed supplement Feed supplement Feed supplement Feed-food-crops Feed-food-crops Feed-food-crops All uses-medical and nonmedical

Pounds

Value in millions of dollars

236,000 258,000 434,000 479,000 779,000 1,200,000 1,800,000 3,311,000

17.0 17.0 19.0 25.0 28.2 39.4 45.4 114.6

The specific antibiotics of major concern in nonmedical usage include tetracyclines, streptomycin, penicillin, and bacitracin along with several others of lesser importance. Consequently, the antibiotics of medical importance are also the antibiotics of nonmedical importance. This fact complicates the role of regulatory agencies as they are presented with the problem of potential public health hazards, such as toxicity, hypersensitivity, and emergence of microbial resistance. The entire aspect of nonmedical usage of antibiotics presents itself in a complex form. It seems appropriate at this time to first examine the current status of the efficacy of antibiotics used nonmedically and then to analyze the public health aspects of such usage.

93

NONMEDICAL USES OF ANTIBIOTICS

II. Antibiotics in Animal Nutrition A. CURRENT STATUS It is generally held today that certain antibiotics are capable of stimulating the growth rate of a variety of livestock and fur-bearing animals. This was the first broad nonmedical use of antibiotics and was given impetus by the investigation of Moore et al. in 1946. Excellent reviews on the use of antibiotics in animal nutrition are available by Jukes (1955), Ferrando and Jacquet (1958), and Luckey ( 1959). These reviews emphasize the fact that antibiotics stimulate appetite, increase food efficiency, reduce requirement for vitamins, increase survival, and, most significant of all, increase the growth rate. The evidence is clear that antibiotics are effective only in the early growing period and in particular in situations in which animals are undergoing stress. Animals that are weak or runts, in poor environmental conditions, or on inadequate diets do much better on antibiotics than do normal animals, reared under good management and fed a complete diet. The animal species generally accepted as requiring antibiotics for maximum production efficiency include poultry, swine, calves, lambs, and fur-bearing animals. Some would also include beef cattle. Since the effectiveness of antibiotics is limited to the early growing period the age periods as shown in the following tabulation have been recommended for feeding (WHO Tech. Rept., 1963). Animal Poultry Swine Calves Beef cattle Lambs Fur-bearinc animals

Ace 8-10 weeks 4-6 weeks 3 months 18 months 2 months 2-3 months

The most frequently used antibiotics for the purpose of animal growth are penicillin, chlortetracycline, and oxytetracycline, primarily, and bacitracin, erythromycin, oleandomycin, spiramycin, streptomycin, and tylosin. All of these have shown growth-stimulatory activity in the animals mentioned in the above tabulation and are used for this purpose commercially in the United States and abroad.

94

HERBERT S. GOLDBERG

Even with the above facts well established there exists, however, current controversy over two aspects of antibiotics in animal feeds. One, a practical problem, concerns levels of antibiotic necessary to the ration and the other, a fundamental problem, concerns the mechanism of action of antibiotic growth stimulation.

B. ANTIBIOTIC LEVELS In the early 1950s the antibiotic level, used in feeds to bring about growth stimulation, varied from 5-20 p.p.m. of the “total ration.” The “total ration” being defined as “the total daily feed intake on a dry-matter basis.” Since then the tendency has been to increase the level of antibiotics in commercial animal feeds. Levels as high as several hundred p.p.m. and higher are currently used. Table I1 indicates the maximum permissible levels in representative TABLE I1 NATIONALREGULATIONS FOR ANTIBIOTIC FEEDSUPPLEMENTS Countrya Austria Belgium

Antibiotics OTC-CTC-penicillin OTC-CTC-penicillinbaci tracin Denmark OTC-CTC-penicillin Fin 1and OTC-CTC France OTC-CTC-penicillinbacitracinb Germany OTC-CTC-penicillin Great Britain OTC-CTC-penicillin Holland Not specified Norway OTC-CTC-penicillin Sweden OTC-CTC-penicillin Switzerland OTC-CTC-penicillinbacitracin United States OTC-CTC-penicillinbacitracinb a b

Animals Pigs-poultry Calves-pigs-poultry

Maximum level (P.P.m. ) 60

Young growing animals Pigs-poultry-fur bearers Pigs-poultry Pigs-poultry-calves Growing pigs-poultry Pigs-poultry-calves Pigs-poultry-calves Pigs-poultry-calves-mink All except dairy cattle Pigs-poultry-calves

50

25 50 200

200 100 100 50 20 50

2000

Ireland, Greece, Israel, G y , and Portugal have no restrictions. Also several other antibiotics.

countries as of 1961. It can be readily seen that the concentration of antibiotics necessary for the growth effect has been universally exceeded. The reasons for this are not too clear and have never been satisfactorily explained on a nutrition basis. In general, levels of antibiotic in feeds have been divided into “low-level feeding,”

NONMEDICAL USES OF ANTIBIOTICS

95

“prophylactic feeding,” and “therapeutic feeding.” Unfortunately, these levels are not clearly defined nor are they well regulated, particularly in the United States. “Low-level feeding is best defined, perhaps, as the minimum level which achieves the growth effect. In most instances 20 p.p.m. should be the maxium necessary for this result. However, the United States accepts low-level feeding as that which occurs at up to 50 p.p.m. Christensen (1956) and Carlson (1957) have judged 50 p.p.m. as being in excess of that needed for the growth effect. It is at this level that the term “prophylactic feeding should apply. The question arises, however, is “prophylactic feeding” needed routinely to prevent infection or should it be used only when needed to stop a suspected disease in a herd or flock? In the United States the “prophylactic level” is assumed to be 100-400 p.p.m. and is used all too often in a routine feeding program. Still higher levels of antibiotic are often used, when necessary, to treat disease. This is properly carried out under veterinary control. However, up to 2000 p.p.m. can be so used, and there is much evidence to indicate that this “therapeutic level” feeding is not always used as intended by statute. The basic concern over the amount of antibiotic in feed is caused by potential public health hazards which may occur when the antibiotic develops a tissue level and is then presented to the consumer in meat. Those levels which result in tissue residues and become implicated in a threat to the public health are those which exceed the level necessary for the simple growth effect. Very few publications of investigations carried out to determine tissue residues in animals fed dietary antibiotics are available. This is unquestionably a neglected area from the standpoint of evaluating any public health aspects of this use of antibiotics. It is undoubtedly true that animals are coming to slaughter with antibiotic residues in their tissues, particularly those which have been on prophylactic or therapeutic dosage. Some data have been obtained in this area and are described. Broquist and Kohler (1954) point out that, following nutritional feeding of chlortetracycline to farm animals in amounts of 10-20 gm.per ton of feed, the antibiotic was not present in the serum or tissues of the animals. When the antibiotic was fed to chickens or pigs for an extended time at levels above 50 gm. per ton, the antibiotic could be detected in the serum. At levels of 1000 gm. per ton, the antibiotic could be recovered from tissue in the order of 1 part

96

HERBERT S. GOLDBERG

per 10,000 parts of tissue, These amounts of chlortetracycline resulting from massive chlortetracycline dosage rapidly disappeared from the tissues when the antibiotic was removed from the diet 1 or 2 days prior to slaughter, or when the meat was cooked. [This latter statement is controversial, Goldberg ( 1959).] Thus far, antibiotics used as feed supplements have not led to untoward effects in man, Residues of bacteriologically active antibiotics are not encountered in the flesh of animals that have received recommended levels of antibiotics ( u p to 20 p.p.m.) in feeds throughout their life span. Furthermore, detectable levels of bacteriologically active antibiotic residues in the flesh of animals fed antibiotic supplements disappear rapidly when antibiotic feeding is discontinued for a few days prior to slaughter. There then would seem to be little public health danger from the use of antibioticsupplemented feed, if feeding were discontinued after growth stimulation occurred and well before the time of slaughter. T. H. Jukes (1955) has summed up the public health hazards of antibiotics used in animal nutrition very clearly as follows. “The prolonged feeding of comparatively high levels of the common antibiotics to animals has not raised problems in public health as regards the commption of animal products. Broquist and Kohler (1954) found no detectable amounts of chlortetracycline in the liver and muscle of chicks receiving chlortetracycline, 200 mg/ kg of diet. Barely detectable amounts were present in the blood serum. At unusually high dietary levels, traces of the antibiotics were found in the liver and muscle. Chicken breast muscle containing 3 mg/g of chlortetracycline was found to be free from the antibiotic after boiling for 15 minutes or after roasting for 30 minutes at 230°C. No toxic reactions are imaginable from the traces of chlortetracycline which might be present in the meat of animals fed high levels of the antibiotic. Indeed, these traces of antibiotic were only of the order of 50 mg/lb or less of muscle tissue even when the chicken received 2000 gm of chlortetracycline per ton of diet and the antibiotic was destroyed by ordinary cooking procedures.” Analyzing the residue of antibiotics from use in feedstuffs has not yet been done on a large scale. Although there are data to show that high-level feeding is required to achieve tissue levels, it is not known how many animals came to slaughter following high-level (prophylactic or therapeutic) feeding.

NONMEDICAL USES OF ANTIBIOTICS

97

C. MODEOF ACTION OF ANTENOTICGROWTH STIMULATION It appears to be widely accepted that the effect of antibiotics upon growth is initiated by modification of the enteric flora. Nevertheless, there is a formidable array of evidence that much beyond the antimicrobial effect is involved. In a limited series of experiments with germfree chicks, Luckey (1956) showed that under some conditions (1-15 p.p.m.) the growth of these chicks was stimulated with antibiotics. However, this work has not been repeated with the same antibiotic levels, and some question exists as to the magnitude of this response. Using somewhat higher levels (25-50 p.p.m.1, Forbes et al. (1959) and Gordon et al. (1957) got no growth response in germfree chicks. There is no dispute, however, over the fact that antibiotic feeding results in a thinning of the intestinal wall (H. G. Jukes et al., 1956; Coats et al., 1955) and alteration of bowel motility (Leaders et al., 1956; Clegg, 1962). This is theorized by some to result in better ab,sorption of nutrients. Several workers have also shown that inactivated antibiotics (without antimicrobial activity) continue to bring about a growth response. This includes heat-, enzyme-, and metal-inactivated penicillin (W. L. Williams et al., 1953; T. K.Jukes, 1955; Taylor and Gordon, 1955). Further evidence for a mode of action beyond an effect on enteric flora is presented by the fact that some antifungal antibiotics produce a growth promotion effect. These include nystatin and griseofulvin (Coats, 1962). It seems unlikely that the antifungal activity of these agents would play a significant role toward influencing the enteric flora of most animals. Evidence substantiating the influence of the enteric flora on growth is best provided by the recent paper of Lev (1962). His work demonstrates the effect of CEostridium welchii toxin on growth rate of chicks. The author indicates clearly that when this organism is inhibited in the gut, growth proceeds at a higher rate. It is suggested that this and other microbial toxins are deleterious to growth and that antibiotics in the feed help control bacteria producing these harmful agents. Luckey (1959) has summarized several modes of action for antibiotic growth stimulation. They are described below because they appear to best represent the available documented opinion.

98

HERBERT S . GOLDBERG

SOMEPROPOSED MODESOF ACTION OF ANTIBIOTIC GROWTHSTIMULATION (modified from Luckey, 1959)

A. Indirect Action 1. Via intestinal microflora a. Increased numbers of “good” microorganisms, such as vitamin synthesizers b. Decrease numbers of “bad” microorganisms, such as vitamin users, toxin producers, and pathogens or potential pathogens 2. External (or intestinal) mileux reaction a. Detoxication b. Chelation activator c. Reduce p H of intestinal mileux B. Direct Action 1. Cells a. Permeability of cell wall b. Biological stabilizer against stress c. Activate anabolic regulator d. Mitotic stimulant 2. Tissues a. Intestinal wall length, weight, and thickness made more efficient b. Increased absorption c. Increased apparent utilization of metabolites d. Decreased energy expenditure

It can be concluded that the mode of antibiotic action as a growth stimulant is multifaceted and that alteration of enteric flora is not a complete explanation.

Ill. Antibiotics in Plant Disease Control ANTIBIOTICSOF IMPORTANCE In a recent review Goodman (1959) listed 25 bacterial diseases of plants that are amenable to antibiotics for treatment or prevention. In addition almost 50 fungal diseases of plants are described that respond similarly to antibiotics. In all of these cases there are only 3 antibiotics which play any significant role. These antibiotics are streptomycin, griseofulvin (Brian et aE., 1946), and cycloheximide (Whiffen et al., 1946). The latter two are antifungal in their activity while streptomycin is able to inhibit many bacterial and some fungal phytopathogens. Although streptomycin is well established as a medical antibiotic, griseofulvin and cycloheximide are less well known. The structures below indicate that these are

NONMEDICAL USES OF ANTIBIOTICS

99

compounds of low molecular weight ( 250450) with cycloheximide a weak acid and griseofulvin a neutral compound.

0 Cy cloheximide

Griseof ulvin

Cycloheximide is the more prominent of these antifungal agents in phytopathology. However, now that griseofulvin is produced by large-scale fermentation as an oral antibiotic for treatment of human fungal disease (Barnett, 1960; D. I. Williams, 1960), it is possible that an extension of its use in agriculture may occur. Streptomycin is primarily effective against bacterial diseases caused by Xanthomonas, Pseudomonm, and Erwinnia spp. and Phytophthora and Peronospora fungi (Zaumeyer, 1956). Cycloheximide is most active against Cocomyces (cherry leaf spot) and turf diseases (Hamilton et al., 1956). Griseofulvin was most recently reported to be active against powdery mildews, melon cankers, and certain Fusarium species (Rhodes, 1962). The fundamental advantage of antibiotics over previous methods of plant disease control has been the fact that antibiotics are absorbed by the plant and are effective within the plant. That is to say the antibiotics are systemic in their action. Prior to the advent of antibiotics, plant disease control was based upon external protectants. Such protectants were applied to surfaces, where they remained until diluted, inactivated, or degraded. They were “preventive” in their action. There is evidence to show that antibiotics are both protective and “therapeutic.” Not only can the antibiotics prevent plant disease, they can also eradicate existing disease by virtue of their ability to act systemically and be translocated. Of course, results vary with the nature of the antibiotic and plant tested. Such influences as phytotoxicity (Goodman, 1962), antibiotic concentration, method of application, temperature, and humidity play an important role in the outcome of this action. Since applications may be made by spraying, dusting, dipping, and soil routes the stability and persistence of the antibiotic will vary as will its

100

HERBERT S. COLDBERG

usefulness in disease control. Another contributing factor is, of course, economic. The current use of antibiotics in plant disease control is undoubtedly limited by this factor. Detailed analysis of stability, persistence, and movement of antibiotics in plants have been made by Pramer (1959), Goodman (1959, 1962), Goodman and Dowler (1960), and Goodman and Goldberg (1960). This use of antibiotics also has a public health aspect since many plants so treated ultimately are used for food. The problem of antibiotic residues from plant disease control is discussed in the next section on antibiotics in food preservation.

IV. Antibiotics in Food Preservation A. CANNING The antibiotic most evaluated as an adjunct to mild heat for the canning preservation of foods has been subtilin. Originally, several optimistic reports (Anderson and Michener, 1950) and ( Burroughs and Wheaton, 1951) indicated this polypeptide antibiotic could preserve foods in cans with the addition of mild heat. It soon became apparent, however, that species of Clostridiurn botulinum, the organism of botulism, an often fatal type of food poisoning, were not destroyed by this procedure (Cameron and Bohrer, 1951). Subsequently, although much work on antibiotics in canned foods was continued, the fear of botulism negated acceptance of any antibiotic agent as the sole source of sterilization of canned products. More recently, however, two additional antibiotics, nisin ( Mattick and Hirsch, 1957), a polypeptide, and tylosin, a macrolide (Greenberg and Silliker, 1962a,b, c ) , have reawakened interest in this subject. These have been tested with some success for ability to reduce thermophilic spoilage bacteria in all types of canned foods including fruits, vegetables, meats, fish, soups, and dairy products. Under this new approach the search for an antibiotic capable of destroying botulinum spores is abandoned and the antibiotics in canned foods are used as a supplement to traditional botulinum destroying methods of processing. In this manner, saprophytic spoilage organisms may be controlled as well as the agent of botulism. In order for agents to be successful against saprophytic spores in canned foods, they must be present in the product throughout its shelf life. Thus, an antibiotic residue is necessarily to be encountered in this procedure. From the available data, it would appear

NONMEDICAL USES OF ANTIBIOTICS

101

that such residues would probably fall in the range of 1-20 p.p.m. (Goldberg, 1959).The actual residue depends, of course, upon the nature of the product, the pH, moisture content, bacterial flora, and on the degree of thermal application. Although many additional antibiotics such as tetracyclines, streptomycin, chloromycetin, and penicillin have been tested for their ability to contribute to preservation of canned foods, they have, in general, been eliminated from canning because of objectionable chemical or physical properties. Consequently, subtilin, nisin, and tylosin remain the antibiotics of choice for inhibition of spoilage bacteria, particularly of the thermophilic variety. B.

DAmY

PRODUCTS

In technologically advanced areas there is little reason to consider the possible use of antibiotics to keep milk fresh. However, fresh milk is unavailable to much of the world's population, partly because of the impossibility of distributing it without adequate pasteurization, refrigeration, and transportation facilities. Consequently, some study has been given to the possible use of antibiotics in this regard. Early observations indicated that penicillin, streptomycin, and other narrow-spectrum antibiotics, although capable of interfering with lactic acid production (souring), were of little value against putrefaction. Various combinations were somewhat more effective, but, as observed with other foods, the broad-spectrum antibiotics proved to be of the greatest value. Several reports indicate that chlortetracycline (CTC) or oxytetracycline (OTC) can substitute to a considerable extent for refrigeration. If 1 p.p.m. of a tetracycline antibiotic is added to raw milk directly after milking, the onset of spoilage is delayed for about one day at 37°C. If the milk is pasteurized, these antibiotics will preserve it without refrigeration from 2, days to several weeks, depending on storage conditions and the level of antibiotics used (Wrenshall, 1959). Residue data for antibiotics in milk preservation indicate some interesting phenomena. Low-level antibiotic residues are remarkably stable in milk. In addition, there is ample evidence that pasteurization temperatures have little or no effect and some constituents of milk offer considerable protective effect. Residue levels for increasing the shelf life of fresh milk can be anticipated at 1-2 p.p.m., and this level is ample for preservative

102

HERBERT S. GOLDBERG

purposes. Studies have been carried out to show that OTC looses only 2.5% of its activity during pasteurization when inoculated at 0.5-1.0 p.p.m. in fresh milk; streptomycin loss and OTC loss are about the same in similar conditions. Cheese products, both processed and natural, may often be spoiled by certain clostridial species. Here the antibiotic nisin has seemed to be most effective and also at a rather low level, less than 5 p.p.m. However, no exact level for nisin can be established since it is found naturally in cheeses. It should be noted here that a recent committee of experts has indicated that hydrogen peroxide is a more practical milk preservative than antibiotics (WHO/FAO Technical Rept., 1960). C. FRESH MEATSAND POULTRY Rather remarkable preservation powers have been reflected in the use of tetracycline antibiotics applied to red meats and poultry. Three methods of introducing the antibiotic have been used with success. The first consisted of dipping steaks and chops in antibiotic solution (Tarr et aZ,, 1952); this retarded bacterial spoilage quite effectively. As many as 20 antibiotics were used but oxytetracycline and chlortetracycline proved best. In a second method, the entire carcass was infused with antibiotic just after slaughter (Weiser et d.,1953). Here again chlortetracycline was able to keep beef successfully at temperatures as high as 80°F. for several days. A third successful method utilized ante-mrtem injection of oxytetracycline for preservation of meat of cattle, lamb, and other species (McMahan et al., 1956). In all cases of tetracycline preservation of meat, it is quite apparent that some low-level residue will reach the consumer. Although residues of OTC are somewhat higher than CTC, there is actually little difference. The initial level in the raw meat ranges from 1 4 p.p.m. Following cooking, a decrease in residue occurs but there is still detectable residue no matter if the meat is cooked rare, medium, or well done. This residue is almost always less than 1.0 p.p.m. (Goldberg, 1959). This point when applied to poultry is somewhat altered. It has been shown that the tetracyclines are the antibiotics of choice for poultry and dipping in antibiotic solution is the preferred method. However, by limiting the dip to 55 p.p.m. concentration, the residue level on the bird is less than 7 p.p.m., and all of this is destroyed

NONMEDICAL USES OF ANTIBIOTICS

103

by cooking (Hines, 1956). The subject of antibiotics in poultry preservation has recently been reviewed ( Ayres, 1962).

D. FRUITS AND VEGETABLES Antibiotic treatment of fruits and vegetables has taken two directions. The first concerns antibiotics in plant disease control. In this instance, streptomycin is clearly the antibiotic of choice (Goodman, 1959). It is applied to apple, pear, peach, and other trees during the growing season to prevent bacterial disease. Among vegetable crops it is applied to beans, tomatoes, cabbage, etc., for the same purpose. In treating the fruit trees, no residue is found in the fruit because the disease treatment is applied before flowering and fruiting. Vegetables, however, are sprayed until time of harvest, and they may show streptomycin residue at detectable levels within the vegetable tissues. The extremely perishable nature of fresh vegetables, especially the green leafy varieties, is one of the most adverse factors encountered in their distribution. Substantial losses due to spoilage are accepted as normal and are a major factor in determining the price consumers must pay for such products. The need for methods of delaying vegetable spoilage has been accentuated by recent trends in distribution involving the prepackaging of these commodities in ready-to-use form. The most prevalent cause of spoilage is bacterial soft rot. Modern methods of handling raw agricultural commodities after harvest materially reduce development of bacterial spoilage. Such methods as hydrocooling and refrigeration, however, only inhibit microbial decay by the influence of temperatures adverse to growth of microorganisms. When perishable produce is removed from these

104

HERBERT S. GOLDBERG

spinach by a l-minute dip in a 500 p.p.m. solution of streptomycin sulfate. Koch and Carroll (1957) reported reduction in decay of spinach with oxytetracycline, streptomycin, polymyxin, and neomycin dip treatments. Cox (1955) and associates also reported complete control of radish pit after 5 days' storage at temperatures of 5"35"C., using 5-minute dip treatments of 50 p.p.m. oxytetracycline solutions. Streptomycin at the same concentration was not as effective and did not give control at 35°C. Carroll and associates (1957) studied decay in a salad mix and each of its components, using dip treatments of antibiotics. Vegetables which deteriorate rapidly appeared to establish the rate of decay for the other components in a salad mix. Degree of spoilage could be judged easily by measuring the volume of liquid exudate. The effects of antibiotics and refrigeration were additive. Oxytetracycline was by far the most effective antibiotic tested. Goodman and associates (1958) confirmed the effects of antibiotics in prolonging the refrigerated shelf life of spinach. They also studied the active antibiotic residues present in various vegetables after treatments with oxytetracycline and streptomycin. Residues of streptomycin were found to be persistent, and there was no assurance that they would be destroyed by cooking. While the information available is still somewhat fragmentary, the future appears to hold considerable promise for the use of antibiotics on vegetable products. Certainly, antibiotics appear capable of contributing to the solution of serious spoilage problems in this area. Realization of these potential values must, however, await clarification of the public health significance of antibiotic residues. Summing up we find that the antibiotics of importance in fruit and vegetable preservation are oxytetracycline, chlclrtetracycline, and streptomycin. The residues are < 5.0 p.p.m. for the tetracyclines and 2-40 p.p.m. for streptomycin.

E. FISHAND SEAFOOD In this area most work has been done by Canadians, Japanese, and English workers concerned with preservation of one of the most perishable of foods (Tarr et al., 1952; Tomiyama et al., 1955; Ingram et al., 1956). As is true with certain other foods, stability of antibiotics is often enhanced, particularly in conjunction with fish skin. Ample studies have shown that only tetracycline antibiotics

105

NONMEDICAL USES OF ANTIBIOTICS

have the capability of controlling spoilage in this product. Application of these antibiotics in the form of ice or dipping fillets in antibiotic solution are both highly effective in increasing storage ability. Although much antibiotic residue is decreased in cooling, it is still necessary to consider the tetracycline residues in concentration of < 1.0-5.0 p.p.m. in fish treated for preservation by antibiotics, Table I11 sums up the current status of antibiotics permitted for food preservation in various countries. It can be quite easily seen TABLE I11 FOODPRESERVATION BY ANTIBIOTICS IN DIFFERENT COUNTRIES

Country

Antibiotics permitted

Tolerance permitted (p.p.m. 1

Used for

Argentina

Chlortetracycline Oxytetracycline

5-10

Meat, poultry, fish

Canada

Chlortetracycline Oxytetracycline

7 5

Poultry Fish preservation in ice Fresh fillets in dipping tanks

10

Great Britain

Chlortetracyclinea 5 Oxytetracyclinea Nisina No limit Nystatin

On the skin but not in the flesh 5

Japan

Chlortetracycline Oxytetracycline

Norway

Chlortetracycline Oxytetracycline

250

United States

Chlortetracycline Oxytetracycline

5

Chlortetracycline Oxytetracycline

7

Chlortetracycline

5

USSR

'Raw fish Cheese and certain canned goods Bananas

Fish preservation in ice; fish for fish pasta; salmon for cannine Slaughterhouse offals for minks in the warm weather Deriod Fish preservation in ice; preservation of shrimps and scallops Poultry preservation in slush-ice tanks Codfish preservation in ice and for transuort

a The use of these antibiotics for the purposes and in the amount stated is a proposal only, made by the Antibiotic Panel of the Ministry of Health.

106

HERBERT S. GOLDBERG

that the experimental results above have not been readily accepted by regulatory agencies of governments due to unanswered public health questions.

V. Antibiotics as Adjuncts in Microbiological Techniques and Procedures

A. TISSUE CULTURE There are generally two broad areas in which the microbiologist has extensively utilized antibiotics in the laboratory. The first is in the selective isolation of specific microorganisms and the second is the characterization of microorganisms for purposes of classification via “antibiograms.” In the area of selective isolation Goldberg (1959) and Cruickshank (1960) have reviewed the subject in detail with primary emphasis on selecting bacteria and fungi of animal or plant pathogenic origin. More recent advances have been in the area of tissue culture growth of virus with attempts to prevent fungal and yeast contamination. Penicillin and streptomycin or chloromycetin alone appear to control satisfactorily the bacterial contamination in tissue cultures. The most recent of the agents to be used in tissue culture to control fungal and yeast contamination is amphotericin B (Gold et al., 1956). It was investigated by Perlman et al. (1961) for effect on several tissue culture cell lines with a variety of media. The results indicated that amphotericin B is not toxic in tissue culture at a recommended concentration of 2.0pg./ml. It has also been shown that normal growth of viruses is not interfered with by amphotericin B. Although this antibiotic is slowly inactivated at 37°C. it does not loose potency in the refrigerator (Table I V ) . As large-scale tissue cultures are produced for growing virus vaccines the need for yeast and fungal control becomes more important. Undoubtedly, amphotericin B will find more widespread use in the future. Nystatin (Hazen and Brown, 1951) continues to be used in tissue culture as an antifungal agent (Table V). It is a polyene antibiotic, as is amphotericin B, and has many similar uses. However, this antibiotic has the disadvantage of being unstable at 37°C. and must be added to tissue culture media to maintain an antifungal level; it thus may be ultimately replaced by amphotericin B.

STABILITYOF AMPHOTERICINB

TABLE IV VARIOUSTISSUE CULTUREMEDIAAT 37°C.

IN

Medium without cells; amphotericin B concentrations (pg./ml.) at: Mediuma

0 day

2 days

4 days

A

5 50 5 50

3.5 50 1.2 43 3.9 44 3.4 43

2.7 29 0.6 37 2.8 37 1.9 39

B C

5 50

D

5 50

a

b

I

7 days Mediuma 1.7 29 0.5 27 2.0 33 1.0 34

A

B C D

Medium with cells;b amphotericin B concentrations ( pg./ml.) at: 0 day 2 days 4 days 7 days 5 50 5 50 5 50 5 50

3.6 46 1.3 41 4.4 50 3.4 42

3.3 16 0.9 33 3.7 39 2.9 40

Composition of media: A-Waymouth‘s MB 752/1 supplemented with 10% (v./v.) calf serum and 0.03% methylcellulose. B-Waymouth‘s MB 752/1 supplemented with 0.5% methylcellulose. CZiegler’s modification of Eagle’s medium containing 10% ( v./v. ) calf serum. D-Eagle’s medium containing 10% (v./v.) calf serum. Cell line: Earle’s L cells NCTC 929 (mouse fibroblast).

2.9 13 0.9 28 3.3 35 2.1 38

r

C m N IA

5

H

108

HERBERT S. GOLDBERG

TABLE V ANTIFUNGAL SPECTRUM

OF

NYSTATIN Minimum inhibitory

concentration (units/ml. ) a

Organism Aspergillus fumigatus Bhtomyces dermatidis (yeast phase) Candida albicam Candida guillermondi Candida krusei Cryptococcus neofomnans Geotrichum luctb H i s t o p h a capsulatum (yeast phase) PetaicilEium spp. Rhodoturulu muciluginosa Saccharomyces cereuisiae Saccharomyces pastorianus Trichophyton mentagrophytes a

6.25 1.56 3.13 3.13 6.25 1.56 6.25 1.56 13.0 1.56 3.13 3.13 6.25

As determined by a twofold dilution test.

Nystatin’s most recent use has been in submerged culture of plant cells (Bryne and Koch, 1962) and with marine fish-cell tissue culture (Clem et al., 1961). In summarizing this use of antibiotics the data above together with the aforementioned recent reviews of Goldberg (1959) and Cruickshank (1960) indicate that viral specimens, chick embryo cultures, and tissue cultures require penicillin and streptomycin or chloromycetin for bacteria-free conditions and amphotericin B or nystatin for yeast- and fungi-free conditions.

CLASSIFICATION B. MICROBIAL Antibiotics have been used in the past as an epidemiologic tool much as phage typing or serology. It is only recently, however, within the last 10 years, that formal classification has been attempted by antibiotic sensitivity patterns or “antibiograms.” The majority of the work published so far has concerned the Pseudomom-Achromobacter group. Shewan et al. ( 1954, 1960) classified Pseudomonas by penicillin sensitivity and divided Achromobacter, Alcaligenes, and Spirillum from Flauobacterium, Pseudomonas, Vibrio, and Aeromonas by penicillin response. The following genera have also been taxonomically evaluated by antibiotic patterns: Coynebacterium (Seaman and Woodbine, 1962), Listeria

NONMEDICAL USES OF ANTIBIOTICS

109

(Jones and Woodbine, 1961), Erysipelothrix ( Wix and Woodbine, 1955), Microbacterium (Turbitt et al., 1959, 1960). Renoux (1960) used erythromycin at 15pg./ml. to differentiate Brucella spp., and Lutz et al. (1958) showed that the Proteus species can be separated by novobiocin. Goldberg and Barnes (1962) have used antibiograms as aids in identifying gram-negative anaerobic rods. They were able to separate Bacteroides from Spherophorus and Fusobacterium genera by the use of penicillin, neomycin, and polymyxin B. Bacteroides were inhibited by penicillin and not by the other two. The Spherophorus and Fusobacterium were inhibited by penicillin and polymyxin B but not by neomycin. Seaman and Woodbine (1962) analyzed a genus which contained both plant and animal pathogens. They concluded that the Corynebacterium from a single habitat could be usefully separated by antibiotics. However, results were unsatisfactory when, for example, the plant and animal pathogens were tested as a single group. One weakness of this method of classification is similar to that inherent in all taxonomic schemes, i.e., lack of consistent, standardized methods with large numbers of strains. It is anticipated that this situation will improve with reawakened interest in the problems of microbial classification (Ainsworth and Sneath, 1962).

VI. Public Health Aspects of Nonmedical Uses of Antibiotics

Untoward effects of antibiotics have been described in numerous reports in the literature of clinical medicine. For the most part, these effects have appeared following long-term therapeutic dosage or have been due to individual idiosyncrasy and drug reaction. The reactions have been emergence of antibiotic-resistant bacteria, hypersensitivity, toxicity, and superinfection or overgrowth of indigenous flora resulting in a pathogenic process, i.e., candidiasis. The question at hand is whether or not low levels of these antibiotics are capable of elucidating the same effects. In most instances the dose of antibiotic ingested from food would be 100-500 times less than a therapeutic daily dose of the same antibiotic. This can be illustrated with chlortetracycline. The level in foods preserved with CTC is 1-10 p.p.m., usually at the lower range. If an individual consumed 1-2 kg. of food per day, the total intake would be 5-10

110

HERBERT S. GOLDBERG

mg. This is about 100300 times less than the therapeutic daily dose of CTC. In order to consider the low levels of these antibiotics each can be reviewed in turn with regard to resistance, superinfection, hypersensitivity, and toxicity rate at low levels. A. TETRACYCLINES The tetracyclines of importance in foods and feeds are chlortetracycline and oxytetracycline. The maximum level to be encountered would seem to be less than 10 p.p.m. according to the preceding data. The foods involved are all of the fresh variety and include meats, poultry and fish, fruits, and vegetables. Many reports have been made on the effect of these antibiotics on intestinal bacteria of laboratory animals. Unfortunately, the data are conflicting, particularly with regard to coliform population and resistance (Schwachman et al., 1951; Goldberg et al., 1958, 1959). However, there have appeared more satisfactory studies with humans on long-term OTC or CTC, and these data are worthy of comment. Schwachman and associates (1951, 1952; Schwachman and Kulczycki, 1958) in several reports have used chlortetracycline or oxytetracycline in treating chronic cystic fibrosis over a period of 8 to 9 years. These antibiotics were given in daily doses up to 25 mg./kg. body weight. Although development of resistance by Staphylococcus and Proteus species occurred and an increase in yeasts was found, the authors continued treatment and were satisfied with results, since only clinically beneficial responses were noted. Sprunt and McVay (1953) used CTC at a daily level of 500mg. for 19 months in geriatric patients as a routine prophylactic. Toxic effects were minimal and did not require cessation of antibiotic. A reduction in respiratory and urinary infections was observed, although no studies on enteric microorganisms were carried out. Other studies support the efficacy of therapeutic daily doses of tetracycline antibiotics as prophylactic agents over long periods. However, bacteriologic analyses were not reported ( Stowens, 1951 ). Studies of long-term, low-level tetracyclines in humans are confined for the most part to attempts to correct growth failure in malnourished children. Joliffe et al. (1956) fed 20 mg. CTC daily for 7 months to undernourished children. When compared with

NONMEDICAL USES OF ANTIBIOTICS

111

controls, these children showed greater weight gains. No untoward effects were reported, although no bacteriology was done. Similarly, Scrimshaw is reported by Carey (1958) to have fed 50mg. CTC daily to children for up to 30 months. No toxic effects were noted. Physical examination and hematological study supported this claim, although no bacteriology was done. Bacteriologic analysis was carried out by Loughlin et al. (1958), a study which evaluated OTC as a growth-promoting agent in 243 young children. Daily 10 and 50 mg. doses were administered for 12 months to several groups of these children. Cultures of Staphylococcus aureus and enterococci were isolated from rectal swabs and checked for OTC resistance prior to and periodically after the study began. A skin test was also employed to determine allergic sensitization to OTC. The results showed complete absence of toxic effects, gastrointestinal upsets, increased resistance, moniliasis, and positive skin tests. More recently a few limited studies have been carried out to investigate specifically the effect of CTC or OTC levels from food preservation on human intestinal flora. Kuwahara et al. (1958) administered CTC to five healthy adults in a daily dose of 0.0-20 mg. for 20-107 days and observed change in flora and skin reaction for sensitivity to CTC. No change was observed in cases given daily doses of 10 and 20 mg., except a tendency toward constipation and a concurrent increase in colon bacilli and enterococci in stool during administration. In all the cases, resistant strains emerged during drug administration, especially among strains of colon bacilli which were found to show resistance to 100 pg./ml. However, these strains disappeared after stopping the administration of CTC. Over a 4-month period, after the end of the administration, skin tests were carried out using CTC solution; the results were negative in all cases. Knothe (1957, 1958) found that coliform resistance developed when more than 25 mg. CTC/kg. of food were present. However, the resistant flora disappeared quickly on cessation of antibiotic in the food. Finally, Goldberg ef al. (1961), working with 46 persons, fed 10 mg. OTC/kg. of food and found that resistant coliforms existed prior to antibiotic administration, that OTC-induced resistance was transitory, that no OTC blood levels appeared, and that hypersensitivity to OTC failed to occur. These experiments were carried out over 14 months, and all individuals acted as their own controls.

112

HERBERT S. GOLDBERG

On the basis of available information, it seems questionable that any hazard exists from ingestion of OTC or CTC at levels in foods that are below 10 p.p.m. in antibiotic residue. With the knowledge that hypersensitivity to these antibiotics is extremely rare, this too presents a doubtful hazard. One relatively unknown area, however, concerns breakdown products of the tetracyclines such as isochlortetracycline. This heat degradation product appears to be harmless; further work is needed in this area.

B.

STREPTOMYCIN

Streptomycin has many characteristics subject to objection when used therapeutically. A t the lower levels it is found useful in food preservation, although many of these criticisms still remain. In almost all studies on intestinal flora in animals using high or low levels, this antibiotic brings about rapid resistance. Streptomycin has been shown to be extremely stable in milk, in tissues of vegetables, and in other plant foods. Furthermore, the toxicity of this antibiotic for the eighth cranial nerve is well documented. Consequently, it would seem that streptomycin can be a public health hazard in foods. Streptomycin in plant disease control is not a hazard so long as field workers do not react allergically when applying streptomycin to plants. Studies on development of hypersensitivity by field workers have been carried out, however, and appear to be of no significant hazard (Goldberg, 1962). Streptomycin in animal feedstuffs also does not appear to be hazardous since oral streptomycin is not absorbed into the tissue of animals and is excreted in feces and urine. C. PENICILLIN This antibiotic has little role in food preservation. Its limited antibacterial spectrum and the prominent ability to stimulate allergic responses in a large number of individuals precludes its use in this manner. On the other hand, this antibiotic plays an important role in animal dietary supplements. Levels are fed that are quite low and little, if any, penicillin gets into animal tissues. The main public health concern is environmental. Several investigators have reported resistant staphylococci in human attendants working in areas where penicillin supplements were used. In addition, the

NONMEDICAL. USES OF ANTIBIOTICS

113

animals appeared to be carriers of penicillin-resistant organisms at a higher percentage than expected (Smith and Crabb, 1957).

D. NISIN-SV~TILIN-BACITRACIN These three antibiotics are all polypeptide in nature and can be considered as a group although they have individual characteristics which contribute to their role in foods and foodstuffs. As a group, they are not absorbed from the gut into tissues and thus their influence on other than intestinal flora is limited. They are of little consequence in hypersensitivity and nontoxic at low levels by the oral route. Nisin and subtilin have a useful role in canned food preservation as a supplement to heating. They can prevent spoilage successfully, and in acid foods, where botulism is not a threat, they can supplement reduced heating processes. Since they are narrow spectrum antibiotics, they do not alter the intestinal flora and, in fact, are acted upon by digestive enzymes of the body. Of the two, nisin has the advantage of occurring in nature in many dairy products and thus is consumed by many, daily, as part of their diet. As long as these antibiotics are used as advocated in canned foods, at levels below 20 p.p.m., and accompanied by botulinum-destroying processing, they appear to lack any hazard to public health. Bacitracin has found a useful role in the zinc form as an animal dietary supplement. Public health does not seem endangered by such use.

E. TYLOSIN~LEANDOMYCIN The macrolide antibiotic, tylosin, has recently been advocated as a supplement to canned food preservation. The claim that it is not used medically is not quite valid since it is closely related to erythromycin and oleandomycin and shares some cross resistance with these medical antibiotics. The spectrum of tylosin is somewhat broader than nisin and subtilin and should be further investigated along with other canning antibiotics. In general, published reports on tylosin and tylosin residues in foods are promising (Denny et al., 1961; Greenberg and Silliker, 1962a, b, c ) . This antibiotic is also used in animal feeds, particularly for swine. It has also been advocated for prophylactic and therapeutic use in pleuropneumonialike organism (PPLO) diseases of poultry. At the present time,

114

HERBERT S. GOLDBERG

tylosin and oleandomycin show a minor role in nonmedical usage of antibiotics.

F. RESEARCHNEEDS Because of the paucity of noncommercial laboratories working in the area of nonmedical use of antibiotics, there are many unanswered questions. The comments in the preceding pages are based upon the author’s own investigations and the published literature. It wouId appear, however, at this time that considerable studies are needed to further results, protect the public health, and expand the worlds food supply. The necessary public health studies fall into three categories.

1. Laboratory Studies Most needed here are acceptable standardized methods for the assay of antibiotics in all types of foods, Obviously, intelligent evaluation of residues cannot be made until such tests are available and standardized. The techniques of the F.D.A., available on request, go a long way in this direction.

2. Animal Studies These must be directed to acute and chronic oral toxicity studies with the antibiotic compounds and their breakdown products. These studies should be oriented toward finding safe levels. If it is clear that levels are going to be present, what are the safe ranges toxicologically? 3. Human Studies

Studies such as those completed by Kuwahara et al. (1958), Knothe (1957, 1958), and Goldberg (1962; Goldberg et aZ., 1961) should be expanded and extended by direct studies on human volunteers. One can take the laboratory and animal data and achieve meaningful results. However, studies on resistance, toxicity, and hypersensitivity should be encouraged in human volunteers whenever possible.

REFERENCES Ainsworth, G . C., and Sneath, P. H. A. (1962). “Microbial Classification.” Cambridge Univ. Press, London and New York. Anderson, A. A., and Michener, H. D. (1950). Food Technol. 4, 188.

NONMEDICAL USES OF ANTIBIOTICS

115

Ayres, J. C. ( 1962). “Antibiotics in Agriculture” (Woodbine, ed.), pp. 244266. Buttenvorths, London. Barnett, S. M. (1960). Wisconsin Med. J. 59, 421. Bonde, R. (1953). Am. Potato J . 30, 143. Brian, P. W., Curtis, P. J., and Hemming, H. G. (1946). Trans. Brit. Mycol. SOC. 29, 173. Brody, H. D., and Francis, F. J. (1956). Pre-Pack-Age 10, 29. Broquist, H. P., and Kohler, A. R. (1954). Antibiotics Ann. 2953-54, p. 409. Burroughs, J. D., and Wheaton, I. E. (1951). Canner 112, 50. Byrne, A. F., and Koch, R. B. (1962). Science 135, 215. Cameron, E. J., and Bohner, C. W. ( 1951). Food Technol. 5, 340. Carey, B. W. (1958). Intern Congr. Biochem. 5th Congr., p. 208. Carlson, C. W. (1957). Feed Age 7, 42. Carroll, V. J., Benedict, R. A., and Wrenshall, C. L. (1957). Food Technol. 11, 490. Christensen, J. J. (1956). First Intern. Conf. Antibiotics in Agr., Proc. Natl. Acad. Sci. 397, 13. Clegg, F. G. (1962). In “Antibiotics in Agriculture” (Woodbine, ed.), pp. 361-369. Buttenvorths, London. Clem, L. W. et al. (1961). Proc. Soc. Exptl. B i d . Med. 108, 762. Coats, M. E. (1962). In “Antibiotics in Agriculture” (Woodbine, ed.), pp. 203-208. Butterworths, London. Coats, M. E., Davies, M. K., and Kar, S. K. (1955). Brit. J. Nutrition 9, 110. Cox, R. S. (1955). Plant Diseuse Reptr. 39, 421. Cruickshank, R. (1960). Brit. Med. Bull. 16, 79. Denny, C. B. et al. (1961). Food Technol. 15, 338. Fernando, R., and Jacquet, J., Jr. (1958). Reu. Hyg. Med. SOC. 6, 77. Forbes, J. J., Parks, J. T., and Lev, M. (1959). Ann. N . Y. Acad. Sci. 78, 321. Gold, W. et al. (1956). Antibiotics Ann. 1955-56, p. 579. Goldberg, H. S., ed. ( 1959). “Antibiotics, Their Chemistry and Non-medical Uses.’’ Van Nostrand, Princeton, New Jersey. Goldberg, H. S. (1962). In “Antibiotics in Agriculture” (Woodbine, ed.), pp. 389-400. Butterworths, London. Coldberg, H. S., and Barnes, E. M. (1962). Abstr. Intersci. Conf. Antimicrobial Agents and Chemotherapy, Am. SOC.Microbiol. p. 46. Goldberg, H. S., Read, B. E., and Goodman, R. N. (1958). Antibiotics Ann. 1957-58, p. 144. Goldberg, H. S., Goodman, R. N., and Lanning, B. (1959). Antibiotics Ann. 1958-59, p. 930. Goldberg, H. S . et al. (1961). Antimicrobial Agents and Chemother. 1961, pp. 80-88. Goodman, R. N. (1959). In “Antibiotic, Their Chemistry and Non-medical Uses” ( H . S. Goldberg, ed.), Van Nostrand, Princeton, New Jersey. Goodman, R. N. ( 1962). In “Antibiotics in Agriculture” (Woodbine, ed.), pp. 165-180. Butterworths, London.

116

HERBERT S. GOLDBERG

Goodman, R. N., and Dowler, W. N. (1960). Intern. Congr. Plant Protect. 2, 1559. Goodman, R. N., and Goldberg, H. S. (1960). Phytopathology 50, 851. Goodman, R. N., Johnston, M. R., and Coldberg, H. S. (1958). Antibiotics Ann. 1957-58,p. 236. Gordon, H. A., Wagner, M., and Wostmann, B. (1957). Antibiotics Ann. 195657 p. 218. Greenberg, R. A., and Silliker, J. H. (1962a). J. Food Sci. 27, 64. Greenberg, R. A,, and Silliker, J. H. (1962b). Food Sci. 27, 60. Greenberg, R. A., and Silliker, J. H. (1962~).Bacteriol. Proc. p. 1963. Hamilton, J. M. et al. (1956). Science 123, 1175. Hazen, E. L., and Brown, R. (1951). Proc. Soc. Exptl. Biol. Med. 76, 93. Hines, L. R. (1956). Proc. Natl. Acad. Sci. U.S. 397, 227. Ingram, M., Barnes, E. M., and Shewan, J. M. (1956). Food Sci. Abstr. 28. Joliffe, N. et al. (1956). Antibiotics Ann. 1955-56, p. 19. Jones, S. M., and Woodbine, M. (1961). Vet. Res. Ann. 7, 39. Jukes, H. G., Hill, D. C., and Branian, H. D. (1956). Poultry Sci. 35, 716. Jukes, T. H. (1955). “Antibiotics in Nutrition,” M. D. Encyclopedia, New York. Knothe, H. (1957). Deut. Med. Wochschr. 82, 1685. Knothe, H. (1958). Arzth. Wochschr. 13, 179. Koch, G., and Carroll, V. J. (1957). Antibiotics Ann. 195657, p. 1010. Kuwahara, S. et al. (1958). Japan. J . Microbiol. 2, 225. Leaders, F., Pittinger, C. B., and Long, J. P. (1960). Antibiot. Chemotherapy 10,503. Loughlin, E. H.et al. (1958). Antibiotics Ann. 1957-58, p. 95. Luckey, T. D. (1956). Proc. Natl. Acad. Sci. U . S. Luckey, T. D. (1959). In “Antibiotics, Their Chemistry and Non-medical Uses” (H. S. Goldberg, ed.), Van Nostrand, Princeton, New Jersey. Lutz, A. et al. (1958). Ann. Inst. Pasteur. 94, 44. McMahan, et al. ( 1956). Antibiotics Ann. 1955-56, p. 727. Mattick, A. T. R., and Hirsch, A. (1944). Nature 154, 551. Moore, P. R., Evenson, A., Luckey, T. D., McCoy, E., Elvehem, C. A., and Hart, E. B. (1946). J. Biol. Chem. 165, 437-441. Perlman, D.et al. (1961). Proc. Soc. Exptl. Biol. Med. 106, 880. Pramer, D. (1959). Adoan. Appl. Microbiol. 1, 75. Renoux, G. (1960). Arch. Inst. Pasteur. Tunis 37, 23. Rhodes, A. (1962). In “Antibiotics in Agriculture” (Woodbine, ed.), pp. 101-121.Buttenvorths, London. Schwachman, H., and Kulczycki, L. L. (1958). Am. J. Diseases Children 96, 6. Schwachman, H., Foley, G. E., and Cook, C. D. (1951). J. Pediat. 38, 91. Schwachman, H. et al. (1952). J. Am. A . 149, 1101. Seaman, A., and Woodbine, M. (1962). In “Antibiotics in Agriculture” ( Woodbine, ed. ), pp. 333-346.Buttenvorths, London. Shewan, J. M., Hodgkiss, W., and Liston, J. (1954). Nature 173, 208. Shewan, J. M., Hobbs, G., and Hodgkiss, W. (1960). J . Appl. Bacteriol. 23, 379.

NONMEDICAL USES OF ANTIBIOTICS

117

Smith, H. W., and Crabb, W. E. (1957). Vet. Res. 69, 24. Smith, L. W. (1953). Phytopathology 42, 475. Sprunt, D. H., and McVay, L. V. (1953). Antibiotics Ann. 1953-54, p. 273. Stowens, D. (1951). Pediatrics 8, 60. Tarr, H. L. A. et al. (1952). Food Technol. 6, 363. Taylor, J. H., and Gordon, W. S. (1955). Nature 176, 312. Tomiyama, T., Kuroki, S., and Nomura, M. (1955). Bull. Japan. SOC. Sci. Fisheries 21, 958. Turbitt, P. A,, Seaman, A., and Woodbine, M. (1959). J . Appl. Bacteriol. 22. Turbitt, P. A., Seaman, A,, and Woodbine, M. (1960). Dairy Sci. Abstr. 22, 543. Weiser, H. H. et al. (1953). Food Technol. 7, 495. Whiffin, A. J., Bohonos, N., and Emerson, R. L. (1946). Phytopathology 44, 509. Williams, D. I. (1960). Practitioner 184, 383. Williams, W. L. et al. (1953). Federation PTOC. 12, 290. Wix, P., and Woodbine, M. (1955). Brit. Vet. J. 111, 432. World Health Org. Tech. Rept. (1960). Milk Hyg. No. 197. World Health Org. Tech. Rept. (1963). Public Health Aspects of the Use of Antibiotics in Food and Feedstuffs No. 260. Wrenshall, L. ( 1959). In “Antibiotics, Their Chemistry and Non-medical Uses” ( H . S. Goldberg, ed.), Van Nostrand, Princeton, New Jersey. Zaumeyer, W. J. (1956). Proc. Intern. Conf. Antibiotics, 1st Conf. pp. 171187.

This Page Intentionally Left Blank

Microbial Aspects of Water Pollution Control K. WUHRMANN Institute of Water Supply, Sewage Purification and Water Pollution Control at the Swiss Federal lnstitute of Technology, Zurich, Switzerland

I. Introduction ........................................... 11. Aspects of Microbiological Waste Treatment . . . . . . . . . . . . . . . A. Sewage as a Growth Medium ........................ B. Transport of Metabolic Substrates to Microorganisms in Purification Systems ................................ C . Kinetics of Substrate Removal from Dilute Media by Mixed Cultures .......................................... 111. The Problem of Slowly Decomposable Substances in Wastes . . . A. Physiological Considerations .......................... B. Process Considerations .............................. IV. The Removal of Nitrogen from Wastes-A Special Contribution of Microbes to Pollution Control .......................... References ............................................

119 120 122 125 129 133 135 137 141 150

1. Introduction At the National Conference on Water Pollution in 1960 Professor G . M, Fair from Harvard University formulated the sanitary engineer’s expectations in regard to microbiology in water pollution control as follows: ..... it is these biological workmen (microbes) to which we look for returning our lakes, streams and tidal estuaries to natural cleanliness by themselves or for removing even the most fractious substances committed to water by household and manufactury in treatment works constructed so as to provide the most favorable environment for the operations of these beneficient microorganisms.” This confidence in the omnipotence of microbes is certainly a great challenge for the microbiologist. Unfortunately, he has to pour some water into the wine, however, because what will be the worth of a philosophy that all organic compounds can be attacked and finally decomposed by microorganisms, when the rates of decomposition might be infinitesimally small? And it is just our present experience that each day numerous substances from the production line of the chemists have to be classified as microbiologically “ h a r d compounds. Assuming the microbes to be the workmen for keeping lakes and streams clean, it is obviously man’s 119

120

K. WUHRMANN

duty, therefore, not to impose on these workmen a task they are simply not able to fulfill-at least not at the pace which the unreasonable behavior of man is requiring from them. The participation of microbiological biocoenoses in so-called ‘‘self-purification” reactions in surface and underground water and the techniques of biological waste treatment represent the two major fields in which microbes are directly involved in water pollution control, Space does not allow coverage of both of these aspects, and it was the choice of the author to concentrate on questions related to the active utilization of microbes for pollution abatement in biological waste purification processes. We have looked upon the subject from a rather pragmatic point of view because it is the main aim of this article to awaken the interest of more microbiologists in the physiological, ecological, and process problems raised by the mixed fermentations utilized in many phases of active water pollution control measures. There has been, and is still, too much engineering and too little microbiology in this field of environmental sanitation. The engineers are not mainly responsible for this situation. It seems to the author that it results much more from the attitude of microbiologists, which has led to the peculiar situation that aerobic and anaerobic mixed fermentations have been run in waste purification on a tremendous scale for nearly a century on an entirely empirical basis. The application of microbiology in water pollution control is, however, a very challenging subject for microbiologists because the ecosystem is relatively simple (at least in comparison to soil) and numerous possibilities for an experimental approach exist. The following sections may give an impression of some of the problems involved. The choice of subjects discussed is somewhat arbitrary; it was the intention, however, to stimulate the discussion of a few questions which are of immediate practical interest.

11. Aspects of Microbiological Waste Treatment Procedures of biological waste treatment are the oldest and also by far the largest application of so-called continuous fermentations. There exists, however, a gap between the theoretical findings in this field of applied microbiology and the traditional techniques of waste purification. It is felt, therefore, that the systematics of procedures, nomenclature, and mathematical treatment of processes used in

MICROBIAL ASPECTS OF WATER POLLUTION CONTROL

121

fermentation technology could shed new light on the empirical and often prejudiced handling of biological waste treatment problems. Certain difficult tasks, such as the elimination of slowly decomposable substances from wastes in which the bulk impurities consist of easily fermentable material, cannot be resolved economically without previous theoretical considerations on the dynamics of continuous processes. But even in modem texts on waste purification no reference is made to the already extensive literature in this field. It is to the merit of Herbert (1961) to have included waste treatment in the discussion on continuous fermentations. Downing and Wheatland (1962) were the first to try a mathematical approach to the activated sludge process on such a theoretical base. Downing, Painter, and Knowles (1963) showed a fruitful application on this approach in a discussion of nitrification in activated sludge plants. We shall come back to this important article in a later section. The paper published by Porges (1960)in Volume 2 of this series gives a comprehensive description of the general layout of an activated sludge plant. Further information on present-day types of sewage works appears in this article and in textbooks such as Imhoff and Fair (1947). Based on the nomenclature used by Herbert ( 1961), all systems of modern biological sewage treatment works are “open continuous systems with feed-back.” In activated sludge processes the microorganisms are returned to the fermentors after separation from the treated liquid in a settling tank. Trickling filters use packed towers with fixed growth. Among activated sludge plants, single or multiphase homogeneous systems (“stirred fermentors” ) and heterogeneous systems (“pipe flow fermentors”) are applied. Both systems work with either single or multiple substrate additions. The unaerobic treatment of the solid residue from waste treatment is traditionally carried out in homogeneous fermentors ( sludge digestion tanks). This phase is still practiced in many places on the basis of batch treatment, and true continuous fermentation with an open system is restricted to modern large plants. More recently, “closed systems” have been introduced into the activated sludge technique with the so-called complete oxidution plants, These installations are used for the purification of small amounts of sewage or industrial wastes (for instance milk processing wastes) from isolated homes, hotels, or small factories. They are

122

K. WUHRMANN

dimensioned in such a way that sludge growth and sludge losses

by autolysis and effluent suspended solids are equilibrated. Application of the mathematical theory of continuous fermentation to the above operation schemes of sewage plants in a quantitative sense is extremely difficult. Some of the reasons might be found on the next pages. However, in spite of the present obstacles, it seems to the author that further research in the line of Herbert (196l), and Downing et al. (1962,, 1963) will be highly rewarding for a more extended understanding and a more efficient application of the principles of the activated sludge process. The following sections have been written to describe some properties which we consider essential for the comprehension of the inherent possibilities and limitations of the mixed fermentation systems in waste treatment plants. AS A. SEWAGE

A

GROWTHMEDIUM

By far, the largest volume of polluted water to be treated in sewage purification plants is of domestic origin. In many cities the admixture of industrial wastes may influence, of course, the composition of the combined sewage considerable by adding new chemicals or by changing the proportion of substances already present. As a basis for the later discussion of the biological treatment processes it is worthwhile to consider briefly the composition of a city sewage from the microbiological point of view. It is a peculiar situation that our information on the kind and concentration of individual compounds in sewage is extremely scarce. To some extent this is certainly due to analytical difficulties. The main reason for this lack of knowledge is, however, the predominantly technical point of view which governs most studies on waste treatment processes (even those of microbiological basis). This situation has favored the use of over-all parameters for measuring “pollution” such as BOD’, oxidizability, and COD2, parameters which give only rather limited or no information on the properties of a waste as a substrate for fermentation. Painter and Viney (1959) and Painter, Viney, and Bywater BOD, = Biochemical Oxygen Demand in five days, (oxygen consumed by the respiration of the microorganisms in a water sample within 5 days at ZO’C). 2 COD = Chemical Oxygen Demand, (oxygen consumed by organic constituents in a water sample in an oxidation reaction with a strong oxidizing agent, i.e. chromic acid or bichromate-sulfuric acid at boiling temperature).

123

MICROBIAL ASPECTS OF WATER POLLUTION CONTROL

(1960) have published analytical data which give for the first time concentration values for defined groups of compounds in domestic sewage. The amounts of individual amino acids in city sewage from India have been investigated earlier by Sastry, Subrahmanyan, and Pillai (1958). Although their figures can hardly be compared with the situation in Western countries, they give an idea of the relative distribution of amino acids in sewage. In Table I some findings of Painter et nl. are summarized. TABLE I COMPOSITION OF WHOLESEWAGE, STEVENAGE, HERTS.,ENGLAND, 1959a

Constituent Carbohydrates total Amino acids Free Bound Higher fatty acids Soluble acids Esters Anionic surface active agents Amino sugars Amides Creatinine

Organic C in solution (p.p.m. 1 30

Organic C in suspension (p.p.m. 1 14

Organic C, total (p.p.m.1 44

3 7 0 23 0 12

0 21.5 73 5 34 4

3 28.5 73 28 34 16

0 0 3

0.7 1.3

0.7 1.3

0

3

Total By analysis 94 211.5 305.5. By addition 78 153.5 231.5 17 27.5 24.2 Unaccounted for ( % ) a From Painter and Viney (1959), mean values of the two samples presented by the authors.

In spite of the gross grouping of compounds, 17% of the organic carbon in solution could not be accounted for, showing that considerable amounts of substances which might or might not be fermentable are still of unknown character. The soluble and insoluble “carbohydrate” group has been further investigated qualitatively and it was found that in the soluble fraction glucose and sucrose dominate ( ca. 50% of the anthrone-positive compounds), followed in concentration by lactose and galactose. Arabinose, xylose, and fructose were also detected. The sewage analyzed by the authors was rather concentrated. By comparison, based on

124

K. WUHRMANN

total organic carbon or organic nitrogen, a population equivalent of sewage flow of only about 120 to 125 liters per capita per day can be estimated. Since the organic carbon to nitrogen ratio is nearly identical with values found, for instance, in sewage of American cities or of the city of Zurich (ca. 400 liters per capita per day), concentrations of some individual compounds in those more diluted waste waters can be calculated from the values in Table I. In Table I1 concentration values of some groups of substances which are of special interest as microbial substrates are assembled. ESTIMATE OF

Component Carbohydrates as glucose Aminoacids as alanine Soluble fatty acids as acetate NH,~

THE

TABLE I1 CONCENTRATION OF MICROBIALSUBSTRATES IN DOMESTIC SEWAGE@ Concentration (mmoles/liter ) in sewage flows ( liters/capita/day ) of: 150 250 400 0.35

0.2

0.13

0.22

0.14

0.08

0.8

0.46

0.3

1.9

1.1

0.7

Based on the analysis by Painter and Viney (1959) and a comparison of Organic C concentrations in sewage of the cities of Zurich (1960) and Stevenage. @

The values in Table I1 represent rough approximations only. They demonstrate, however, quite clearly that city sewage is a poor medium in comparison to substrates usually made up for the culturing of microorganisms. This is an important observation for all further considerations concerning the kinetics of substrate removal in the purification process and the estimate of growth rates of the organisms involved. Most organic industrial wastes contain higher concentrations of individual substances and, accordingly, represent better fermentation substrates provided these substances are easily attacked by the microflora in the treatment works as is generally the case with wastes from the food and fermentation industries. It is evident from Table I1 that in domestic sewage the carbon

MICROBLAL ASPECTS OF WATER POLLUTION CONTROL

125

source is the growth-limiting factor for a heterotrophic flora. This has direct practical implications in two respects: ( a ) The small quantities of carbon compounds might lead to the expectation that with suitable amounts of microorganisms, oxidation of the pollutants will be a matter of very short fermentation time. All sewages contain, however, a considerable amount of substances rather resistant to microbial attack (e.g., certain detergents!). It will be shown later that the preferential utilization of the favorable carbon sources is a disadvantage for the biological degradation of such “ h a r d substances. ( b ) The carbon/nitrogen ratio is very low. It is evident from this fact that the problem of elimination of the fertilizing ions (NH;, NO 2, NO : 3 also phosphorus compounds) by incorporation into sludge organisms is impossible to solve. Other means obviously have to be found for the solution of this problem, which will be considered again in a later section.

B. TRANSPORT OF METABOLIC SUBSTRATES TO MICROORGANISMS IN PURIFICATION SYSTEMS The only reason why the activated sludge process can be operated under economical conditions is the peculiar property of the organisms in this system to form aggregates heavy enough to settle in a sedimentation basin. No satisfactory explanation for this behavior is available, although some papers discussing this problem have been published (reviewed by McKinney, 1956). We just have to accept the fact that, by flocculation of bacteria in an activated sludge plant, particles with a thickness of something between 0.5 to several millimeters are formed. The operation of a trickling filter also depends on the formation of organism films adhering to the filter stones. Such pellicles may grow to cell layers more than 1 mm, thick, depending on filter operation, kind of waste, and filter depth. As a consequence, the majority of organisms in these aggregates are largely dependent on the diffusion rates of the substrates from the surrounding liquid through the cell masses for their metabolism. In view of the low concentration of individual substances in most wastes (including oxygen), the amounts transported per unit time will be rather small. It is of interest therefore to have an idea of the relationship of substrate transport rates and utilization rates within these cell aggregates.

126

K. WUHRMANN

The special problem of oxygen supply has been discussed by the author (Wuhrmann, 1957, 1960) on the basis of previous investigations by Warburg (1923), Fenn (1927), and Gerard (1931). Although some indispensable data on the oxygen consumption by individual cells under various nutritional conditions are lacking, an order of magnitude of tolerable diameters of cell aggregates can be calculated, assuming a minimum supply of the gas to the innermost cell of a floc. The equations given in Fig. 1 have been derived for some geometric forms of compact cell associations.

d 2 = (c, - c ; )

240 -

FIG. 1. Diffusion of metabolites into aggregates of living cells. Symbols: = concentration of substrate at the surface of the aggregates and around a cell in the center of the aggregate, respectively (mg/cm3); d = diameter or thickness of the aggregate (cm); D = diffusion coefficient of substrate (cmZ/s); a = specific consumption rate of substrate by cells (mg/cm3 . s ) . (From Wuhrmann, 1960.) c,, ci

Assuming a respiration rate of lo-* mg 02/cm3 of cells per second a t 15"C., a relationship according to Fig. 2 can be calculated. A diffusion coefficient of 5 x 10-o cm.2/second for oxygen in the floc material and a cell volume of 1 p3 (including the slime layer) has been assumed. It is easily seen from Fig. 2 that, for instance, at normal oxygen tensions of about 2 mg./liter in mixed

MICROBIAL ASPECTS OF WATER POLLUTION CONTROL

127

liquors of activated sludge plants, the innermost cell in a spherical floc will just be respiring at a critical oxygen tension of 0.1 mg./ liter when the diameter of the floc does not exceed roughly 500 p. Assuming similar critical conditions for cells at the bottom of

Concentration grudient C , - C j , p.P.m. 0 FIG. 2. Relation between floc size and oxygen supply to the innermost cell at various oxygen concentration gradients. D (assumed) = 5 x 10-6

crnz/s; temperature

= 15°C. (From Wuhrmann,

1960.)

trickling filter pellicles, the thickness of the cell layers should not exceed about 100 p. In both treatment systems the cell aggregates are generally much thicker than these dimensions. We have to

128

K. WUHRMANN

conclude, therefore, that a considerable percentage of organisms enclosed in the flocs or pellicles are supplied with oxygen far below their need and that some of them might even be submitted continuously to nearly complete anaerobiosis. An indirect proof for anaerobic conditions in seemingly fairly well-aerated activated sludge or trickling filter slimes may be seen in the reduction of nitrates and nitrites, regularly occurring in treatment plants. Similar considerations are valid for the transport of other substrates from the medium to the cells within aggregates. Because the diffusion coefficients for most organic molecules utilized by the organisms are smaller than that for oxygen, and because the concentration gradients of individual substrates may not even reach the level of oxygen, the nutrition of cells in the central parts of flocs, dependent on these substrates, will be greatly limited by diffusion rates. Monod (1942) has shown that there is no lower concentration limit for substrate use by cells for growth. It is obvious, however, from the equations in Fig, 1 that the quantities of energy substrates reaching the cells within ftocs must be far below the amount required for even half-growth rate, considering the absolute concentration normally found in sewage (Tables I and 11). This means that a large part of the cells enclosed in flocs or trickling filter slimes contribute very little to the purification activity of the whole biomass. The organisms in trickling filters are a t a particular disadvantage and it is comprehensible that the purification efficiency per volume of filter bed can be 6 to 10 times lower than that of an activated sludge aeration basin, although the total sludge mass per volume may be the same (for weights of biomass in trickling filters see Tomlinson, 1946). It would be a reasonable expectation from this discussion that sufficient turbulence to disrupt all organism aggregates in the aeration tank of an activated sludge plant would cause a higher metabolic activity of the biomass and hence a better efficiency in purification. Nothing like this has been demonstrated, however, experimentally. Emphasizing diffusion rates, such effects should nevertheless be sizable and further critical experiments seem justified. The much higher per volume efficiency of so-called high-rate filters in comparison to standard filters may be partly explained by the fact that their operation conditions allow the formation of only very thin films of organisms on the filter stones.

MICROBIAL ASPECTS OF WATER POLLUTION CONTROL

129

C. KINETICSOF SUBSTRATE REMOVALFROM DILUTE MEDIA BY MIXEDCULTURES Treatment of organic wastes has one and only one objective: the compkte removal of unwanted organic compounds from the polluted water. Therefore, the kinetics of the concentration change of these substances in contiact with the biomass in the treatment plants is highly important, It was for a long time a dogma that dissolved decomposable pollutants disappear from a suspension of activated sludge according to an exponential function. This idea, originating in observations of the change of BOD in the course of the purification process, gave rise to many speculations concerning the mechanisms of substrate removal and the reactions between the organisms and the waste substances. When pure compounds are assayed individually, it can be shown easily that their concentration change, when in contact with activated sludge ( Wuhrmann, 1956) or trickling filter slimes (Water Pollution Research Lab., 1956), is nearly linear with time. This constant removal rate can be followed to the lowest concentration limits set by the analytical methods available. Figure 3 illustrates this type of reaction. It implies automatically a proportionality between the amount of organisms in contact with the substrate and the elimination rate. Experiments prove this relationship satisfactorily (Fig. 4 ) . The domingting role of diffusion as a limiting factor for substrate supply is probably the most rational explanation for the observed “zeroorder” reaction. The system may be much more complicated, however, and interactions between growth effects and enzyme reaction kinetics (involved in the active transport of substrates into the cells) should also be considered. Furthermore, we have to emphasize that the various species of organisms, present in the mixed culture, exert their individual reaction rates with a given substance. The approximately zero-order removal is also found when substrate mixtures are assayed for the individual components. Figure 5 represents the result of such an experiment. The BOD of the supernatant solution was also included in the observations and it is interesting to see that this parameter decreases exponentially with time (in accord with accepted opinions). There is, however, no visible correlation between the BOD readings and the quality change of the substrate as a function of time. The stepwise disappearance of individual compounds from a

130

K. WUHRMANN

I

..I! Tr

250

'C

200

Butirate C,= 1150mg./liter

L Q)

.L

\

2 I5O

C

0 .c

z

c C

Tryptophan C, = 1870 mg./liter

al C

8 al

5

100

+

u)

n 2

v)

50

Glucose 0

0

I

2

3 4 Aeration time, hours

5

6

7

FIG. 3. Concentration decrease of pure compounds in suspensions of activated sludge as a function of contact time. Sludge concentrations as indicated; temperature = 20°C.; pH = 7.5. No nitrogen source added. ( From Wuhrmann, 1956.)

MICROBIAL ASPECTS OF WATER mLLunoiv CONTROL

131

waste in the course of the purification process is understandably much less pronounced in a continuous flow system, where mixing and short circuiting will occur, than in the batch experiments mentioned above. The principle nevertheless remains unchanged and leads to the following conclusions.

I = 20"

350 -

c .-0 c

0

..E

-

W

00 Sludge concentration mg /liter dry solids

FIG.4. Elimination rates of various substrates in suspensions of activated sludge as a function of sludge concentration. Substrate concentrations as indicated. Note: rates for organic N are only valid for the initial concentration. Temperature = 20°C.; pH = 7.5. (From Wuhrmann, 1956.)

1. The quality change of a waste during the purification process is a discontinuous function of treatment time. 2. Over-all parameters such as organic C, organic N, and COD (not to speak of BOD) are unsuitable for the detection of this quality change. Rather detailed analyses would be necessary to characterize the true sequence of events in a treatment plant. Some interesting consequences arise from these observations in regard to the physiological effects of a waste on the biocoenosis in receiving water bodies as a function of its degree of treatment. The biological response to the quality of the treated waste depends entirely on the presence or absence of definite compounds or groups of compounds, other ecoIogical conditions being equal. Visible

132

K. WUHRMANN

growth of heterotrophic bacteria like Sphaerotilus natans, for instance, requires a medium containing carbohydrates, carboxylic acids, or other easily fermentable carbon sources. In a continuous flow system (river) the absolute concentration of such substrates 0.4

100

0.3

500

W

s

.L

rn

W

._ \

L

W

0

c ._ -

\

!OO

5 0.2 0

E" 0" 0

E E

m

100

0.I

0

c

0

Contact time, minutes

FIG. 5 . Simultaneous elimination of pure compounds by activated sludge from a mixed substrate, and BOD decrease of the supernatant. Sludge concentration = 1470 p.p.m. dry solids; temperature = 20°C.; pH = 7.5. (From Wuhrmann, von Beust, and Ghose, 1958.)

can be very low for the induction of mass developments of the organism. Experience with natural or model water courses indicates that heavy growth will occur at substrate concentrations in the order of less than 2 p.p.m. utilizable organic carbon. From the organic carbon compounds present in domestic sewage about 60 to

MICROBIAL ASPECTS OF WATER POLLUTION CONTROL

133

70% seems to be readily available for Sphaerotilus as carbon source. Hence, diluting a raw sewage with a carbon content of 70 p.p.m. (i.e., ca. 50 p.p.m. available carbon) twenty-five times with clean river water will result in a mixture still promoting extensive growth of the bacterium. This can, in fact, be easily demonstrated in suitable assays in model rivers (Wuhrmann, 1954). Treatment of this same sewage with activated sludge until all of the more readily fermentable compounds are oxidized may result in an organic carbon content of about 10 p.p.m. in the final effluent. Only traces of substances available for Sphaerotilus will be left. When this effluent is diluted in a river to give the same organic carbon concentration as with the above mentioned twentyfivefold dilution of raw sewage, this should produce a medium unfit for Sphaerotilus. Experiments in model rivers confirm this hypothesis to a large extent as may be seen from Table 111. In this example, the effluent of a primary sedimentation basin and the final effluent of an activated sludge plant ( twenty-five- and threefold dilutions, respectively) were simultaneously assayed in model rivers with identical ecological conditions (flow velocity, river bed, temperature, etc. ). The mean composition of the water mixtures within the &weeks observation period is given in the column headings of the table for the two channels. Comparison of the analytical findings with the biological observations demonstrates the entirely different effect of the two waters from the ecological point of view although their organic C, organic N, BOD, COD, etc., were quite similar. No better proof could be given for the famous saying, “Dilution is no solution against pollution”! 111. The Problem of Slowly Decomposable Substances in Wastes

It is an increasing concern of the persons responsible for water pollution prevention that, in spite of all efforts in the realization of waste treatment projects, the absolute amount of organic pollutants gaining access to surface waters is steadily increasing. Growth of population and expansion of industries, on one hand, and the incomplete degradation of waste compounds in treatment works, on the other hand, counteract the lightening of the burden on our streams intended with the construction of purification plants. The so-called “refractory” materials still present in even highly

134

K. WUHRMANN

TABLE I11 BIOLOGICAL RIVERTESTON THE BIOCOENOLOGICAL EFFECTOF TREATED AND UNTREATED DOMESTICSEWAGE(SEASON: SEPTEMBER + OCTOBER1958) Analysis of channel water below waste outfall (average of 6 weeks) Test channel 3a

Component

Test channel 3 (4 % settled sewage in ground water; u = 28 cm/sec)'

( 33% biologically

treated sewage in ground water; v = 28 cm/sec)

BOD (p.p.m. 0) Organic C (p.p.m. C ) Organic N (p.p.m. N )

3.7 2.6 0.41

2.9 3.2 0.44

NH,+ (p.p.m. N )

0.47

0.2

NO2-+ NO3- (p.p.m. N )

1.0

3.6

Microflora and microfauna (3 0 meters below waste addition)

Macroscopic aspect Relative mass of Heterotrophs Autotrophs Bacteria Sphaerotilus natans Protozoa Carchesium sp. Vorticella sp. Euplotes sp. Diatoms Cocconeis sp. Gomphonema sp. Nitzschia sp. Tabellaria ( capucina) Navicula sp. Green algae Vaucheria sp. Tribonema sp. Spirogyra sp. Hormidium sp. Stigeoclonium sp. u

= Flow velocity in channel.

Test channel 3 ( 4 % settled sewage in ground water; v = 28 cm/sec)

Test channel 3a (33% biologically treated sewage in ground water; v = 28 cm/sec)

Grossly polluted

Clean

60%

-

40%

100%

+++++ + -

+++ ++ +++ +++ ++ + ++++ ++++ + -

+f f

++ ++ ++ -

++ +

MICROBIAL ASPECTS OF WATER POLLUTION CONTROL

135

purified effluents of municipal sewage works (see, for instance, Bunch, Barth, and Ettinger, 19sO), together with numerous resistant synthetic organic chemicals from industrial wastes, are additive along the water courses, rendering water reuse more and more difficult. In many regions this problem is not yet very menacing because the presently existing high-dilution ratios in receiving water bodies mask detrimental effects. In densely populated and industrialized areas, however, the action of refractory pollutants is so evident that the further use of the water is already doubtful. It is impressive to learn from the observations of Middleton and Rosen (1956) and from the data published in the compilation of the National Water Quality Network ( 1958-1959) what quantities of organic compounds are transported by surface waters or may even be found in ground water. In view of the confidence in the possibilities of microbial action, as expressed by G. M. Fair (1960), it is the task of the microbiologists to clearly delineate the aid which can reasonably be expected from microbes for the solution of these problems and to indicate unmistakably where processes other than fermentation have to be applied, The term “refractory material” is rather vague from a physiological point of view. Some groups of compounds, such as straightchain or branched, unsaturated hydrocarbons, nitrated or chlorinated cyclic compounds, heterocyclic nitrogen compounds, and polyethers, which are found in chemical wastes, detergents, fuel, pesticides, etc., may indicate a few of the substances involved.

A. PHYSIOLOGICAL CONSIDERATIONS Different types of interactions between uncommon substrates and the organisms in a treatment plant can be imagined. ( a ) Organisms are present in the mixed culture which use the compounds in question as sole source of carbon as soon as favorable substrates such as carboxylic acids, alcohols, and carbohydrates are no longer available in the medium, This reaction would imply some sort of “diauxte” (Monod, 1942). Diauxic growth has only been observed, however, with substrates whose metabolism requires enzymes having the same precursor, True diauxie seems to be restricted, therefore, to substances of related constitution. This does not completely exclude such a reaction in waste treatment plants, but no experimental evidence exists which would prove a breakdown of waste compounds one by one.

136

K. WUHRMANN

In the literature on the microbial decomposition of hydrocarbons (see the extensive review by Fuhs, 1961), some indications are found that more favorable carbon sources may compete with the hydrocarbons for oxidation. No experiments correlating growth of the organisms with the concentration change of individual substances in the medium have been published, however, which would confirm a multiphase attack on the substrate mixture. It may be mentioned here that Gaudy ( 1962 ) has described experiments which were intended to demonstrate diauxic utilization of carbohydrate mixtures fed to activated sludge. However, the results of this study do not prove convincingly the interpretation given by the author, and the question whether diauxie really exists in a mixed fermentation (considering the entire biocoenosis as an “organism”) remains unanswered, ( b ) It is the author’s opinion that most uncommon compounds in wastes are metabolized simultaneously with the other constituents in the medium. This assumption is based on the fact that the utilization of such substrates, generally requiring enzymic adaptation, is possible only when sufficient nutrients for active growth of the organisms are present. The decomposition of dinitro-u-cresol by Corynebacterium simplex, published by Gundersen and Jensen (1956), is a good example. Although the strain could utilize the compound as carbon and nitrogen source, its dissimilation was much faster in the presence of amino acids. Furthermore, the property to oxidize dinitro-o-cresol could only be maintained in subcultures when the additional substrates were present. In Table IV another example from our own experience shows that the oxidative dissimilation of trinitrololuene by an adapted strain of Pseudumonas (isolated from soil) also depends on the presence of a favorable growth medium. The strain is capable of attacking the compound to some degree in the resting stage after adaptation, but the decomposition rate is negligible. Similar observations on the behavior of detergents in mixed culture (activated sludge or river water) have been reported by Bogan and Sawyer (1954) and by Sawyer, Bogan, and Simpson (1956). When they added synthetic sewage as a supplemental nutrient to natural river water, the microbial degradation of n-dodecyl sulfate in the water was considerably accelerated in comparison to the rate in the river water alone. The more complete

MICROBIAL ASPECTS OF WATER POLLUTION CONTROL

137

milieu obviously favored the growth of the microorganisms in the system which then utilized the detergent ( an easily decomposable one) simultaneously with the other nutrients. TABLE IV OXIDATIVE DISSIMILATION OF TRINITROTOLUENE (TNT) BY A STRAIN OF PSEUDOMONAS AS A FUNCTION OF THE NUTRIENT VALUEOF THE MEDIUM^ Casein hydrolyzate (vitamin free) in the basal mineral medium with a-TNT ( p.p.m.) 0 0.03 0.1 0.3 1 3 10 30 a

a-TNT decomposed in 16 hours at 22°C.

(%I 24.5 26.0 27.0 31.6 42.6 74.2 95.5 99.0

Germanier and Wuhrmann, unpublished results.

When enzymic adaptation of the mixed cultures in treatment plants is a major factor for the decomposition of “ h a r d components in a waste, it should theoretically be possible to increase the rate as well as the absolute amount of elimination of such substances by an amelioration of the nutrient value of the medium. Unfortunately, no experiments supporting this assumption are known to the author, and it is suggested, therefore, that some systematic investigation should be started on the behavior of slowly decomposable compounds in media of various quality for mixed cultures. The above discussion also leads to the conclusion that the operation of a fermentation process influences, to a great extent, the degree of breakdown of certain waste constituents.

B. PROCESSCONSIDERATIONS The property of “resistance” to microbial degradation in a treatment plant may be attributed to either one or both of the following factors. 1. Only a few microorganisms exist in the mixed biocoenosis of the plant which possess the enzymes for the partial or complete breakdown of a compound. Furthermore, such organisms may be highly exacting in their growth requirements.

138

K. WUHRMANN

2. The specific rate of utilization of resistant compounds (i.e., quantity decomposed per unit cell mass and unit time) may be very low even after complete enzymic adaptation of a large percentage of the organisms, In many instances more readily available substrates may compete with the utilization of “hard’ substances. Considering the theory of continuous fermentation, the two factors act differently in the process mechanism. a. When the removal of a special substrate component depends on the presence of only a few species in a mixed biocoenosis, the problem of washout of these bacteria from the system is of greatest importance, especially when they are slowly growing types. I t is evident that maintaining a special organism in a mixed flora at a desired numerical level requires a sufficient growth rate SO that production of the organism exceeds the loss with the waste sludge. In agreement with theoretical considerations, the mass of sludge to be wasted in the steady state, at given conditions of sewage strength increases with decreasing mixed liquor concentration levels and aeration time. Emphasizing the generally accepted operation scheme in activated sludge plants which prefers relatively dilute organism suspensions (in general 1000 to 3000 p.p,m. in the mixed liquor), it is clear that only species with high-growth rates are able to build up a noticeable population in the system. Slowly growing types must be washed out sooner or later in spite of the continuous feedback. The problem is similar to the situation in treatment plants where, for instance, nitrification is to be achieved, and the same theoretical considerations are valid which have been developed by Downing, Painter, and Knowles (1963) for nitrifying bacteria in the activated sludge process. In this fundamental paper the authors prove for any continuous fermentor (pipe flow or completely mixed systems) that a relatively slow-growing organism, entering the system in small numbers, can only achieve a constant population density, appreciably higher than the inflow concentration, at a high mixed liquor concentration and a low excess sludge production. I t is evident from this study, which will be quoted more extensively in the next section, that the general tendency to operate a treatment plant at the lowest sludge concentrations, compatibIe with the required BOD removal, is strictly opposed to the requirements imposed by compounds needing a special flora for their decomposition.

MICROBIAL ASPECTS OF WATER POLLUTION CONTROL

139

b. The linear relationship between the elimination rate of a fermentable substance and the concentration of the biomass (Fig. 4 ) implies automatically that, in the presence of slowly degradable waste components, a maximum quantity of biological sludge and long contact times should be available for the treatment process. Therefore, the requirements for the removal of resistant compounds in biological purification plants converge in any case to the same operation conditions, that is, the mixed liquor concentration should be maintained at such a level that a minimum of excess sludge is produced, and the contact time in the aeration basin should be as long as possible. Owing to the interactions between essential operational variables, however, the practical possibilities in sewage works for guiding the process are rather limited. Long detention times and high mixed liquor concentrations, for instance, contradict themselves because the fairly low nutrient values of most wastes limit the quantity of biomass that can be supported. A point will be reached invariably where sludge growth and sludge losses by autolysis and as suspended solids in the effluent are equivalent. Increasing the detention time beyond this stage will diminish the concentration and the activity of the sludge because of a relative increase of the portion of inert material (“humified organic solids; mineral particles from the raw sewage). With domestic sewage containing about 60 to 80 p.p.m. organic carbon (BOD of ca. 120 to 140 p.p.m.) this critical point is reached at an aeration period of about 16 to 24 hours where a mixed liquor concentration of ca. 3000-4000 p.p.m. represents steady state conditions, Although the theory of fermentation gives clear-cut operational directions for the elimination of “ h a r d compounds from wastes, it is too optimistic to believe that the capabilities of heterotrophic microorganisms could solve all problems of waste treatment. This may be exemplified with observations on the biological purification of a chemical waste. Table V contains some results from pilot plant-activated sludge plants fed exclusively the waste from a large factory producing organic intermediates and dyes. Although the plants were operated at equilibrium conditions for sludge growth and sludge loss, and the possible maximum of mixed liquor concentration was maintained, a considerable percentage of organic compounds (representing nearly half of the initial organic C and

140

K. WUHRMANN

practically all of the organic N ) reappeared in the final effluent. For BOD, the efficiency of the treatment was very satisfactory, however. It is rather doubtful whether any increase of the aeration time would have been of help for a better removal of the organic compounds, and, moreover, an increase of the sludge concentration was physiologically impossible. This experiment demonstrates, therefore, that inherent limitations in the composition of certain RESULTSOF

THE

TABLE V ACTIVATEDSLUDGE TREATMENT OF

A

CHEMICALWASTE^

Operational data: mean values of daily observations during a 6-week period Aeration time ( mixed liquor) (hours ) : 12 8 4 Sludge concentration mixed liquor ( p.p.m. ) : 2330 3300 3400 0, concentration in mixed liquor (p.p.m. 0,): 3.55 4-5 2-3 0 0 Ob Excess sludge: Sludge recirculation ( % ) : 100 100 100 Analytical data: average values from 15 composite samples (24-hours sampling time) in the operation period of 6 weeks In

Out

In

Out

In

Out

39 1OC 276 276 1Oc 276 BOD (p.p.m. 0,) : 83 145 145 62 145 65 Organic C (p.p.m. C ) : 15.4 14.4 Organic N (p.p.m. N ) : 15.2 15.4 14.6 15.4 a Mixed liquor concentration at maximum equilibrium value. Temperature 17"C., pH (inflow) 8.3. b Effluent contains considerable quantities of suspended solids ( range. 25-35 p.p.m. ). 0 Range: 5-15 p.p.m. This BOD represents nothing else but the endogenous respiration of the organisms suspended in the effluent!

wastes make it theoretically impossible to remove all of those substances which do not represent efficient carbon sources for a majority of species in a treatment plant. Unfortunately, numerous examples like the above one could be cited. In spite of the omnipotence of the microbial world to attack organic chemicals, it must be concluded, therefore, that certain purification problems cannot be solved without additional techniques such as adsorption or precipitation processes.

MICROBIAL ASPECTS OF WATER POLLUTION CONTROL

141

IV. The Removal of Nitrogen from Wastes-A

Special Contribution of Microbes to Pollution Control

Mineralization of organic impurities in wastes by microbial oxidation is no final solution to waste disposal. The fertilizing effect of some of the end products of waste decomposition on autotrophic aquatic plants may result in the formation of new organic material by photosynthesis comparable to the mass already oxidized in the treatment plant. This so-called secondary pollution is mainly caused by the nitrogen and phosphorus compounds leaving the treatment works. The relative importance of the fertilizing effect of nitrogen and/or phosphorus in lakes or rivers depends on the local situation. In arid regions, where reuse of water is imperative, an additional problem arises. The gradual build-up of the concentration of nitrogen compounds (especially NO, NO, and NH;) will eventually limit recycling of used water. Thus, an urgent need exists for coupling oxidative waste treatment with processes for the complete elimination of, for instance, P and N from effluents. The inorganic N as NHE NO, and NO, which makes up more than 90% of the total N in final effluents, may be eliminated by physicochemical methods. Economical considerations, however, show that microbiological processes will suit the needs much better. For an extensive review of the problems involved we refer to the transactions of the seminar on “Algae and Metropolitan Wastes” (1960) held at R. A. Taft Engineering Center, Cincinnati. The special subject of nitrogen elimination has been discussed recently by the author ( Wuhrmann, 1963), emphasizing the various possible methods. Within the frame of this article the elimination of nitrogen by bacterial denitrificution is of special interest because this problem involves a chain of microbial processes which must be accomplished in a continuous flow line although the reaction steps are partly of opposing nature. In this process all of the nitrogen compounds in a waste have to be oxidized during aerobic biological purification to NO, and NOST In a subsequent anaerobic fermentation these oxides are reduced by denitrifying bacteria to Nz or NzO. The final reaction products escape into the atmosphere.

142

X. W H R M A N N

Nitrogen balances of biological sewage purification plants of the activated sludge or trickling filter type show remarkable nitrogen deficits even under conventional operation conditions. These losses increase visibly with increasing concentration of NO3 in the final effluents. The earlier discussion on oxygen supply to cell aggregates (Section 11, B ) suggests that in a seemingly well-aerated milieu, which allows the extensive growth of the autotrophic and highly aerobic nitrifiers, anaerobic loci exist where denitrification might occur. This experience indicates that it is only a question of process design to favor already existing microbial reactions for eliminating most of the nitrogen in the incoming waste. The extensive literature on the physiology of nitrifying organisms has been reviewed by Meiklejohn (1954) and Lees (1954). Growth rates of nitrifying organisms are further reported by Skinner and Walker ( 1961). The biochemistry of bacterial denitrification was discussed recently in review papers by Taniguchi (1961) and Nason ( 1962). The microbial reaction steps required are summarized in the scheme of Fig. 6. Oz

Oxidot. org. compounds NH:-NO;-NO;

-4

Pr. Sed.

Aerator

-

Respir.

NO; \ .,,_/NZ.N20

I---+-\

Anaerob.

--

Sludge reclrculalion

FIG.6. Flow diagram for an activated sludge plant with denitrification,

The task is to find the optimum operation conditions for the nitrification as well as for the denitrification phase, presupposing that the normal oxidative purification process, taking care of the organic pollutants, remains undisturbed. 1. Nitrifimtion Phase

It is interesting to remember that the process of microbial nitrification was first detected in experiments with sewage purification [Schloesing and Muntz (1877) in their work on sewage purification by soil percolation]. Since then the oxidation of ammonia to NO, and NO3 in trickling filters and activated sludge plants has become common knowledge. However, the desirability of these oxida-

MICROBIAL ASPECTS OF WATER POLLUTION CONTROL

143

tion steps in waste treatment has been a matter of continual discussion. For the removal of nitrogen from wastes by biological means, nitrification is a prerequisite. I t is therefore of primary concern to optimize growth conditions for nitrifying organisms as well as to determine the time required for the complete oxidation of all the NH,f in solution. In practice the following facts must be emphasized. a. Two types of organisms have to be present in the biocoenosis (Nitrosomonas species for the reaction NH4f NO, and Nitrobucter species for the step NO, NO3 ). b. The growth-limiting substrate of the Nitrobacter species is the metabolic product of the Nitrosommas species. C . Both organism groups are embedded in the large mass of heterotrophic bacteria forming the bulk of the sludge in the purification plants. These heterotrophs have population dynamics entirely different and independent from the nitrifiers. This last point greatly complicates the fermentation system because it is not the growth of the nitrifiers which is essentially responsible for the increase of the biomass during the oxidation period, but the heterotrophic component of the mixed population. Hence, the quantity of organisms to beqreleased from the system as excess sludge in a steady state depends more or less on the growth of the heterotrophic organisms and is not a function of the growth of the nitrifiers which we are interested in maintaining and increasing in number as much as possible. In conventional activated sludge treatment of domestic sewage, the mass increase of the sludge AS is nearly independent of the mixed liquor concentration S in the aeration basin, whereas the population increase ( c m - c m o ) of a special organism m (for instance Nitrosomonas) is approximately proportional to its initial concentration cmoand to the concentration x of its growth-limiting substrate (NH; in case of N i t r o s o m m s ; NO, in case of Nitrobacter). Increasing the plant load with organic pollutants (which are transformed to sludge solids at a more or less constant proportion) means, therefore, an increase in AS, producing invariably a “dilution” of the population density of organism m. When the sludge mass AS is wasted continuously to maintain a wanted value of mixed liquor concentration S, a partial washout of the organism m must take place. This washout will occur even when the growth

-

144

K. WUHRMANN

rate of the nitrifiers during sewage treatment is sufficient to build up a population in pure culture under similar conditions. It is easy to see, however, that a population increase of organism m will take place when at a given plant load (and hence at a fixed value of AS) the sludge concentration S is increased, thus lowering the proportion AS/S. Downing et al. (1963) express this interaction by c,-c,~ C O ,

AS

3 --

S

giving a straightforward mathematical proof for this formulation. Introducing into this relation the yield equation for the growth of organisms in terms of substrate consumption c,-c,~

=Em (x-x~)

and the substrate concentration relationship in a feedback system like an activated sludge process

+

(1 p ) xo = xc+ p * x the concentration change of the critical substrate for organism m as a function of the sludge growth (i.e.) in terms of the quantity of wasted sludge) is, according to Downing et al.,

k,

t = In (1

+

G)

'

xd(AS+S)+x(AS*p-S)

where

k,

maximum growth rate constant of organism m when all nutrients are in excess p feedback in the system as a fraction of inflow quantity S mixed liquor concentration AS mass increase of mixed liquor concentration in one pass t time X concentration of limiting substrate for organism m at which growth rate is half maximum

MICROBIAL ASPECTS OF WATER POLLUTION CONTROL

145

concentration of limiting substrate in the inflow of the fermentor x concentration of the limiting substrate in the effluent xo concentration of the limiting substrate after mixing of inflow and feedback sludge Em yield constant, mass of cells formed per unit substrate utilized ~4

This equilibrium equation can also be used for calculating the population development of organism m from a starting point (where only the number of organism m which flows into the system is present) until its population equilibrium in the mixed liquor is reached. Downing showed, in fact, that an increase of Nitrosom o m must occur in the mixed population when a high mixed liquor concentration is carried, and, vice versa, that a washout of this organism is inevitable at low mixed liquor concentrations (Fig. 7 ) . The formation of NO, by Nitrobacter from the NO, produced by N i t r o s o m m has to be considered on the basis of the same mathematical relationship, Owing to the interdependence of the two reaction steps and, hence, of the growth rates of the two organisms, it is evident that the entire system is mathematically rather complicated. The authors’ way of thinking is without a doubt very promising (not only in the special case of nitrifying mixed populations). It is clear, however, that the application of this mathematical approach to the description of mixed fermentation systems stands or falls with detailed knowledge on the ecology and physiology of the organisms involved, Considering the present situation, we have to admit that much fundamental data are lacking in this respect and much more systematic research is needed before fermentations with mixed cultures, aiming at a definite fermentation product or at the dominance of a definite organism in the biocoenosis, can be planned on a sound basis. 2. Denitrification In contrast to the nitrification stage, the denitrification in the combined process is not limited by the number of denitrifying organisms in the system. According to experience a large percentage of the bacteria which make up normal activated sludge consist of facultative aerobes which may reduce NO2- and NO, if proper

K. WHRMANN

146

-E -4 z 5: 2 .-z a

I00

ul

C 0

r

0

-e

Sludge mass other than Nitrosomonas

50

C

.-

c

C

1 Nitrosomonas

0)

C

0

0

20 Time (days) 40

f

(a)

-

I

2E

ul

100

Sludge mass other than Nitrosomonas

0

0

E

51 e .-

Nitrosomonas

f

z

r

50

--e C

.C U C

0)

0

0

I

I

10

20 Time (doys)

(b)

FIG. 7. Examples of predicted variations in the concentrations of Nitros o m o m in the aeration unit of an activated sludge plant under constant operation conditions. Assumptions: BOD, of sewage L = 250 p.p.m.; mixed liquor aeration time t = 3h; growth rate constant of Nitrosomonas k, = 0.2 day-1; saturation constant for NH: X = 1 p.p.m. Yield constant Em = 0.05; NH: in settled sewage xi = 80 p.p.m.; initial concentration of Nitrosomonas cm0 = 50 p.p.m. ( a ) Activated sludge in mixed liquor = 2000 p.p.m. ( b ) Activated sludge in mixed liquor = 6000 p.p.m. (From Downing, Painter, and Knowles, 1963.)

MICROBIAL ASPECTS OF WATER POLLUTION CONTROL

147

conditions are provided. Two critical conditions must be emphasized, however. a. Provision of hydrogen donors to the denitrifiers for reduction of all NO, and NO, in solution. For some time it was believed that in a nitrifying mixed liquor sufficient quantities of respiration substrates, required as oxygen acceptors, would no longer be available. Based on this prejudice, rather complicated flow systems have been devised for feeding the denitrifying organisms with hydrogen donors (see for instance Wuhrmann, 1957b; Bringmann et al., 1959). It was later found in the author’s laboratory, however (Wuhrmann, 1960), that the endogenous reserve material in the cells can be easily mobilized for denitrification and that the quantity of these substrates is by far sufficient for the reduction of the relatively small quantities of nitrites and nitrates in nitrified sewage. The rate of reduction is of course somewhat dependent on the kind of substrate available (Fig. 8 ) . For practical purposes, however, these differences are not relevant, 2. Ecological conditions for denitriikation. In view of the practical application of the process, the sensitivity to oxygen of the reduction of nitrites or nitrates is the most pertinent factor to be considered. The presently available information on this point is rather conflicting, It is generally assumed that denitrification is an anaerobic process which is greatly inhibited by rather low oxygen tensions in the medium. Several strains isolated from activated sludge were affected, however, to a variable extent and no general role could be established (literature in Mechsner and Wuhrmann, 1963). Recent findings ( Wuhrmann and Mechsner, 1964) indicate that the pH of the environment is a clue factor for the action of oxygen on the reducing processes, and many of the previously contradictory results may be due to this interaction. At the pH normally found in activated sludge systems (usualIy around neutrality), oxygen inhibition of denitrification seems to be maximum. In contrast, denitrification rates at pH values below 6 are nearly independent of oxygen tension. This explains the practical observation that traces of oxygen in the mixed liquor of a sewage treatment plant sharply diminish the denitrifkation rate although the literature does not report such a pronounced oxygen sensitivity of the pure strains. From the point of view of plant operation the requirement for

148

K. UTUHRMANN

nearly complete anaerobiosis in the denitrification phase is rather inconvenient because the mixed liquor leaving an aeration basin carries appreciable amounts of oxygen. Fortunately, the respiration of the sludge uses up this oxygen in a fairly short time (depending

Minutes

FIG.8. Reduction of nitrate by activated sludge from a nitrifying plant, N atmosphere; temperature = 25°C.; pH = 7.3. Substrates: A, 8 pmoles NO,; B, 8 pmoles NO34.4 pmoles glucose; C, 8 pmoles NO3- + 4.4 pmoles glucose 0.4 mg. Difco Tryptose. Sludge volume, 2 ml. (From Wuhrmann, 1960.)

+

+

on the endogenous respiration rate of the sludge), thus conditioning itself for the reducing reactions. Considering the opposing ecological conditions in a continuous process which combines strictly aerobic steps with highly reducing

149

MICROBIAL ASPECTS OF WATER POLLUTION CONTROL

and anaerobic phases in the same flow chain, the entire process of nitrogen removal by denitrification is surprisingly simple to operate. An arrangement according to Fig. 6 gives quite convincing results as may be seen from Fig. 9. Experience shows that a further perfection of the process depends mostly on the improvement of the nitrification step which for the moment is the less reliable phase in the reaction chain. 3(

2!

2c

g -

1

I!

P IC

C

IOC

E

5(

2

ac

c 10

20

July

- Jutllef

10

20

Augur1

- AoPI

10

20

September - Septernbre

I0 October

20

- Octobre

10 November

20

- Novembre

FIG.9. Continuous denitrscation in activated sludge treatment of domestic sewage, according to flow diagram of Fig. 6. Hydraulic load of aeration basin = 10 m3/m3 . d; BOD load, aeration basin = 1.2 kg BOD/m3 * d; mixed liquor concentration (aeration basin and denitrification basin) = 3000 p.p.m.; recirculation rate = 100%; temperature in aeration basin = 15.5"C.; detention time in denitrification basin = 2 hours. For comparison the values for total N and N elimination in a parallel plant with conventional flow diagram and identical operation parameters are indicated with dashed lines.

150

K. WUHRMANN

Nitrogen losses by microbial denitrification from soil and water are well known, The realization of this process in an entirely artificial continuous flow system with relatively short detention times is encouraging. It supports the hope that other combined fermentation chains may be found for difficult purification problems. Already well-known multiphase processes, operating rather uneconomically at present, such as the methane fermentation of the solid residues from sewage works (“sludge digestion”), may also be improved considerably by a proper application of the principles of continuous fermentation.

REFERENCES “Algae and Metropolitan Wastes.” ( 1960). Trans, Seminar. Taft Eng. Center, Cincinnati. Bogan, R. H., and Sawyer, C. N. (1954). Sewage lnd. Wastes 26, 10691080. Bringmann, G., Kuehn, R., and Wagner, B. (1959). Gesundh. lngr. 80, 364-367. Bunch, R. L., Barth, E. F., and Ettinger, M. B. (1960). 3rd Manhattan Conf. on Biol. Waste Treatment, Paper No. 8, New York, 1960. Downing, A. L., and Wheatland, A. B. (1962). Trans. Inst. Chem. Engrs. (London) 40, 91-103. Downing, A. L., Painter, H. A., and Knowles, G. (1963). J . Proc. Inst. Sewage Purif. pp. 2-25. Fair, G. M. (1960). Proc. Natl. Conf. Water Pollution pp. 452-453. Washington, D. C., 1960. Fenn, W. 0. (1927). J. Gen. Physiol. 10, 767-779. Fuhs, G. W. (1961). Arch. Mikrobiol. 39, 374-422. Gaudy, A. F. (1962). Appl. Microbiol. 10, 264-271. Gerard, R. W. (1931). Biol. Bull. 60,245-268. Gundcrsen, K., and Jensen, H. L. (1956). Acta Agr. S c a d . 6, 100-114. Herbert, D. ( 1961 ). In “Continuous Culture of Microorganisms,” Monograph No. 12, pp. 21-53. SOC. Chem. Ind., London. Imhoff, K., and Fair, G. M. (1947). “Sewage Treatment.” Wiley, New York. Lees, H. (1954). In “Autotrophic Microorganisms” (B. A. Fry and J. L. Peel, eds.) pp. 84-98. Cambridge Univ. Press, London and New York. McKinney, R. E. ( 1956). In “Biological Treatment of Sewage and Industrial Wastes” (J. McCabe and W. W. Eckenfelder, eds.), Vol. 1, pp. 88-100. Reinhold, New York. Mechsner, Kl., and Wuhrmann, K. (1963). Pathol. Microbiol. 26, 579-591. Meiklejohn, J. ( 1954). In “Autotrophic Microorganisms” (B. A. Fry and J. L. Peel, eds.) pp. 68-83. Cambridge Univ. Press, London and New York. Middleton, F. M., and Rosen, A. A. (1956). Public Health Re@. 71, 11251133.

MICROBIAL ASPECTS OF WATER POLLUTION CONTROL

151

Monod, J. ( 1942). “Recherche sur la croissance des cultures bactBriennes.” Hermann, Paris. National Water Quality Network. (1958-1959). U.S. Public Health Sera Publ. NO. 663. U. S . Dept. of Health, Education, and Welfare, Washington, D. C. Nason, A. (1962). Bacteriol. Rev. 26, 16-41. Painter, H. A., and Viney, M. (1959). J . Biochem. Microbiol. Tech. Eng. 1, 143-162. Painter, H. A., Viney, M., and Bywater, A. (1960). J . Proc. Inst. Sewage Purif. pp. 3-11. Porges, N. (1960). Advan. Appl. Microbiol. 2, 1-30. Sastry, C. A., Subrahmanyan, P. V. R., and Pillai, S. C . (1958). Sewage Ind. Wastes 30, 1241-1247. Sawyer, C. N., Bogan, R. H., and Simpson, J. R. (1956). Ind. Eng. Chem. 48, 236-240. Schloesing, T., and Miintz, A. (1877). Compt. Rend. Acad. Sci. 84, 301. Skinner, F. A., and Walker, N. (1961). Arch. Mikrobiol. 38, 339-349. Taniguchi, Sh. ( 1961 ). 2. Allgem. Mikrobiol. 1, 341-375. Tomlinson, T. G. (1946). J . PTOC. Inst. Sewage Purif. pp. 1-12. Water Pollution Research Lab. ( 1956). Stevenage Herts. Ann. Rept. 1955. H.M.S.O., London. Warburg, 0. (1923). Biochem. 2. 142, 317. Wuhrmann, K. (1954). Sewage Ind. Wastes 26, 212-220. Wuhrmann, K. ( 1956). In “Biological Treatment of Sewage and Industrial Wastes” (J. McCabe and W. W. Eckenfelder, eds.), Vol. 1, pp. 49-65. Reinhold, New York. Wuhrmann, K. ( 1957a). 2. Allgem. Pathol. Bakteriol. 20, 567-576. Wuhrmann, K. (1957b). Schweiz. 2. Hydrol. 19, 409-427. Wuhrmann, K. (1960). 3rd Manhattan Conf. on Bwl. Waste Treatment Papel. No. 3, New York, 1960. Wuhrmann, K. ( 1963 ) , Verhandl. Intern. Ver. Limnol. 15, 580-596. Wuhrmann, K., van Beust, F., and Chose, T. K. (1958). Schweiz. 2. H y drol. 20, 284-310. Wuhrmann, K., and Mechsner, K1. (1964). Path. Microbiol. 27 (in press).

This Page Intentionally Left Blank

Microbial Formation and Degradation of Minerals MELVINP. SILVERMAN AND HENRY L. EHRLICH Pittsburgh Coal Research Center, U . S . Bureau of Mines, Pittsburgh, Pennsylvunia and Depadment of Biology, Rensseluer Polytechnic Institute, Troy, New York 153 I. Introduction ........................................... 154 11. Geological Concepts .................................... 111. Mechanisms of Microbial-Mineral Interactions .............. 157 158 A. Enzymic Processes .................................. 159 B. Nonenzymic Processes ............................... IV. Survey of Microbial Interactions with Inorganic Substances .... 161 170 V. Sulfur Mineral Deposits ................................. A. Elemental Sulfur ................................... 170 B. Metal Sulfides ..................................... 174 186 C. Metal Sulfates ..................................... 187 VI. Iron and Manganese Deposits ............................ A. Occurrence and Associated Biological Activities ......... 190 B. Mechanisms of Microbial Interactions with Iron and Man192 ganese Minerals .................................... 197 C. Economic Importance ............................... 198 VII. Conclusion ............................................ 198 References ............................................

1. Introduction It has been characteristic of microbiology and other disciplines that the tempo of man’s research activities more often than not has been in direct proportion to his needs and his welfare. Thus the diseases of man received early emphasis by microbiologists. Man’s dependence upon agriculture for his food supply gave impetus to soil microbiology with special emphasis on plant diseases and soil fertility and resulted in establishing the importance of microorganisms in the cycles of carbon, nitrogen, phosphorus, and sulfur in nature. Much less attention was paid to other natural activities of microorganisms except where such obvious occurrences as the evolution of HZS, Hz, and CH, from stagnant bodies of water or the deposition of iron oxides in swamps and bogs demanded attention. By and large microbiologists concerned themselves with the metabolism of organic matter and virtually ignored the inorganic environment. Happily, not all were so engaged; the classic studies of microbiologists such as Winogradsky, Beijerinck, Kluyver, Van Niel, and 153

154

MELVIN P. SILVERMAN AND HENRY L. EHRLICH

others on the microbial metabolism of inorganic compounds coupled with the ability of these researchers to discern the broader implications and unifying concepts of their findings laid a solid foundation for subsequent studies of the geological activities of microorganisms. Man's present concern with relatively unexplored regions of the earth such as the oceans and polar regions, his efforts to discover additional sources of mineral wealth to replace those threatened with depletion, and the possibility of discovering extraterrestrial life during his imminent exploration of outer space have all focused renewed attention on the activities of microorganisms in the inorganic environment. This essay is not intended as an exhaustive survey of the literature but considers those papers which provide a basis for our present knowledge of geomicrobiology. The role of microorganisms in transformations of minerals in nature is discussed with the main emphasis on mineral concentrations of economic importance to the mining industry. Microbial transformations of more diffuse mineral accumulations as they affect soils and soil fertility have been discussed by Alexander (1961) and will not be considered here. Fossil fuels, though classed as minerals, are also outside the scope of this review. Excellent reviews of petroleum microbiology ( Beerstecher, 1954) and coal microbiology (Rogoff et aZ., 1962) have been published.

II. Geological Concepts Geochemists divide the planet earth into three zones: the atmosphere, the hydrosphere (rivers, lakes, oceans), and the lithosphere (the remaining solid mass of the earth). The portion of the earth occupied by life forms is termed the biosphere and embraces the lower portion of the atmosphere, the hydrosphere to depths of about 11,000 m., and the upper portion of the lithosphere to a maximum depth of about 2000 m. (Mason, 1952). It is within this relativeIy narrow zone that the influence of microorganisms on minerals is exerted. The time in the history of the earth at which biological activities first became important in mineral transformations is uncertain. It must be assumed that the earth's crust formed long before the first life forms appeared. Therefore, early mineral formation and degradation must have proceeded without biological intervention. Some evidence for extensive geomicrobial activity in proterozoic,

FORMATION AND DEGRADATION OF MINERALS

155

paleozoic, and mesozoic times is suggested by isotope fractionation studies (Thode et al., 1962) and observations of fossil and living microorganisms from geological deposits of these eras ( Cayeux, 1937; Dombrowski, 1960; CAock, 1923; Reiser and Tasch, 1960; Tasch, 1960). Whether living microorganisms isolated from ancient deposits truly represent ancient life is controversial ( Dombrowski, 1963; Meyer, 1963). Within the lithosphere, the major rock formations are either igneous, sedimentary, or metamorphic. Igneous rocks arise from deep-seated molten material (magma). As magmatic solutions cool, in situ or upon rising from the depths under pressure, different igneous rocks form by virtue of a sequence of differential crystallizations from the parent magma; rocks with the highest melting point crystallize first, those with the lowest last. Near the end of the crystallization process many valuable elements originally present in minor amounts are concentrated in the residual magmatic and hydrothermal fluids. Final crystallization produces valuable mineral deposits of hydrothermal origin. Sedimentary rock, as the name implies, arises from the consolidation by heat and pressure of sedimentary deposits. Continued heat and pressure causes recrystallization of sedimentary rock to form metamorphic rock. Admixture of magmatic fluids with sedimentary rock or recrystallization of igneous rock under heat and pressure also give rise to metamorphic rock. However formed, the rocks are relatively stable in their immediate environment. But the dynamic movement of rocks and fluids within the earth's crust as well as removal of overlying strata by erosion alter the environment. At or near the surface, the interplay of physical, chemical, and biological forces (weathering) breaks rock down into small particles and water-soluble compounds, which eventually deposit as sediments at varying distances from the parent rock. This highly simplified account of rock formation and weathering is illustrated in Fig. 1. All rocks, regardless of mode of formation or status as ores, are affected by water. It plays a major role in mineral transformations. Water is indispensable for the support of microorganisms and other life forms, and any discussion of the role of microorganisms in mineral formation and degradation must take this into account. It is the solvent in which inorganic compounds will dissolve or precipitate according to their solubility and tendency to hydrolyze

156

MELVIN P. SILVERMAN AND HENRY L. EHRLICH

at a given concentration. Water also serves as a major transport vehicle, carrying minerals in solution or exerting mechanical force on particulate matter. During weathering, the combined action of chemical, physical, and biological forces on the mineral constituents of rocks may produce different chemical species with altered solu-

- -

Lithosphere

Sedimentary rack

Magma

FIG.1. Formation and transformation of major rock types.

bility and stability in water. When economically important minerals of ore-bearing or gangue rocks are altered to water-soluble chemical species they may be transported by water away from the parent material. Subsequent precipitation by chemical or biological means in locally heavy concentrations followed by rock-forming processes results in the formation of new ore deposits. The water-insoluble minerals of the residual rock are left behind as gangue rocks, usually of little or no commercial value. Analogous processes involving highly soluble gangue minerals and insoluble ore minerals will result in the enrichment of residual rocks with ore minerals and the formation elsewhere of gangue rocks. These relationships are outlined in Fig. 2,.More detailed accounts may be found in standard geology texts (Bateman, 1950; Mason, 1952). Mineral deposits may be classified in relation to the surrounding host or gangue rocks. Syngenetic deposits were formed simultaneously with the host rock, while epigenetic mineral deposits were

FORMATION AND DEGRADATION OF MINERALS

157

formed subsequent to the deposition of the gangue. Mineral deposits formed independently of any other rocks or minerals in their immediate environment are authigenic. An original mineral accumu-

FIG.2. Effect of water on enrichment and depletion of ore-bearing and gangue rock.

lation at a given site is a primmy deposit. If, on the other hand, it is derived from a primary mineral, it is called a secondary deposit.

111. Mechanisms of Microbial-Mineral Interactions Biological mineral transformation is common to all cellular life, but quantitatively significant transformations are restricted to a few groups of organisms (Ehrlich, 1963d). All living cells cause smallscale transformations of trace elements necessary for enzyme activation and function. These processes cause scarcely any alterations in the distribution of minerals on earth. But certain groups of

158

MELVIN P. SILVERMAN AND HENRY L. EHRLICH

microorganisms, whether directly or indirectly, transform relatively large amounts of mineral matter on a scale sufficient to influence its geological distribution. A central theme of this essay is the means by which these organisms effect mineral transformations, with special emphasis on minerals of economic importance. Microorganisms which may participate in these processes include bacteria, fungi, algae, and protozoa.

A. ENZYMIC PRO~ESSES 1. Redox Reactions

Large-scale microbial mineral transformations can be brought about by direct enzymic oxidations or reductions. The importance of these processes to appropriate microorganisms resides in the following. Chemosynthetic autotrophs oxidize minerals to obtain both the energy and reducing power required for COZ assimilation. Photosynthetic autotrophs assimilate Cot, with the help of the radiant energy of sunlight; the reducing power needed is furnished by the oxidation of minerals. The reduction of minerals by certain heterotrophs furnishes the oxidizing power required for the anaerobic oxidation of organic nutrients and the production of useful energy. The salient features of these processes are summarized in Fig. 3. Because of the magnitude of the requirements for energy and reducing or oxidizing power by these organisms, large quantities of minerals must be transformed, 2. Digestion of Metal Complexes Some inorganic ions in aqueous solution, particularly ions of iron, copper, manganese, zinc, calcium, and magnesium, form chelates or complexes with certain organic chemicals, such as amino acids, proteins, organic acids, and others (Alexander, 1961; Baer, 1955; Halvorson, 1931 ) . Complexing agents, in general, stabilize inorganic ions in solution and keep them from precipitating or, in some instances, from being rapidly oxidized or reduced ( Halvorson, 1931). Since many heterotrophic bacteria and fungi are capable of using one or another of the organic complexing agents as carbon, nitrogen, or energy source, they are capable of freeing complexed inorganic ions. The released ions precipitate as water-insoluble hydroxides, oxides, or salts due to spontaneous reaction with water or other dissolved substances at an appropriate pH and Eh (Alex-

FORMATION AND DEGRADATION OF MINERALS

159

ander, 1961; Clark and Resnicky, 1956; Harder, 1919; Pringsheim, 1949a; Starkey, 1945a, b) . Complexing agents may also help to dissolve sparingly soluble inorganic substances by complexing one or more of the products of dissociation, thus shifting the chemical equilibrium in favor of

Chemosynthet ic autotroph

photosynthetic outot roph

Heferotroph

FIG.3. Major functions of large-scale microbial mineral transformations. Adapted from Ehrlich (1963d).

solution (Bromfield, 1958b; I>uff and Webley, 1959; Schatz, 1962). Since naturally occurring complexing agents are of biological origin, their formation by microbes can help to bring minerals into solution. Microbial activity on complexing agents may play an important role in the formation of bog ores (Harder, 1919), but this has been questioned (Pettijohn, 1949).

1. Role of

B. NONENZYMIC PROCESSES Metabolic Products

Mineral transformations can be effected not only by direct enzymic interaction but also by interaction with end products of me-

160

MELVIN P. SILVERMAN AND HENRY L. EHRLICH

tabolism (Alexander, 1961; Waksman, 1932,). Heterotrophic microorganisms may form inorganic acids, such as HzS, HzCOa, HN03, or H2SO4.They may also form organic acids, ammonia, organic bases, and inorganic ions, like sulfide, sulfate, carbonate, phosphate, and phosphite. All these substances may react nonbiologically with dissolved or solid inorganic matter. The inorganic and organic acids may dissolve insoluble minerals, such as limestone, gypsum, anhydrite, silicates, and others. A recent study by Webley et al. (1963) has shown that certain bacteria, actinomycetes, and fungi associated with silicate rocks can dissolve synthetic Ca, Mg, and Zn silicate and wollastonite ( CaSi03) in pure culture, presumably with their acid metabolic products, Ammonia and organic bases may precipitate heavy metal ions as hydroxides by raising the pH of the solution. Inorganic anions may precipitate alkaline earths and heavy metals by the formation of insoluble salts. Gaseous products of microbial metabolism, such as COZ, 0 2 , and H2S, may also effect mineral transformations. COz may form carbonic acid in water, which at acid pH may dissolve limestone and other carbonates, or at alkaline pH may precipitate carbonates. 0 2 , evolved in photosynthesis, may cause autoxidation of some substances like ferrous and manganous ions. H2S will react with heavy metal ions like ferric iron, by reducing the oxidized form and precipitating the sulfide salt.

2. Adsorption to Cell Surfaces Another nonenzymic mechanism whereby microorganisms may react extensively with their mineral environment is the adsorption of mineral matter to the cell surface. This is considered to be an important function of the sheathed bacteria ( Chlamydobacteriales ) and certain flagellates (Pringsheim, 1946, 1949a, b; Skerman, 1959). The chemical composition of their cell surface is supposed to have a unique affinity for iron and manganese (Zappfe, 1931), which become fixed at the cell surface in the form of hydroxides or salts (Alexander, 1961; Pringsheim, 1949a, b; Thimann, 1955). In fact, the sheath of some of these bacteria has been described as consisting of Fe( OH)3 and inorganic oxide (Dondero, 1961). It is known that iron and manganese oxides are good adsorbents themselves, iron oxides for anionic and manganese oxides for cationic substances, and part of the adsorption process probably is not ascribable to the cell surface proper but to the inorganic precipitate upon

FORMATION AND DEGRADATION OF MINERALS

161

it (Zappfe, 1931). However, it is interesting to note that certain organisms are described that accumulate iron preferentially (Pringsheim, 1946). It has been claimed that iron and manganese accumulation in the sheaths or stalks of some bacteria is a build-up of the end product of enzymic iron and manganese oxidation, particularly in the case of Gallionella (Harder, 1919; Lieske, 1911, 1919). This requires further investigation.

IV. Survey of Microbial Interactions with inorganic Substances

Table I summarizes microbial activities on inorganic compounds which may lead to mineral formation or breakdown. The table does not represent a complete survey of such reactions. Neither are all reactions necessarily of geologic importance, but they are illustrative types that might be important. Most of the reactions listed are oxidations or reductions involving inorganic ions. Many are part of a multiple reaction system involving oxidation or reduction, and salt formation or hydrolysis. Only one of these combinations of reactions may be enzyme catalyzed, but that one is usually rate determining for the over-all reaction sequence. For instance, sulfate is not readily reduced to H2S in the absence of microorganisms at ordinary temperatures, Eh, and pH (Jones et al., 1956). But in the presence of Desulfovibrio spp. or Clostridium nigrificans, sulfate is rapidly reduced to HZS, which will nonbiologically precipitate heavy metals dissolved in the reaction mixture. This may lead to the accumulation of amorphous metal sulfides, which may later change into crystalline forms. As a special example of this the case of sulfate-reducing bacteria may be cited, which Adler ( 1963) holds responsible for precipitating uranium oxide by reducing uranyl ion with biogenic H2S. The uranyl ion may be of magmatic or hydrothermal origin or may be derived by leaching of primary uranium deposits. The precipitation of CaC03 as limestone or marl is another example of a sequence of biological and nonbiological reactions. Its formation may be dependent on enzymic production of COZ and NH3 from organic nitrogen compounds, including urea, with subsequent precipitation of CaC03 at neutral or alkaline pH by nonenzymic means; or it may depend on the microbiological reduction of CaS04 with organic carbon to form, in part, H2S and CaC03;

Element

Microorganism

Ag

Desulfovibrio

As

F. ferrooxiduns Heterotrophic bacteria, Achromobacter, Pseudomonas, Xanthomonus M . lactilyticus

Bi

Ca, Mg

TABLE I MICROBIALINTERACTIONS WITH INORGANIC SUBSTANCES Physiological activitya

M. lactizyticus

Thiorhodaceae. Thiobacteriaceae, Beggiutoa, Achromatium, Desulfovibrio, Other heterotrophs, Algae, Foraminifera

2 AgCl + SO,,.

+ 8 H + + 8 e-

Ag,S

I

+ 4 H,O + 2 C1-

As.$, oxidized to AsO,3-; AsO,3-; SO,z-( ? ) * As033- oxidized to AsO,3-

AsO,3- reduced to As033-

References Baas Becking and Moore (1961) Ehrlich ( 1 9 6 3 ~ ) Quastel and Scholefield ( 1953); Turner (1949, 1954); Turner and Legge (1954) Woolfolk and Whitelev ( 1962)

+ 3 e- + 3 H+ = Bio 4-3 H,O + 5 e- + 6 H + = BiO + 3 HzO

Woolfolk and Whiteley (1962)

Ca precipitated as carbonate; Mg precipitated as carbonate (bacteria only)

Ashirov and Sazanova ( 1962); Bavend a m ( 1932); Breed et al. ( 1957); Butlin ( 1953); Cay e w ( 1937); Cushman (1940); Glock (1923); Moore (1958); Nadson ( 1928) ; Setchell (1926); Skerman ( 1959); Taylor (1950)

Bi( OH), Bi03-

Element

TABLE I (Continued) Physiological activitya

Microorganism

+ HC03+ HC0,-

References

Thiobacteriaceae, Acid-producing heterotrophs Algae

CaC0, + H + = Ca2+ MgCO, + H + = Mg2+

Cd

Desulfocibrio

CdCO,

co

Desulfovibrio

Nadson (1927a, b ) Miller (1950) Miller ( 1950)

cu

T . ferrooxidans, F. ferrooxidans

Desulfovibrio, C . nigrijicans

Cu2+ and S0,Z-

M . lactilyticus

Cu( OH),

Bryner et al. (1954); Bryner and Jameson ( 1958); Ehrlich (1962) Baas Becking and Moore (1961); Miller ( 1950) Woolfolk and Whiteley (1962)

Fe

T . ferrooxidans Ferrobacillus spp. Gallionella Leptothrix ochruceu Sphaerotilus Protozoa Algae

+ SO,,- + 8 H + + 8 e- = CdS + 4 H,O + CO,a8[2 CoC03.3 Co(OH),] + 98 H + + SO,,- + 8 e = CoS + 58 H,O + I COs2Cu,S + 4 H,O = 2 Cu2+ + 6 H + 3. H,SO, + 10 eCuS + 4 H 2 0 = Cu2+ + 6 H + + H,SO, + 8 e -

i

i

reduced to CuS; Cu,,S9; Cu2S

+ H + + e-

Fez+ = Fe3+

= CuOH + H,O

+ e-

Adsorption, precipitation

Parker ( 1947 ) ; Paine et al. (1933);

Kinsel (1960); Kucera and Wolfe (1957); Leathen and Braley ( 1954) ; Lieske ( 1911) ; Mulder and van Veen ( 1963); Praeve ( 1957); Pringsheim ( 1946) ; Temple and Colmer (1951)

w

8

5 8z

tl

M 0

*

*I

!2

=I

8 $

!* m

E:

+

8

Element

TABLE I (Continued) Physiological activitya

Microorganism

M . kctilyticus B. circulans B . polymyxa

I

Desulfovibrio, C . nigrijicans

Mn

Fe3+

+ e-

Fe3+

+ SO,2- + 8 H+ + 9 e-

= Fen+ = FeS + 4 H,O

A erobact er Bacillus Coynebacterium + Chromobactedun Pseudomonos MetaUogenium fungus Mn2+ + 2 H,O = MnO, 4 H+ 2 eCladosporium curouiaria Helminthosporium Cephalosporium Arthrobacter Diatoms T . thiooriduns \ BaciUus MnOa+4H++2e-=Mn2++2H20 M. kctdyticus Yeast I

+

+

I

+

References B m d e l d (1954a, b); Roberts (1947); Woolfolk a d Whiteley (1962) Berner (1962); Breed et al. ( 1957); Campbell et al. ( 1957); Starkey (1945a) Alexander ( 1961) ; Brodeld ( 1956); Brodeld and Skerman ( 1950); Ehrlich ( 1963a) ; Kindle (1932); Timonin ( 1950); Zavarzin (1961, 1962)

Garey and Barber ( 1952); Hochster and Quastel (1952); Perkins and Noviellj ( 1958,1962);Wool-

folk and Whiteley ( 1962); Vavra and Frederick ( 1952)

TABLE I (Continued) Element Mo

Ni

Physiological activitya

References

T. ferrooxidans

Microorganism

MoS2 oxidized to H2Mo04,pentavalent Mo, and H,S04

M . lactilyticus

Mo6Oz16-

Bryner and Anderson ( 1957); Bryner and Jameson (1958) Woolfolk and Whiteley (1962)

T. ferrooxidans

NiS

Desulfouibrio

P

S

+ 12 H + + 6 e-

+ 4 H,O

= Ni2+

+

= 6 MOO,,,

+ 8 H + + S0,Z- + 8 e-

+ 8 e- = NiS + 4 H 2 0 + CO,2+ 8 e- = NiS + 6 H,O = HP0,2- + 2 H+ + 2 e-

NiCO, S042- + 8 H+ Ni( OH), + SO4,- + 10 H +

+ H,O

Soil microorganisms

HP0,z-

Rhodotomla, PeniciUium

Ca,( PO,),

C . butyricum, E . coli

HP042- reduced to HP0,2- and HP022-

Thiobacteriaceae Thiorhodaceae Chlorobacteriaeae Beggiatoaceae S. nutans Achrmatium Leucothrixc

+ 6 H20

H2S

+ 2 H + = 3 Ca2+ + 2 HP0,2-

H2S=So+2H+ +2e= SO,2- + 10 H + + 8 e-

+ H,O

Razzell and Trussell (1963b) Miller (1950) Alexander (1961); Adams and Conrad (1953) Myskow ( 1960); Nikitia (1959) Alexander (1961); Rudakov ( 1929); Tsubota (1959) Alexander ( 1961) ; Breed et al. (1957); Butlin (1953); Peck (1962); Skerman et al. (1957); Starkey (1956); Vishniac and Santer ( 1957); Vishniac and Trudinger (1962)

E 5

g

5u

iE!

cl

8

Z

8

! c,

g

TABLE I (Continued) Element

Microorganism

Physiological activitya and S,032- oxidized to polythionates and sulfates

Heterotrophs

SO

Fungi

Organic sulfur oxidized to sulfate

Thiorhodaceae Chlorobacteriaceae Thiobacteriaceae Yeast Filamentous fungi Actinomycetes Aerobic bacteria Desulfovibrio C . nigrificans B . megaterium P . zelinskii Yeast

E . coli D. desulfuricans C. nigrificans M . lactilyticus

SO+ 4 H,O = SO,2-

I

+8H+ +6e-

SO,z-

+ 10H+ + 8e-

SO,,-

+ 8 H + + 6 e- = H,S + 3 H,O

= H,S

+4H,O

References Guittoneau (1927); Rippel (1924); Starkey (1934, 1956) Alexander ( 1961); Starkey (1956) Alexander ( 1961); Breed et al. ( 1957 ) ; Butlin (1953); Peck ( 1962) ; Rippel (1924); Starkey 1956 ) ; Vishniac and Santer ( 1957) Alexander ( 1961 ) ; Bromfield ( 1953 ) ; Campbell et al. (1957); Hilz et al. 1959);Peck( 1962); Postgate (1951); Shturm (1948); Wilson et al. ( 1961 ) Alexander ( 1961 ) ; Coleman (1960); Mager ( 1960) ;Postgate (1956); Postgate and Campbell ( 1963 ) ; Wainright (1961); Woolfok ( 1962) ; Woolfolk and Whiteley ( 1962)

Element

Sb Se

References

Bacteria, actinomycetes, fungi All microorganisms

Polysulfides reduced to thiosulfate and sulfide

Alexander ( 1961); Woolfolk (1962)

SO + 2 e -

Starkey (1956)

T . ferrooxidans

Oxidation of Cu,Sb,S,

M. selenicus

HzSe

M. lactilyticus

SeO+Ze-+H+=SeH-

C . pasteurianum D . desulfuricans Neurospora C . albicans Baker’s yeast

Si

TABLE I (Continued) Physiological activity@

Microorganism

Diatoms, radiolaria

Fungi, bacteria, actinomycetes

r

+ 2 H + = H,S

+ 4 H,O

= Se042-

Bryner et al. (1954)

+ 10 €I ++ 8 e-

HSeO3-+4e-+5H+=SeO+3H2O

Assimilation into frustules or tests

Dissolution of silicates

Brenner (1916); ZoBell (1946) Woolfolk and Whiteley ( 1962) Nickerson and Falcone (1963); Woolfolk and Whiteley ( 1962); Zalokar ( 1953) Lewin (1954, 1955, 1957); Trask (1950); Vinogradov and Boichenko ( 1942 ) Duff and Webley (1959); Duff et al. (1963); Webley et al. ( 1963)

::

=! h

3

E u

=!

%

8

K

z

m

$

m

w

3

Element Te

TABLE I (Continued) Physiological activitya

Microorganism M. lactilyticus D. desulfuricans S . faecalis C . diphtheriue

I

HTe0,-

+ 5 H + + 4 e-

= Teo + 3 H,O

M. lactilyticus

HTe04- + 7 H + + 6 e - = T e o + 4 H 2 0

U

M . lactilyticus

UO,( OH),

V

M . lactilyticus D. desulfuricans C . pasteurianum T . ferrooxidans

zn

Metal chelate a b 0

H,VO,

+ 2 e- + 2 H+ = U ( OH),

+ 2 e- + 2 H + = VO( OH) + H,O

ZnS + 4 H,O

= Zn2+

+ SO,,- + 8 H+ + 8 e-

+

+ 91 H +

Desulfwibrio

&[2ZnCO3.3 Zn( OH),] SO,,+ 8 e- = 5&H,O E C032-

Heterotrophic microorganisms

Oxidation of chelating agent with precipitation of metal moiety

+

+ ZnS

References Thomas et al. (1963); Tucker et al. ( 1962); Woolfolk and Whiteley ( 1962) Woolfolk and Whiteley (1962)

w

Woolfolk and Whiteley ( 1962) Woolfolk and Whiteley ( 1962)

Malouf and Prater (1961) Baas Becking and Moore (1961); Miller (1950) Starkey and Halvorson (1927)

Oxidative or reductive half-reactions listed most nearly describe the particular microbial activity cited. Proof of sulfate production lacking. But see Harold and Stanier (1955).

r

FORMATION AND DEGRADATION OF MINERALS

169

or it may depend on the photosynthetic consumption of bicarbonate from Ca( HCOB)z in solution, resulting in the precipitation of CaC03 (Bavendamm, 1932; Nadson, 1928; ZoBell, 1946). Another example of an inorganic reaction sequence, in part biologically catalyzed, is the precipitation of dissolved iron by algae. Here the evolution of 0 2 in photosynthesis promotes autoxidation of ferrous iron to form hydrated ferric oxide, which precipitates and may be adsorbed by the algae themselves (Pringsheim, 1946, 1949a). Table I also records reactions in which the role of microorganisms is passive. In these reactions they act as accumulators or concentrators of mineral matter, without direct enzymic intervention in the process. The accumulation may involve adsorption of iron and manganese compounds, as discussed in Section 111, B, 2, or it may involve the collection and agglutination of calcareous and siliceous particles at the cell surface, as among certain Foraminifera (Cushman, 1940). From the standpoint of orogenesis, both types of processes may lead to massive mineral accumulations in situations where simple precipitation would not occur because of undersaturation, stability of collodial suspension, or the unreactiveness of particulate matter. Table I, with few exceptions, does not refer to specific ore-forming or ore-degrading reactions, Examples of these for sulfur, iron, and manganese are discussed at length in Sections V and VI. For a number of reactions in Table I, the geobiochemical importance is poorly understood, if known at all, especially reactions involving selenium, tellurium, bismuth, and phosphorus. Although reactions of phosphorus in soil have been studied (Alexander, 1961), they do not deal with the biogeochemistry of large accumulations of phosphorus mineral in nature, The question of a biogeochemical role in the formation or transformation of phosphoritic deposits or phosphorite nodules, which are all of marine origin, remains to be resolved (Teodorovich, 1961).

V. Sulfur Mineral Deposits The relative stability of inorganic sulfur compounds, under environmental conditions found in nature, dictates in large measure the form in which sulfur-containing minerals occur. Stable ionic and molecular species that predominate are H2S, HS-, S=, So, HSOI-, and SO1= (Garrels and Naeser, 1958). As a consequence, most

170

MELVIN P. SILVERMAN AND HENRY L. EHRLICH

mineral sulfur deposits occur as elemental sulfur and the salts of sulfides and sulfates. The cycle of sulfur in nature is the result of the activities of plants, animals, and microorganisms as well as nonbiological processes. But only the more important geomicrobial transformations will be considered here. The major sulfur species involved and their transformations are outlined in the following scheme:

s= 1'

-so

so,= I

Hydrogen sulfide of biological, volcanic, or hydrothermal origin may be oxidized aerobically or anaerobically to extracellular elemental sulfur by chemoautotrophic species of Thiobacillus, or aerobically to intracellular elemental sulfur by autotrophic and heterotrophic members of the Beggiatoaceae and Sphaerotilus. Photoautotrophic green and purple sulfur bacteria will also form elemental sulfur anaerobically, but only when hydrogen sulfide is present in ample supply; otherwise oxidation usually continues with sulfate the final product. Thiobacillus species are responsible for most large-scale biological oxidation of elemental sulfur to form sulfate. Once formed, sulfate is extremely stable to chemical reduction. Most suIfate reductions to hydrogen sulfide in nature are the direct result of the activities of the anaerobic dissimilatory sulfate reducers, Desulfovibrio spp. and Clostridium nigrificans. More detailed discussions of these and other microorganisms and the metabolism of sulfur compounds may be found in reviews by Lees (1960), Peck (1962), Postgate (1959, 1960), Starkey (1956), Van Niel (1954), and Vishniac and Santer (1957). The influence of microorganisms on the formation or degradation of deposits of sulfur, sulfides, or sulfates may be deduced by studying these processes as they occur in nature at the present time, or by examining older geological formations and applying our knowledge of the capabilities of microorganisms to a reconstruction of events as they probably occurred. A. ELEMENTAL SULFUR 1. Formation of Deposits The deposition of elemental sulfur in Lake Ixpaco in Guatamala was studied by Ljunggren (1960). Hydrogen sulfide of volcanic origin is oxidized to elemental sulfur and sulfuric acid by members

FORMATION AND DEGRADATION OF MINERALS

171

of the Beggiatoaceae numbering about ~OO,OOO/C~.~ of lake mud. The water is acid ( p H 2.27) and the mud contains 30 to 60% sulfur with total reserves estimated at considerably more than 100,000m3. Chemo- and photoautotrophic bacteria in Lake Sernoe in Russia are responsible for a threefold increase in the rate of hydrogen sulfide oxidation (Ivanov, 1957a). The lake is fed by springs rich in fumarolic H2S, and sulfur is deposited at the rate of 150 kg. per day in the mud or as encrustations during H2S oxidation by Thiobacillus thiuprus; most of the bacteria are located in the surface layers of the mud (Sokolova, 1962,). The photosynthetic green and purple sulfur bacteria do not precipitate sulfur in this lake but oxidize H2S completely to sulfate. However, in several Cyranaican Lakes in the North African desert, conditions are such that photosynthetic bacteria do precipitate elemental sulfur from H2S (Butlin, 1953; Butlin and Postgate, 1954). The H2S in these lakes is largely biogenic, being produced by sulfate-reducing bacteria in the calcium sulfatesaturated waters. The photosynthetic green and purple sulfur bacteria, in turn, provide organic matter suitable as hydrogen donors for bacterial sulfate reduction. Thus a balanced ecological system operates and produces sulfur in such abundance that up to 200 tons can be mined per year. The formation of biogenic sulfur deposits is not restricted to lakes and ponds. Active bacterial hydrogen sulfide formation can be observed under the anaerobic conditions prevailing in deep underground mineral waters. Sulfate-reducing bacteria reduce the abundant CaS04 formations of gypsum and anhydrite, as well as sulfates dissolved in seam waters (Ivanov, 195713; Kuznetsova and Pantskhava, 1962; Volkova and Toshinskaya, 1961). The bacteria are widely distributed in waters associated with Carboniferous or Permian strata but are often absent in waters in Devonian strata where the high CaC12 content together with very high total mineral content of up to 270 gm. salt per liter are bacteriostatic (Kuznetsova and Pantskhava, 1962). In the Carpathian sulfur deposits, which are under active investigation, most of the sulfate-reducing bacteria are found in the underground mineral waters associated with the sulfur ore and in the porous or fractured sulfur-bearing limestones and gypsum strata (Ivanov, 1960, 1961a, b; Ivanov and Kostruva, 1961; Ivanov and Ryzhova, 1961; Ryzhova and Ivanov, 1961). The rate of formation of HZS is barely detectable in the spring months, increasing in summer to 1 to 3.8 mg. H2S per liter per day (Ivanov

172

MELVIN P. SILVERMAN AND HENRY L. EHRLICH

and Kostruva, 1961; Ivanov and Ryzhova, 1961) and appears to depend upon the availability of water-soluble hydrogen donors provided by the influx of surface waters. The latter contain dissolved organic matter. They may, while percolating down through the porous overlying sedimentary strata and limestones, dissolve additional organic matter ( Ivanov, 1961a; Ivanov and Ryzhova, 1961). Incubation of sulfate-reducers with these sedimentary limestones or gypsums results in H2S production. Growth is supported by organic matter extracted from these rocks, by molecular hydrogen, but not by natural gas or methane (Ryzhova and Ivanov, 1961) . Elemental sulfur then forms where oxygenated surface waters descend to meet H2S-rich subterranean mineral waters. While sulfur formation may occur by strictly chemical reactions, biological oxidation is undoubtedly responsible for much of the elemental sulfur formed in these deposits. Halophilic strains of Thiobacillus thiopam, capable of precipitating sulfur from H2S while tolerating low oxidation-reduction potentials, have been found in large numbers where active sulfur deposition is taking place (Ivanov, 1957b; Sokolova, 1960, 1961). In seam waters containing 1.2 gm.H2S per liter, T . thiqmms forms up to 0.5 gm. elemental S per liter per day with about one-third of the H2S oxidized completely to sulfate (Sokolova, 1960). The discovery that dissimilatory sulfate-reducing bacteria fractionate the stable isotopes of sulfur has provided geochemists with a sensitive method for determining whether sulfur deposits are of biogenic origin (Jensen, 1962). Investigations with Desulfovibm'o have established that S*204- is preferentially reduced over S3404', resulting in an increased S32/S34 ratio in the hydrogen sulfide product (Harrison and Thode, 1958; Jones and Starkey, 1957, 1962; Kaplan and Rittenberg, 1962a; Nakai and Jensen, 1960; Thode et al., 1951). Fractionation appears to take place during enzymic rupture of a S-0 bond in the intermediate step involving reduction of sulfate to sulfite (Harrison and Thode, 1958). The magnitude of the fractionation is not constant but varies according to cultura1 conditions. In general, fractionation is enhanced under suboptimal conditions that diminish the rate of sulfate reduction (Harrison and Thode, 1958; Jones and Starkey, 1957, 1962; Kaplan and Rittenberg, 1962a). Subsequent chemical or biological oxidations of isotopically enriched H2S do not significantly alter the isotope ratio of the product. Little or no fractionation occurs during the oxida-

FORMATION AND DEGRADATION OF MINERALS

173

tion of H2S and other partially oxidized or reduced inorganic sulfur compounds by Thiobacillus thiomidarzs, Thiobacillus concretivorus, or Chromatiurn (Jones and Starkey, 1957; Kaplan and Rittenberg, 1962a,b). Saccharomyces cereviseae, however, causes substantial fractionation during sulfite reduction to H2S but is probably not involved in H2S production on a geologically significant scale. The variability inherent in bacterial sulfate reduction is reflected in the isotopic composition of biogenic sulfur or sulfide deposits. These are characterized by generally higher but more widely spread S32/S34ratios when compared with meteoric sulfur considered representative of the primordial sulfur of the earth. Nonbiogenic deposits, on the other hand, show lower S32/S34 ratios more comparable to meteoric sulfur, with a much narrower spread in values. Diffusion of isotopically unenriched sulfur into biogenic deposits may also contribute to the wider spread in S32/S34 ratios of the latter. A number of elemental sulfur deposits have been studied and it is now abundantly clear that sulfate-reducing bacteria played a major role in the formation of the sulfur deposits of Texas and Louisiana salt domes (Feely and Kulp, 1957; Jones et al., 1956; Thode et aZ., 1954), as well as Sicilian deposits (Dessau et aZ., 1962) ,

2. Degradation of Deposits Once formed, elemental sulfur deposits exposed to oxygen are subject to the oxidative activities of bacteria such as Thiobacillus thiooxidam and Thiobacillus thioprus. Underground sulfur ores of the Rozdol deposit in Russia contained few T. thiooxidans upon pumping out the H2S-bearing seam waters. More were found in limestone-bearing ore lying directly beneath Quaternary formations permeable to oxygen-bearing surface waters. The largest numbers occurred in ore which had lain on the surface for 2 years (Karavaiko, 1959). Evidence of incipient bacterial sulfur oxidation was reported by Karavaiko ( 1961). Although oxidation appeared to be absent as judged by over-all pH changes, T . thiooxidans was detected in distinctly acid microzones in fissures and weathered surfaces of the carbonate-containing sulfur ore being mined in open trenches. Other laboratory studies ( Sokolova and Karavaiko, 1962) demonstrated that carbonate-containing ores were oxidized more rapidly by T. thioparus than by T. thiooxkhm. However, with carbonate-free ores, the more acid tolerant T. thiooxkhns prevailed.

174

MELVIN P. SILVERMAN AND HENRY L. EHRLICH

Sterile ore was not oxidized in air, and it was concluded that the oxidative process in Rozdol ores is bacterial, being initiated by T. thioparus with subsequent oxidation due to both species. The mode of attack on elemental sulfur by Thiobacillus species is not known with certainty, Thwbacillus thiooxidans produces organic wetting agents such as ninhydrin-positive material (Jones and Starkey, 1961). Schaeffer and Umbreit (1963) isolated a wetting agent which they identified as phosphotidylinositol. Tenacious physical attachment of cells to sulfur particles has also been observed (Schaeffer et al., 1963) and might account for growth during vigorous agitation (Newburgh, 1954; Starkey et al., 1956). Vishniac and Santer (1957) proposed a preliminary reduction of elemental sulfur to sulfide at the cell surface with sulfide then entering the cell, However, the more recent isotope fractionation studies of Kaplan and Rittenberg (1962b) with T . concretivow suggest that preliminary reduction to sulfide does not occur and that elemental sulfur per se penetrates the cell membrane.

B. METALSULFIDES 1. Metal Sulfide Formation A number of valuable metals occur in nature as simple or mixed sulfide minerals. Economic accumulations of the sulfides of iron, copper, silver, zinc, lead, cobalt, nickel, molybdenum, arsenic, cadmium, and mercury have been exploited by the mining industry. Metal sulfides may represent the major constituent of a given ore or may be associated in lesser amounts with other ores in which case they are often recovered as by-products (Bateman, 1950). Many metal sulfide deposits are of undoubted magmatic or hydrothermal origin. Others, however, are associated with sedimentary rock and have been the object of considerable controversy as regards syngenetic versus epigenetic origin ( Cheney and Jensen, 1962; Davidson, 1962a, b; Garlick, 1961; Love, 1962; Sales, 1962). Proponents of syngenesis argue that metal sulfides were deposited at the same time as the pyrite and other constituents of sedimentary rock. Epigenesists concede that the pyrite may have formed at the same time as the sedimentary rock but argue that other ore metals, introduced by hydrothermal ore-bearing fluids at some time after lithification of the sediment, formed metal sulfides by replacement reactions with pyrite. Evidence supporting either side can be summoned and the controversy remains to be resolved,

FORMATION AND DEGRADATION OF MINERALS

175

There is no need to invoke drastic conditions of temperature, pressure, or pH incompatible with life processes for the synthesis of sulfide minerals. Chemical syntheses of bornite (Cuthbert, 1962), chalcopyrite (Roberts, 1963), pyrite, marcasite, and pyrrhotite (Rosenthal, 1956) have been reported using H2S and appropriate metal compounds at room temperature and atmospheric pressure. The virtual monopoly of sulfate-reducing bacteria in large-scale H2S production from sulfate has already been noted. Biogenic sulfide formation, especially of iron sulfides, cannot be disputed. Among the major habitats of sulfate-reducing bacteria are surface muds of the littoral zones of oceans and inland bodies of water (ZoBell, 1946). The dark bottom deposits of the Black Sea (Sorokin, 1962; ZoBell, 1946) and the black muds of tidal flats and littoral zones of oceans furnish visible evidence of biological iron sulfide deposition. Iron compounds, either in solution or as colloidal or particulate material in the mud, react with biogenic H2S to form an amorphous, hydrated, black iron sulfide known as hydrotroilite (FeSenHzO). Hydrotroilite is not stable and is converted to the more stable iron disulfides, pyrite, or marcasite just below the upper layers of recent sediments (Emery and Rittenberg, 1952). Kaplan et al. (1963) regard bacterial sulfate reduction as the single most important reaction resulting in the transformation of sea-water sulfate to pyrite. The reactions of hydrotroilite leading to pyrite formation are not clear but appear to be controlled by the concentration of ferrous ions available for reaction with H2S. High concentrations of very soluble FeS04 react with H2S to form pyrite above pH 7, and marcasite below pH 7 (Rosenthal, 1956). Berner (1962) found that pyrite and black iron sulfide were formed from the less soluble goethite (HFe02) and H2S at pH 4, whereas only black iron sulfide and elemental sulfur were produced from these reactants at p H 7-9. Berner postulates that at the neutral pH of typical marine sediments the passage of geological time might allow the small amount of ferrous ions in equilibrium with hydrotroilite to react with elemental sulfur to form pyrite. Sugden (1963) visualizes pyrite formation in the natural environment as the result of reactions of hydrotroilite with H2C03, Ca(HC03)2,or H2S. Biogenic formation in nature of metal sulfides other than iron has not yet been established, but laboratory experiments suggest that it is possible. Miller (1950) demonstrated the formation of sulfides

176

MELVIN P. SILVERMAN AND HENRY L. EHRLICH

of antimony, bismuth, cobalt, cadmium, iron, lead, nickel, and zinc by sulfate-reducing bacteria. No mineralogical identification of the products was made, but H2S, liberated upon acid treatment, was titrated. Baas Becking and Moore (1961) prepared covellite (Cus) from chrysocolla ( CuSi03.2H20)or malachite [CuC03*Cu(OH)z], digenite (Cuz.nS) from CuzO, argentite (Ag2S) from AgCl or AgC03, galena (PbS) from PbC03 or 2PbC03-Pb(OH)2, and sphalerite (ZnS) from metallic zinc or smithsonite ( ZnCOs) by bacterial sulfate reduction in artificial sea water. Positive mineralogical identification was made by X-ray diffraction. Copious metal sulfide precipitates were also formed when nickel or cobalt carbonates were incubated with cultures, but identifiable X-ray POWder diagrams could not be prepared. During covellite formation from malachite, native copper appeared as an intermediate product. Similarly, the transient appearance of native silver was noted with both AgCl and Ag2C03. Booth and Mercer (1963) showed that Desulfouibrio desulfuricans, D. orientis, and C . nigrificans were unable to adapt to concentrations of ionic copper greater than 50 p.p.m. These authors, therefore, rejected the idea of a significant role of sulfate-reducing bacteria in the deposition of copper sulfide ores. It must also be considered that heavy metals are present in sea water in only minute quantities, and some mechanism for their mode of concentration would have to be found to permit their precipitation by bacteria. It is not inconceivable that insoluble heavy metal compounds may have entered ancient marine muds as sedimentary particles derived from eroded and weathered terrestrial ores. Certainly the works of Miller (1950) and Baas Becking and Moore (1961) demonstrate that respectable amounts of insoluble rather than ionic forms of heavy metals are amenable to bacterial metal sulfide formation. Sulfate-reducing bacteria may play a more direct role in metal sulfide precipitation than merely acting as microbial H2S generators. Bacterial cultures containing 70-100 mg. H2S per liter averaged 94% precipitation of added solubIe molybdenum, whereas, in the absence of bacteria, 300-1000 mg. H2S per liter averaged only 43% precipitation (Kramarenko, 1962). Accumulations of metal compounds and H2S in microzones at or near the cell surface may be sufficiently large to exceed the solubility product of metal sulfides with the result that precipitation occurs even though the over-all solubility product would dictate otherwise.

FORMATION AND DEGRADATION OF MINERALS

177

2. Degradation of Deposits Metal sulfides, when exposed to oxygen, will oxidize to the corresponding metal sulfates and sulfuric acid. The rate of this process can be accelerated manyfold by the activities of acidophilic ironoxidizing bacteria closely resembling members of the genus ThwbacilZus (Table 11). a. Iron-Oxidizing Thiobacilli. Iron-oxidizing thiobacilli are widely distributed, having been found along with Thiobm'llus thiooxidans and T . concretiuorus in acid waters associated with deposits of metal sulfides and sulfide-bearing coals in the United States (Bryner et al., 1954; Colmer and Hinkle, 1947; Corrick and Sutton, 1961; Leathen, 1952), Mexico (Bryner and Jameson, 1958), Canada (Razzell and Trussell, 1963a,b; Lazaroff, 1963), Scotland (Ashmead, 1955), Spain (Razzell and Trussell, 1963a), Portugal ( Audsley and Daborn, 1962a, b; Miller et al., 1962), Sweden and Germany (Marchlewitz and Schwartz, 1961), Russia (Lyalikova, 1960a, b, 1961; Zarubina et al., 1959), and Japan (Ito et al., 1960). They appear to be indigenous to habitats in which metal sulfides and oxygen occur under acid conditions. They have not been reported elsewhere, although Gleen ( 1950) has demonstrated biological oxidation of acid iron solutions percolated through soil. Three species have been described: Thiobacillus ferrooxidans (Colmer et al., 1950; Temple and Colmer, 1951), Ferrobacillus ferrooxiduns (Leathen et al., 1956; Leathen and Braley, 1954), and Ferrobacillus sulfooxidans (Kinsel, 1960). All grow autotrophically in acid mineral salts media with ferrous iron as the sole energy source, but were originally described as differing in their relation to sulfur compounds as alternate energy sources: T . ferrooxidans utilizing thiosulfate, F . sulfooxidans growing on elemental sulfur, and F. ferrooxidans using neither of these. The separation of the iron-oxidizing thiobacilli into different genera and species has been questioned (Ivanov and Lyalikova, 1962; Lyalikova, 1960b; Marchlewitz and Schwartz, 1961; Unz and Lundgren, 1961). With the exception of the organism isolated by Leathen and co-workers (1956) and one strain isolated by Corrick and Sutton (1961), all other isolates have been reported to utilize thiosulfate, elemental sulfur, or both (Beck, 1960; Bryner and Jameson, 1958; Colmer et al., 1950; Colmer, 1961; Corrick and Sutton, 1961; Kinsel, 1960; Razzell and Trussell, 1963a; Unz and Lundgren, 1961).

178

MELVIN P. SILVERMAN AND HENRY L. EHRLICH

TABLE I1 METALSULFIDEMINERALS OXIDIZED BY IRON-OXIDIZING THIOBACILLI Mineral

Formula

References

Arsenopyrite

FeS,.FeAs,

Ehrlich ( 1 9 6 3 ~ )

Bornite

Cu,FeSd

Bryner et al. (1954); Ivanov (1962); Razzell and Trussell (196313); Sutton and Corrick (1963, 1964)

Chalcocite

cu2s

Bryner et al. ( 1954); Ivanov (1962); Razzell and Trussell ( 1963b); Sutton and Corrick (1963, 1964)

Chalcopyrite

CuFeS,

Bryner et al. (1954); Bryner and Anderson ( 1957); Bryner and Jameson ( 1958); Ivanov et aE. ( 1961); Ivanov (1962); Malouf and Prater (1961); Razzell and Trussell (1963b); Sutton and Corrick (1963, 1964)

Covellite

cus

Bryner et al. (1954); Marchlewitz et al. ( 1961); Razzell and Trussell ( 1963a); Sutton and Corrick (1963, 1964)

Enargite

3 Cu2S.As,S,

Ehrlich ( 1 9 6 3 ~ )

Galena

PbS

Ehrlich ( 1961)

Marcasite

FeS,

Leathen et al. (1953a,b); Silverman et al. (1961)

Millerite

NiS

Razzell and Trussell (196313)

Molybdenite

MoS,

Bryner and Anderson ( 1957); Kramarenko (1962)

Orpiment

As,%

Ehrlich ( 1 9 6 3 ~ )

Pyrite

FeS,

Bryner and Anderson (1957); Bryner and Jameson (1958); Ivanov et al. (1961); Ivanov (1962); Leathen et at. ( 1953a,b); Marchlewitz et a2. ( 1961); Silverman et al. (1961); Sutton and Corrick (1963, 1964)

Sphalerite

ZnS

Ivanov et al. ( 1961); Ivanov ( 1962); Malouf and Prater ( 1961)

Tetrahedrite

Cu8Sb,S,

Bryner et al. (1954)

FORMATION AND DEGRADATION OF MINERALS

179

Strains of F . ferrooxiduns from Leathen's collection oxidized elemental sulfur (Silverman and Lundgren, 1959b) or both sulfur and thiosulfate (Unz and Lundgren, 1961). Furthermore, a strain of T. ferrooxiduns from Colmer's collection also utilized both sulfur and thiosulfate (Unz and Lundgren, 1961). Hence it is quite possible that future research will show that all iron-oxidizing thiobacilli are varieties of the same species and that the name Thiobacillus ferromiduns, by virtue of priority, will be retained. To avoid confusion we have adhered to the nomenclature employed by individual investigators when referring to specific organisms. But for the sake of convenience, we have used the term iron-oxidizing thiobacilli when referring to these organisms as a group, there being no taxonomic inferences intended. Physiologically, the iron-oxidizing thiobacilli are chemoautotrophs, utilizing reduced inorganic iron or sulfur compounds for energy with COZ and inorganic nitrogen as carbon and nitrogen sources. The attempts of many workers to obtain heterotrophic growth have been unsuccessful, except for the recent report of growth of a strain of Ferrobacillus ferrooxiduns on glucose (Remsen and Lundgren, 1963). Inorganic nitrogen may also be replaced by several amino acids, peptone, and urea (Bryner and Jameson, 1958; Kinsel, 1960; Lundgren et ul., 1962; Remsen and Lundgren, 1963). They are nutritionally undemanding, growing in simple mineral salts media (Beck, 1960; Leathen et ul., 1951; Silverman and Lundgren, 1959a; Temple and Colmer, 1951). Trace element requirements presumably are satisfied by impurities. They are obligate aerobes and are usually mesophilic in their relation to temperature (Kinsel, 1960; Razzell and Trussell, 1963a; Silverman and Lundgren, 1959a). Optimal temperatures for growth vary among different strains and have been reported at 20"-25°C. (Leathen et al., 1956), 28°C. ( Silverman and Lundgren, 1959a), and 32°C. (Kinsel, 1960). Marchlewitz and Schwartz (1961) studied 13 different strains of iron-oxidizing thiobacilli and reported very good growth of 12 strains at 25"C., 13 strains at 31"C., 4 strains at 35°C. and 37"C., and 3 strains at 45°C.; none grew at 50°C. The optimal temperature for growth does not necessarily coincide with the optimum for iron oxidation. Silverman and Lundgren ( 1959b) observed maximal rates of iron oxidation by resting cell suspensions at 37"C., although no growth of their strain occurred at that temperature. The ironoxidizing thiobacilli are acidophilic, resembling Thiobacillus thio-

180

MELVIN P. SILVERMAN AND HENRY L. EHRLICH

oxidans in their acid requirements. Good growth occurs at pH values ranging from about 2.5 to 4.5 (Beck, 1960; Kinsel, 1960; Leathen et al., 1956; Razzell and Trussell, 1963a; Silverman and Lundgren, 1959a) but falls off below pH 2 (Razzell and Trussell, 1963a). Cells do not survive long above pH 6.5 (Ehrlich, 1962). Rapid iron oxidation by resting cell suspensions occurs in the pH range 2.5-4.2 (Silverman and Lundgren, 1959b) in agreement with pH requirements for growth. In addition to acid conditions, Lazaroff (1963) reported that rather high sulfate concentrations are required for iron oxidation. He suggests that since the requirement is not catalytic, sulfate may be involved in the entrance of ferrous ions into the cell, or in the synthesis of a sulfur compound required for energy transfer subsequent to iron oxidation. The organisms are remarkably resistant to or can adapt to high concentrations of metal ions; up to 10,000 to 15,000 p.p.m. Cu, 40,000 p.p.m. Zn, and 40,000 p.p.m. Fe have been tolerated (Booth and Mercer, 1963; Malouf and Prater, 1961; Marchlewitz et al., 1961). b. Mechanisms of Metal Sulfide Oxidation. Several agents can participate in the oxidation of metal sulfide minerals; these are oxygen, acid ferric sulfate solutions, and iron-oxidizing thiobacilli. Figure 4 illustrates the ways in which these agents are capable of oxidizing sulfide minerals, with their individual contributions determined by their relative efficiency as oxidizing agents and the susceptibility of different sulfide minerals to oxidation under different conditions. Minerals such as galena ( PbS), chalcopyrite ( CuFeSs), bornite ( Cu6FeS4), and sphalerite (ZnS) are oxidized 4

N

0

.. .-0 al L

c 0

8

FIG.4. Role of ferric sulfate, oxygen, and iron-oxidizing thiobacilli in metal sulfide oxidation.

FORMATION AND DEGRADATION OF MINERALS

181

relatively easily; pyrite ( FeS2 ) is moderately oxidizable; while covellite (CuS ), chalcocite ( Cu2S), and molybdenite (MOSS) are more difficult to oxidize (Ivanov, 196.2). The question of which oxidizing agent is the more important in nature is difficult to determine because the iron-oxidizing thiobacilli, when found in metal sulfide deposits, usually occur in the presence of oxygen and acid ferric sulfate solutions. The role of ferric sulfate in mineral sulfide oxidation may be described as follows. The ferric sulfate originates from pyritic materials which are believed to oxidize in aerated waters according to the over-all Eqs. (1)and ( 2 ) :

+ 3% 0,+ H,O + FeS0, + H$O, 4 FeS04 + 0, + 2 H,SO, + 2 Fez( SO,), + 2 H20 FeS,

(1)

(2)

Reaction ( 2 ) proceeds very slowly in the absence of a catalyst but rapidly in the presence of iron-oxidizing thiobacilli. Additional ferric sulfate may be contributed by the oxidation of mixed sulfide minerals such as chalcopyrite ( CuFeS,) or bornite ( Cu5FeS4).The ferric sulfate then can oxidize metal sulfides to form the corresponding metal sulfates while being reduced in turn to ferrous sulfate. It is particularly noteworthy that when the metal sulfide being oxidized by ferric sulfate is pyrite or marcasite, the net result is an increase in ferrous sulfate which may be generated by reaction ( 3 ) (Stokes, 1901) : FeSz

+ Fez(SO,),

+= 3 FeS0,

+2 S

13)

With increased ferrous sulfate available as energy source, the population of iron-oxidizing thiobacilli should increase with a concomitant increase in ferric sulfate. Increased quantities of ferric sulfate in turn can oxidize additional iron sulfides to produce even greater quantities of ferrous sulfate. Thus, an ever increasing cycle of oxidations and reductions ensues, resulting in the oxidation of large quantities of metal sulfides. The importance, therefore, of the iron-oxidizing thiobacilli in ferric sulfate oxidation of mineral sulfides lies in regenerating ferric sulfate from spent oxidant. The bacteria accomplish this at a much faster rate than oxygen alone. In addition to their role in regenerating ferric sulfate from spent ferrous sulfate solutions, the iron-oxidizing thiobacilli undoubtedly are involved in direct oxidative attack on metal sulfide minerals independent of the action of ferric sulfate. This has been demon-

182

MELVIN P. SILVERMAN AND HENRY L. EHRLICH

strated best with the iron-free minerals covellite, chalcocite, tetrahedrite, molybdenite, orpiment, and synthetic CuzS in the absence of added iron (Bryner et al., 1954; Bryner and Anderson, 1957; Ehrlich, 1962, 1963c; Razzell and Trussell, 1963a, b). The exact mechanism remains unknown. Further research is needed to assess the relative contribution of direct bacterial attack to mineral sulfide oxidation. The crystal structure of a given mineral is an important factor governing its susceptibility to oxidation. Pyrite ordinarily is very susceptible to bacterial attack but some well-crystallized museum grade specimens are resistant ( Leathen et al., 1953b; Silverman et al., 1961). It is possible that well-ordered crystal lattices are less susceptible to oxidation than imperfect lattice structures or those incorporating impurities. Bacterial oxidation of sulfide minerals is influenced by the accompanying gangue minerals, especially when the latter are alkaline. The resultant neutralization of acid leach solutions not only has an adverse effect on the activities of the acidophilic ironoxidizing thiobacilli but also interferes with the activity of acid ferric sulfate solutions by precipitating the iron as insoluble oxides. Precipitated iron and other metallic ions may coat the surface of unreacted metal sulfides and prevent ready access of bacteria or other oxidizing agents ( Razzell and Trussell, 1963b). Preliminary acid treatment to remove or neutralize alkaline material can circumvent this problem. Silverman et al. (1961) demonstrated that the removal of calcite ( CaC03) from a pyrite concentrate allowed bacterial oxidation to proceed. Sutton and Corrick (1963, 1964) obtained similar results when copper sulfide minerals containing calcite or siderite ( FeC03) were neutralized. The association in nature of Thiobacillus thiomiduns or T . concretivorus with the iron-oxidizing thiobacilli is somewhat puzzling. Thiobacillus thiomiclans is incapable of oxidizing metal sulfide minerals with the exception of marcasite (Leathen et al., 1953a; Silverman et al., 1961; Sutton and Corrick, 1961b; Temple and Delchamps, 1953) . Thiobacillus concretivorus cannot oxidize pyrite or copper sulfide minerals (Sutton and Corrick, 1963, 1964). When marcasite is absent, one must postulate the presence of elemental sulfur or another oxidizable sulfur compound. Elemental sulfur may well be an intermediate product of metal sulfide oxidation. Sullivan ( 1930a) reported an increase in carbon disulfide-soluble material

FORMATION AND DEGRADATION OF MINERALS

183

upon acid ferric sulfate leaching of copper sulfide minerals. Sato (1960) measured Eh and pH during metal suEde oxidation using simple sulfide minerals as single electrodes. He concluded that oxidation proceeds by a stepwise migration of metal ion into the surrounding solution leaving behind a mineral that becomes progressively enriched in sulfur until elemental sulfur remains. The identification of isolates as T. thwoxiduns in some instances may be premature. Thiobacillus thiooxicEans and the iron-oxidizing thiobacilli are morphologically indistinguishable, the chief distinction being oxidation by the latter of ferrous ions. Since both are capable of growth on elemental sulfur in acid mineral solutions, identification of primary isolates as T. thiooxiduns solely on this basis cannot be regarded as certain unless they can be shown not to oxidize iron as well. Thiobacillus thiooxiduns, when present, serves a useful function by producing sulfuric acid from reduced inorganic sulfur compounds and helping to maintain an acid environment conducive to the oxidative activities of the various agents that attack metal sulfides. In addition to T . thiooxidans and T . concretivorus, slime-encapsulated bacteria, yeasts, and other fungi, protozoa, and algae have also been found in many sites inhabited by the iron-oxidizing thiobacilli (Ehrlich, 1962, 1963b; Lackey, 1938; Marchlewitz et al., 1961; Razzell and Trussell, 196313). These organisms display unusual tolerance to conditions of high acidity and high metal ion concentrations normally considered too toxic for them, but their geochemical role, if any, is not well understood. Flagellated protozoa and amebae, present along with yeasts in the underground waters of a copper mine, were observed to feed on the iron-oxidizing thiobacilli, suggesting the operation of a balanced ecological system in which carbon fixation is dependent upon chemosynthetic rather than photosynthetic autotrophy (Ehrlich, 1963b). It should be obvious from the foregoing that iron-oxidizing thiobacilli play an important role in the solubilization of metal sulfides, making them available for secondary deposition elsewhere. Several interesting studies (Kramarenko, 1962; Lyalikova, 1960a, 1961) on the distribution of iron-oxidizing thiobacilli in and about Russian metal sulfide deposits indicate that the bacteria are most often found in highly irrigated zones of these deposits. The presence of pyrite as well as fissures, cracks, and dislocations in the ore favored their development. On the other hand, the absence of

184

MELVIN P. SILVERMAN AND HENRY L. EHRLICH

cracks and fissures in massive metal sulfide deposits, the alkaline nature of associated minerals or surrounding rock formations, or the cold temperatures and immobilization of water in the permafrost regions of the far north were unfavorable for their development. One of the more important ore-forming processes in which microbial action might play an important role is supergene enrichment (Bateman, 1950). It occurs when relatively poor sulfide mineral deposits lie partly within the zone of oxidation above the water table and partly below the water table where molecular oxygen is excluded in the hypogene zone. Iron sulfide minerals in the zone of oxidation are oxidized to sulfuric acid and ferric sulfate. This acid ferric sulfate solution reacts with other metal sulfides within the zone of oxidation to form soluble metal sulfates which trickle down through the deposit to the primary sulfide minerals of the hypogene zone. Here the enriched metal sulfate solution in contact with H2S or the primary metal sulfides forms secondary or supergene enrichments of sulfide minerals. Many rich secondary ore deposits of great economic value have been formed by this process. It is strikingly evident that the conditions within the zone of oxidation essential for the formation of supergene sulfide mineral deposits are also ideally suited for the activities of the iron-oxidizing thiobacilli. They may well have been instrumental in accelerating the process, although there are no reports in the literature on the bacteriology of sulfide mineral deposits undergoing active supergene enrichment. c. Economic Considerations. Acid ferric sulfate solutions are commonly employed in the mining industry to leach valuable metals from metal sulfide ores ( Sullivan, 1930a,b; 1931). The leaching operation is in reality a redox reaction in which minerals are oxidized at the expense of ferric sulfate. Chemical leaching is profitable when applied to many grades of ore, but the cost of replacing spent, acid, ferrous sulfate solutions, or regenerating them by chemical means, prevents its application to ores of marginal grade. The discovery of the iron-oxidizing thiobacilli and the elucidation of their role in oxidizing metal sulfides and ferrous ions in sulfuric acid solutions presented a means whereby these bacteria could be used to regenerate spent leach solutions and opened the way for more profitable leaching of ores as well as waste rocks from mining operations. Malouf and Prater (1961) described one such operation where mine wastes, accumulating at the rate of 200,000

FORMATION AND DEGRADATION OF MINERALS

185

tons per day and containing only traces of copper, could be leached profitably employing leach solutions rich in iron-oxidizing thiobacilli. Zimmerley et al. (1958) patented a cyclic leaching process employing bacterial regeneration of spent leach solutions for use in leaching copper, molybdenum, zinc, chromium, and titanium ores. Portuguese pyrites rich in uranium have been leached for their uranium content employing a similar process ( Audsley and Daborn, 1962a, b; Miller et al., 1962). It is interesting to note that efficient natural leaching processes, developed empirically at several mines years before the existence of iron-oxidizing thiobacilli was known, use conditions conducive for the activity of these bacteria (Razzell and Trussell, 1963b; Taylor and Whelan, 1943; Weed, 1956). Other possible uses for bacteria in metallurgical operations have been discussed by Sutton and Corrick (1961a). It is known that certain tissues of selected species of higher plants tend to accumulate anomalous concentrations of metals from soil overlying ore deposits. In addition, high concentrations of ore minerals often determine which species will live under these conditions. These facts have been exploited in geochemical prospecting for hidden ore deposits (Hawkes, 1957). It may well be that specific microbial species will predominate in such soils whereas other normal soil flora will be conspicuous by their absence; or, common soil microorganisms may exhibit a higher tolerance for metal ions than they do in other soils. Kendrick (1962) found 31 species of fungi with very high tolerance to copper in a Canadian copper swamp. Certainly the restricted habitat of iron-oxidizing thiobacilli would be useful in locating sulfide mineral deposits. A beginning may have been made along these lines when Kramarenko (1962) reported that the distribution of T. thiooriduns and T. ferrooxidans was confined to underground waters associated with concentrated ore formation; they were absent in waters with dispersed ore mineralization, Microbial oxidation of sulfide minerals, while beneficial for the extraction of metals from ores, may also have detrimental effects by increasing stream pollution. Pyrite and marcasite, associated with coal and adjacent rock strata, upon exposure to oxygen, water, and iron-oxidizing thiobacilli oxidize to sulfuric acid and iron sulfate. Ashmead (1955) concluded that four-fifths of the acid produced in two British collieries could be attributed to bacterial action. The magnitude of acid production can readily be appreciated from the

186

MELVIN P. SILVERMAN AND HENRY L. EHRLICH

calculations of Hodge (1937), who estimated that from one to three million tons of sulfuric acid entered the Ohio river and its tributaries in a single year. The acid corrodes mining machinery and equipment (Ash et al., 1955), kills aquatic flora and fauna, corrodes bridges, locks, dams, and concrete structures, dissolves rocks and minerals, and contributes to the hardness of water. The acid-soluble iron sulfates that accompany the acid discharges eventually hydrolyze and precipitate from solution to form a red to yellow coating of basic ferric sulfates and hydrated oxides of iron. Intensive research aimed at understanding and controlling acid mine drainage was undertaken and is summarized in several reports (Braley, 1954; Leathen, 1952; Lorenz, 1962; Moulton, 1957; Temple and Koehler, 1954). It was as a direct outgrowth of these studies that the iron-oxidizing thiobacilli were discovered and studied, and it became apparent that these bacteria might be useful in removing pyritic sulfur from coal prior to its combustion in power plants so as to reduce air pollution caused by volatile oxides of sulfur. Zarubina et al. (1959) reported that T . ferromidans removed up to 27% of the pyrite from Russian coals. Silverman and co-workers (1963) used F. ferrooxidans to remove more than 80% of the pyrite from some American coals. Bacterial pyrite removal from coal depended upon coal rank, particle size, associated alkaline material, and various pretreatments of coal.

C. METALSULFATES 1. F o r m t i o n of Deposits Sulfate deposits occur most frequently as calcium (gypsum, anhydrite) or magnesium salts (epsomite) and less frequently as the salts of barium (barite), iron (melanterite, coquimbite), and copper ( chalcanthite ) ( Latimer and Hildebrand, 1940; Teodorovich, 1961). Their formation is generally thought to be nonbiological by geologists, being formed in many instances by the evaporation of marine or brackish waters (Bateman, 1950). It is conceivable, however, that microbial oxidation of inorganic sulfur compounds to sulfuric acid might have contributed to sulfate deposition. The secondary reaction of sulfuric acid with carbonate salts would form sulfate salts. For example, the oxidation of a limestone-bearing sulfur ore by T . thiooxidans resulted in the formation of gypsum crystals (Karavaiko, 1962) according to reactions ( 4 ) and (5):

FORMATION AND DEGRADATION OF MINERALS

187

Other carbonates whose cations form relatively insoluble, stable sulfates would also be suitable material for microbial sulfate formation. Nevertheless, it must be admitted that microbial formation of sulfate deposits in nature remains conjectural. 2. Degradation of Deposits The stability of sulfates to chemical reduction under the normal environmental conditions of the biosphere is well recognized (Garrels and Naeser, 1958), and the sole process by which sulfate reduction occurs below temperatures of several hundred degrees is through the activity of dissimilatory sulfate-reducing bacteria (Jensen, 1962). The influence of the latter on the formation of sulfur and sulfide deposits has already been discussed. What we wish to stress here is their virtual monopoly with respect to widespread degradation of sulfate. The activities of sulfate-reducing bacteria are contingent upon adequate supplies of sulfate, a suitable hydrogen donor, and anaerobiosis. Adequate sulfate supply is no problem; it exists in almost limitless quantity in nature. This fact, coupled with the ability of the bacteria to thrive at high or low temperatures (Barghoorn and Nichols, 1961; Butlin, 1953), to tolerate a wide range of salt concentrations, and to utilize molecular hydrogen as well as organic hydrogen donors ensures the ubiquitous distribution of these organisms in the biosphere. Recent evidence from isotope fractionation studies of sedimentary sulfides of the Sudbury basin suggests that sulfate-reducing bacteria were of geological significance in Proterozoic times, some 2000 million years ago (Thode et al., 1962). Thus, the conclusion is inescapable that they have played and will continue to play an important role in the geology of inorganic sulfur compounds on our planet.

VI. Iron and Manganese Deposits The geochemistry of iron and manganese is closely related because of the widespread and often simultaneous occurrence of the two elements and the complementarity of properties of some of their compounds. For this reason, iron and manganese will be considered together.

w

TABLE 111 BIOGEOCHEMISTRY OF SOMEIRON AND MANGANESE MINER~~S Ore-building Formula Source organismsa

Name Carbonates Rhodochrosite Siderite Oxides Pyrolusiteb Manganiteb Hausmanniteb Pyrochroite Manganosite Goethite, lepidocrociteb Hematite6

MnC03

Sedimentary

FeC03

Sedimentary, hydrothermal

MnOz-HzO

Sedimentaryc

Mn20,. H20 MnA Mn(OH)Z MnO Fe203- H20

SehenhTc Sedimentaryc Sedimentary" Sedimentary0 Sedimentary6

Sulfate-reducing bacteria Bacteria

co 03 Ore-dissolving organismsa Bacteria, fungi

2

Bacteria, fungi

.d

2

B 8

I

Bacteria, algae,

Bacteria

2

Bacteria

Sulfides Alabandite

Fe,04 2 Fe203.3 H,O

Sedimentaryc Sedimentary"

MnS

Ign@JUS, hydrothermal, sedimentary

b-

3 i!

fungi, protozoa

fungi, protozoa Magnetite0 Limoniteb

Ez

?a

?a

r

BQ

TABLE I11 (Continued)

Name

Formula

Troilite

FeS

Sedimentary

Hydrotroilite

FeS .nHzO

Igneous, hydrothermal, sedimentary Igneous, hydrothermal, sedimentary Igneous, hy drothermal, sedimentary

Pyrite, marcasite Pyrrhotite

Sulfates Melanterite Coquimbite

FeS04*7H,O Fez( SO,),-Q HzO

Silicates Thuringite

7 F e 0 - 3 (Al, Fe),0,*5 SiO,.nH,O

Ore-building organismsa

Source

Sedimentary Sedimentary

Ore-dissolving organismsa

\ Sulfate-reducing bacteria

Thiobacilli, ferrobacilli

?d

?d

?d

?d

Igneous, ?d sedimentary Chamosite 4 Fe0-A1,03-3 SiO,.3 H,O Igneous, ?d sedimentary GreenaliteFe, . . .Fez . . . (Si,O,,) (OH),,*2 H,O Igneous, ?d glauconite sedimentary See text for specific studies and fuller identification of organisms. 0 In most instances, the kind of manganese or iron oxide formed or broken down was not identified. 0 Occasionally formed by hydrothermic process; also authigenic. d No specific microbial action known, but such action is deemed possible.

?a

?a

5

8 z

8

! m %-

r

v)

?a

@

w

a, (D

190

MELVIN P. SILVERMAN AND HENRY L. EHRLICH

A. OCCURRENCE AND ASSOCIATED BIOLOGICAL ACTIVITIES Table I11 lists some of the common iron and manganese minerals, their mode(s) of formation and transformation, and the types of organisms which may be involved. I t is evident from the tabulation that carbonates, oxides, and sulfates of iron and manganese are most frequently of sedimentary origin, whereas their sulfides and silicates may be of sedimentary, igneous, or hydrothermal origin. The criteria, whereby geologists characterize the mode of origin of a mineral, are described in most introductory geology texts (e.g., Bateman, 1951). 1. Carbonates The manganese and iron carbonates, rhodochrosite and siderite, may occur separately or in association with each other and magnesite (MgCO,) (Teodorovich, 1961). They are found in sedimentary deposits, but siderite has also been reported from hydrothermal deposits ( Bateman, 1951; Teodorovich, 1961) . They are unstable except in reducing environments of slightly acid to neutral pH. The ability of sulfate-reducing bacteria to produce MnC0, in the laboratory has been reported by Thiel (1925), but mineralogical identification of the product was not made. I t is not known whether such microbial activity could account for some rhodochrosite formation in nature. FeC03 precipitation by heterotrophic bacteria under anaerobic conditions has also been noted (Harder, 1919; Van Hise and Leith, 1911), but again mineralogical identification of the product was not made. Microbial contributions to siderite formation might be inferred from the observation that siderite occurs in some sand-silt-clay littoral or shallow water strata with disseminate pyrite and abundant organic matter (Bateman, 1951; Teodorovich, 1961), conditions favoring the growth of COz- and HzS-producing bacteria. Reduction of ferric to ferrous iron during bacterial decay of organic matter, and absorption of COz by plants, may also aid siderite formation (Harder, 1919). Microbial intervention in MnCO3 and FeC03 oxidation to oxides has been reported on a laboratory scale. Synthetic MnC03 oxidation by bacteria and fungi has been observed by a number of workers (Beijerinck, 1919; Leeper and Swaby, 1940; Thiel, 1925; Zavarzin, 1961, 1962), none of whom named the oxide of manganese formed except Beijerinck, who called it MnOz. The oxidation of FeCO,

FORMATION AND DEGRADATION OF MINERALS

191

solution by Leptothrix was reported by Winogradsky (1888). Oxidation by Gallionella ferruginea of FeC03 precipitate formed on iron filings was reported by Sartory and Meyer (1947). While MnC03- and FeC03-oxidizing bacteria were not tested on rhodochrosite and siderite, respectively, it seems very likely that they could promote oxidation of these minerals in nature in the light of the foregoing observations. 2. Oxides Oxides of manganese and iron, separately and in combination, are of fairly widespread natural occurrence ( Bateman, 1951; Robinson, 1929). Many are associated with sedimentary formations, but some with hydrothermal deposits. Biogeochemical formation of sedimentary and authigenic deposits has been widely claimed (Baars, 1950; Beijerinck, 1919; Bromfield, 1956, 1958a; Butkevitch, 1928; Deaugard, 1929; Ehrlich, 1963; Gillette, 1961; Gruner, 1922; Harder, 1919; Kindle, 1932; Leeper and Swaby, 1940; Lieske, 1919; Mann and Quastel, 1946; Mulder and van Veen, 1963; Sartory and Meyer, 1947; Thiel, 1925; Winogradsky, 1888; Zapffe, 1931; Zavarzin, 1961, 1962). The basis for these claims rests partly on finding fossil or live bacteria and algae in iron and manganese deposits, and partly on laboratory demonstrations that such organisms can cause iron and manganese oxide formation. Mineral types of iron and manganese oxides with which microorganisms have been associated include limonite (Deaugard, 1929; Dietz, 1955; Stauffer and Thiel, 1949), goethite (Buser and Gruetter, 1956; Stauffer and Thiel, 1949), 6-Mn02 (Buser and Gruetter, 1956), and amorphous manganese oxide (Bromfield, 1958a). It is quite likely that initial biogenic iron and manganese oxides undergo structural modification to such minerals as hematite, magnetite, hausmannite, and others through aging over geologic time. Iron and manganese oxides are usually broken down through microbial reduction to ferrous iron and manganous manganese (Bromfield, 1954a; Clark and Resnicky, 1956; Ehrlich, 1963a; Garey and Barber, 1952; Mann and Quastel, 1946; Perkins and Novielli, 1962; Roberts, 1947; Vavra and Frederick, 1952). Specific organisms that have been shown to be active in iron and manganese reductions include members of the Bacillus macerans-polymyxa group and Thiobacillus thiooxiduns. However, other, as yet unidentified, organisms are probably also involved, Bacillus circulans, B a c i l l ~ ~

192

MELVIN P. SILVERMAN AND HENRY L. EHRLICH

megaterium, and Aerobacter aerogenes reduce ferric lactate ( Bromfield, 195413). The mineralogical form of the oxides reduced in these experiments were in general undefined. 3. Sulfides The genesis of iron sulfide ores, such as troilite, hydrotroilite, pyrite, marcasite, and pyrrhotite, by bacterial sulfate reduction was discussed in Section V. Similar biogeochemical formation of alabandite (MnS) has not been reported. It is probably not readily formed biogenically because of its instability at acid pH and in air (Teodorovich, 1961). Microbial oxidation of iron sulfides was discussed in Section V. Biogeochemical oxidation of alabandite has not been reported. 4. Sulfates Biogeochemical formation of iron sulfates, such as melanterite and coquimbite, has not been recorded but seems highly probable in view of iron sulfate formation by the iron-oxidizing thiobacilli. Yellow to red deposits of “yellow boy” in “sulfur” or “red water” streams, which are the result of action by these bacteria, are said to consist of basic ferric sulfates and ferric hydroxides (Leathen et al., 1953a) or hydrated iron oxide (CoImer et al., 1950; Silverman et al., 1961). Very acid pH and a high concentration of sulfate, such as could exist in microenvironments of intense microbial activity on iron sulfides in ore bodies, could favor an accumulation of melanterite and coquimbite.

5. Silicates No definitive work on the formation of iron and manganese silicates by biogeochemical means exists. However, iron and manganese incorporation or adsorption by diatoms has been observed (Kindle, 1932; Molisch, 1911) and may indicate a biogeochemical mechanism contributing to sedimentary iron and manganese silicate formation. OF MICROBIAL INTERACTIONS WITH IRON B. MECHANISMS

AND

MANGANESE MINERALS

The taxonomy of microorganisms active on iron and manganese compounds has been reviewed at various times (Cholodny, 1926; Ellis, 1919; Mulder and van Veen, 1963; Pringsheim, 1946, 1949a, b;

FORMATION AND DEGRADATION OF MINERALS

193

Skerman, 1959; Starkey, 1945a; Winogradsky, 1922; Wolfe, 1963). Table IV lists genera of iron and manganese bacteria recognized by “Bergey’s Manual of Determinative Bacteriology” (Breed et a,?., 1957). The interaction of these microorganisms with iron and manganese compounds can be direct or indirect. TABLE IV OR SOLUBILJZE IRON BACTERIA WHICH PRECIPITATE AND MANGANESE COMPOUNDS~ Genus

Metal precipitated

Arthrobacter Clonothrix Clostridiumb Crenothrix Desulfovibrio Ferribacterium Ferrobacillus Gallionella Leptothrix

Mn Fe, Mn Fe Fe, Mn Fe Fe, Mn Fe Fe Fe, Mn

Metallogenium Micromonospora Naumaniella Ochrobium Siderobacter Siderocapsa Sideromonas

Mn Fe Fe Fe Fe Fe Fe, Mn

Sideronema Siderosphaera Siderophacus S p haerotilus Thiobacillusc Toxothrix

Fe, Mn Fe Fe Fe Fe Fe

Genus Aerobacter Bacillus Escherichia Thiobacillusa

Metal dissolved Mn Fe, Mn Mn Mn

Mode of nutrition Heterotrophic Heterotrophic Heterotrophic Heterotrophic Heterotrophic Heterotrophic Autotrophic Autotrophic Heterotrophic, autotrophic Heterotrophic Heterotrophic Heterotrophic Heterotrophic? Heterotrophic Heterotrophic? Autotrophic, heterotrophic Heterotrophic Heterotrophic Heterotrophic Heterotrophic Autotrophic HeterotroDhic Mode of nutrition Heterotrophic Heterotrophic Heterotrophic Autotrophic

a Compilation based on Alexander ( 1961 ) ; Breed et al. ( 1957 ) ; Perkins and Novielli (1962); Skerman (1959); Zavarzin (1961). Only C . nigrificans. 0 Only T . ferrooxidans. Only 2’. thiooxidans. I J

194

MELVIN P. SILVERMAN AND HENRY L. EHRLICH

1. Indirect Action In indirect action some microorganisms may oxidize or reduce iron and manganese compounds without enzymic interaction, or they may simply accumulate already oxidized iron and manganese. For example, they may cause oxidation and precipitation of iron or manganese by generating an oxidizing environment through 0 2 generation or CO, consumption. Some algae ( Cyanophyceae, Chrysophyceae, Volvocales, Chlorococcales, Euglenineae, Conjugales, Ulothricales) are particularly active in this way, precipitating iron and manganese oxides, which may encrust the cell surface (Pringsheim, 1946, 1949a). Adsorption of preformed iron and manganese oxides may be due to a special binding capacity for these compounds (Pringsheim, 1949a). Many of the sheathed bacteria ( Crenothrix, Clonothrix, Leptothrix ), one of the stalked bacteria ( Gallionella), and some protozoan flagellates ( Anthophysa, Siderodendron, Bikosoeca, Siphomonas) are thought to function in this way, although they may also participate in the formation of these oxides (Lieske, 1911, 1919; Molisch, 1911; Praeve, 1957; Pringsheim, 1946; Sartory and Meyer, 1947; Schorler, 1904). In addition, microorganisms may produce a reducing environment by forming metabolic end products which can act as reductants of iron or manganese oxides (Pringsheim, 1949a; Starkey, 1945a). 2. Direct Action In direct action, microorganisms may interact enzymically with iron or manganese compounds. For example, iron-oxidizing thiobacilli oxidize ferrous iron to ferric iron at acid pH (Beck, 1960; Kinsel, 1960; Leathen and Braley, 1954; Leathen et al., 1953b; Razzell and Trussell, 1963a; Silverman and Lundgren, 1959b; Temple and Colmer, 1951), where autoxidation of iron is extremely slow. Gallionella can oxidize iron enzymically at neutral pH (Kucera and Wolfe, 1957; Lieske, 1911; Sartory and Meyer, 1947), where autoxidation is rapid and difficult to distinguish from biological oxidation. Proof of iron oxidation by Gallionella rests on the observation of growth in the absence of organic carbon, where iron is the only possible energy source for growth. Leptothrix ochracea can also oxidize iron in the absence of an organic carbon source (Lieske, 1919); it is described as a facultative autotroph (Skerman, 1959).

FORMATION AND DEGRADATION OF MINERALS

195

Enzymic manganese oxidation is very probable. A strong indication of such activity was provided by experiments with soil which showed that enzyme inhibitors, like sodium azide, prevented disappearance of manganous manganese and the build-up of oxides of manganese (Mann and Quastel, 1946). Another strong indication of enzymic manganese oxidation was given by experiments with a combination of pure cultures of a Corynebacterium and a Chromobacterium and with a mutant of the Corynebacterium, which formed oxides of manganese (Bromfield, 1956). In both these instances, autoxidation due to microenvironmental conditions generated on cell or other surfaces, although a remote possibility, cannot be ruled out entirely (Lieske, 1919; Molisch, 1911). If energy-yielding enzymic manganese oxidation does occur, it presents a puzzle as to its function since heterotrophic organisms, with few exceptions (Praeve, 1957; Sartory and Meyer, 1947), are implicated in this activity. The answer may be that this oxidation provides energy for the assimilation of organic carbon by a nutritional process that has been termed mixotrophic (Lieske, 1919). Enzymic ferric iron reduction to ferrous iron has not been unequivocally demonstrated, although very suggestive evidence of such activity has been published (Bromfield, 1954a, b; Roberts, 1947). Enzymic reduction of manganic oxide to manganous manganese appears probable from the reports of several workers (Baars, 1950; Ehrlich, 1963a; Garey and Barber, 1952; Mann and Quastel, 1946; Perkins and Novielli, 1962; Vavra and Frederick, 1952). The specific enzyme systems in bacterial MnOz reduction have so far not been characterized. However, those of animal tissues and yeasts have been studied with MnOz acting as electron acceptor under anaerobic conditions (Hochster and Quastel, 1952). Ferric iron would probably have a similar function. Enzymic interaction with iron and manganese compounds may manifest itself in another way. Organic salts or complexes of iron and manganese may be attacked by microorganisms so as to destroy the organic moiety of the compounds. This frees the metal ion which, owing to hydrolysis or other chemical reaction, may form an insoluble compound or precipitate ( Starkey, 1945a). The organic moieties of such compounds may be proteins, amino acids, or other organic acids. Chemical equations describing the biological oxidation of iron

196

MELVIN P. SILVERMAN AND HENRY L. EHRLICH

and manganese may be formulated as follows. Ferrous iron at acid pH appears to be oxidized according to reaction ( 2 ) : 4 FeSO,

+ 0, + 2 H,SO,

+ 2 Fez( SO,),

+ 2 H,O

The reaction between FeS04 and 0, has been shown to be stoichiometric for the iron-oxidizing thiobacilli (Beck, 1960; Silverman and Lundgren, 1959b). At neutral pH, iron may be oxidized according to reaction ( 6 ) (Baas Becking and Parks, 1927) : 4 FeC03 + 0, + 6 H,O + 4 Fe( OH), + 4 CO, (6) Manganese oxidation at neutral or alkaline pH may proceed according to reaction (7) (Alexander, 1961) : Mn+ +

+ 2 H,O -+ MnOz + 4 H + + 2 e-

(7) Some observations have been made on the nature of the ironoxidizing enzyme systems of the iron-oxidizing thiobacilli. Vernon et al. (1960) detected cytochromes c and al, but no cytochrome b in cell-free extracts, and proposed a transfer of electrons from ferrous ions to oxygen via cytochrome c and cytochrome al. Blaylock and Nason (1961, 1962, 1963) prepared a particulate iron oxidase system from Ferrobacillus ferrooxidans with an absolute requirement for oxygen. It contained an iron-cytochrome c reductase as the first enzyme and cytochrome oxidase as the terminal enzyme of the system. Although cytochromes of the b, c, and a types were detected¶ only cytochromes c and a were reduced by ferrous ions. The path of electrons from ferrous iron to oxygen was postulated to occur via iron-cytochrome c reductase and cytochrome oxidase in the sequence shown in (8): Fe + + + cytochrome c + cytochrome a + 0,

(8)

Similar studies of the enzymes involved in microbial manganese oxidation remain to be made, as do studies of the enzymes involved in the reduction of iron and manganese. The problem of the energetics and efficiency of iron oxidation in relation to COz fixation has been studied and analyzed (Beck, 1960; Lyalikova, 1958; Silverman and Lundgren, 1959b; Silverman and Rogoff, 1961; Temple and Colmer, 1951). Normally, 0 2 consumption during iron oxidation is extremely rapid with Q O z N values in the range 2000 to 5000, but it varies with availability of 0 2 , age of cells, and their physiological condition. The free-energy efficiency of COz assimilation in young, vigorous cultures ranges from 20 to 30% but decreases to 3 to 5% in old cultures.

FORMATION AND DEGRADATION OF MINERALS

197

The energetics of biological manganese oxidation have not been considered so far, mainly because the precise reaction involved in the process is still unknown. If reaction ( 9 ) Mn+++%O2$2OH-+MnO2+H20

(9)

is assumed to describe the process, the AF is -36.8 kcal. (Goldberg and Arrhenius, 1958). This is of the same order of magnitude as the energy liberated during FeC03 oxidation at neutral p H ( AF is -40 kcal.; Baas Becking and Parks, 1927). C. ECONOMIC IMPORTANCE Biogeochemical action on iron and manganese compounds may be economically important in mineral conservation and in new hydrometallurgical extraction methods, especially for low grade ores. While the need for new workable extraction methods for iron ores is not yet as great as for manganese ores, it may arise in the not too distant future, as high grade iron deposits become depleted and no new ones are discovered, Rich manganese ore reserves in the United States have always been inadequate for present-day needs in steel, alloy, and dry-cell battery manufacture. As a result, much manganese ore has been imported ( Bateman, 1951). The possibility of bacterial extraction of manganese from low grade domestic ores has been reported (Perkins and Novielli, 1958, 1962). In addition, suggestions of mining manganese nodules, a rich manganese ore in the oceans, have been made (Cowen, 1960; Mero, 1962). Interaction of iron and manganese with appropriate bacteria in public water supplies presents a purification problem (Chambers and Ingols, 1956; Ellis, 1919; Ingols and Wilroy, 1962; Zapffe, 1931). Although the foregoing references suggest some methods of control, additional methods of even greater efficacy remain to be worked out. The relation of bacteria to iron corrosion has been extensively studied and discussed (Baumgartner, 1962; Booth and Tiller, 1960; Kuznetsov and Verzhbitskaya, 1961; Starkey, 1956; Tiller and Booth, 1962). It will not be considered further. The use of iron and manganese bacteria for mineral prospecting does not seem to have been widely considered except in the case of the iron-oxidizing thiobacilli (Kramarenko, 1962). Further ecological studies are needed to provide additional information on other possible useful indicators of iron and manganese ore deposits.

198

MELVIN P. SILVERMAN AND HENRY L. EHRLICH

VII. Conclusion It is apparent that microorganisms have profoundly influenced the transformations of some economically important minerals. But vast gaps in our knowledge of geomicrobiology remain to be filled. It is the task of future research to bring into proper perspective the role, both past and present, of microorganisms in transformations of the inorganic environment, and to indicate ways in which their activities can be exploited in the future.

REFERENCES Adams, F., and Conrad, J. P. (1953). Soil Sci. 75, 361-371. Adler, H. H. (1963). Econ. Geol. 58, 839-852. Alexander, M. ( 1961). “Introduction to Soil Microbiology.” Wiley, New York. Ash, S. H., Dierks, H. A., Felegy, E. W., Huston, K. M., Kennedy, D. O., Miller, P. S., and Rosella, J. J. (1955). U.S . Bur. Mines Bull. 555, 46 pp. Ashirov, K. B., and Sazanova, I. V. (1962). Mikrobiologiya 31, 680-683. Ashmead, D. ( 1955). Colliery Guardian 190, 694-698. Audsley, A., and Dabom, G. R. (1962a). Trans. Inst. Mining Met. 72, 235-246. Audsley, A,. and Daborn, G. R. (196213). Trans. Inst. Mining Met. 72, 247-254. Baars, J. K. (1950). Trans. 4th Intern. Congr. Soil Sci. Amsterdam 1, 192-195. Baas Becking, L. G. M., and Moore, D. ( 1961). Econ. Geol. 56, 259-272. Baas Becking, L. G. M., and Parks, G . S. (1927). Physiol. Reu. 7, 85-106. Baer, F. E., ed. (1955). “Chemistry of the Soil.” Reinhold, New York. Barghoorn, E. S., and Nichols, R. L. (1961). Science 134, 190. Bateman, A. M. (1950). “Economic Mineral Deposits,” 2nd ed. Wiley, New York. Bateman, A. M. (1951). “The Formation of Mineral Deposits.” Wiley, New York. Baumgartner, A. W. (1962). J. Petrol. Technol. 14, 1074-1078. Bavendamm, W. (1932). Arch, Mikrobiol. 3, 205-276. Beerstecher, E. ( 1954). “Petroleum Microbiology.” Elsevier, Amsterdam. Beck, J. V. (1960). 1. Bacteriol. 79, 502-509. Beijerinck, M. W. (1919). Zentr. Bakteriol. Parasitenk. Abt. 11 49, 470-471. Berner, R. A. (1962). In “Biogeochemistry of Sulfur Isotopes” (M. L. Jensen, ed. ), pp. 107-120. National Science Foundation Symposium, Yale Univ., New Haven, Connecticut. Blaylock, B. A,, and Nason, A. (1961). Federation Proc. 20, 46. Blaylock, B. A., and Nason, A. (1962). Federation PTOC. 21, 49. Blaylock, B. A., and Nason, A. (1963). J . Bid. Chem. 238, 3453-3462. Booth, G. H., and Mercer, S . J. (1963). Nature 199, 622. Booth, G. H., and Tiller, A. K. (1960). Trans. Faraday SOC. 56, 1689-1696.

FORMATION AND DEGRADATION OF MINERALS

199

Braley, S. A. (1954). Summary Rept. Commonwealth of Pennsylvania Dept. Health ~ndustrialFellowship No. 326B, 279 pp. Breed, R. S., Murray, E. G. D., and Hitchens, A. P., eds. (1957). “Bergey’s Manual of Determinative Bacteriology,” 7th ed. Williams & Wilkins, Baltimore, Maryland. Brenner, W. (1916). Jahrb. Wiss. Botan. 57, 95-127. Bromfield, S. M. (1953). J. Gen. Microbiol. 8, 378-390. Bromfield, S. M. (1954a). 1. Soil. Sci. 5, 129-139. Bromfield, S. M. (1954b). 1. Gen. Microbiol. 11, 1-6. Bromfield, S. M. (1956). Australian J. Biol. Sci. 9, 238-252. Bromfield, S. M. (1958a). Plant Soil 9, 325-337. Bromfield, S. M. (1958b). Plant Soil 10, 147-160. Bromfield, S. M., and Skerman, V. B. D. (1950). Soil Sci. 69, 337-338. Bryner, L. C., and Anderson, R. (1957). Ind. Eng. Chem. 49, 1721-1724. Bryner, L. C., and Jameson, A. K. (1958). Appl. Microbiol. 6, 281-287. Bryner, L. C., Beck, J. V., Davis, D. B., and Wilson, D. G . (1954). Ind. Eng. Chem. 46, 2587-2592. Buser, W., and Gruetter, A. (1956). Schweiz. Mineral. Petrog. Mitt. 36, 49-62. Butkevitch, V. S. (1928). Wiss. Meeresinst. Ber. 3, 7-80. Butlin, K. R. (1953). Research (London) 6, 184-191. Butlin, K. R., and Postgate, J. R. (1954). In “Biology of Deserts” (J. L. Cloudsley-Thompson, ed. ), pp. 112-122. Symposium Inst. Biol., London. Campbell, L. L., Jr., Frank, H. A., and Hall, E. R. (1957 j . J . Bacteriol. 73, 516-521. Cayeux, L. (1937). Compt. Rend. SOC. Geol. France 10, 115-116. Chambers, H. H., and Ingols, R. S. (1956). Water G Sewage Works 103, 284-285. Cheney, E. S., and Jensen, M. L. (1962). Econ. Geol. 57, 624-627. Cholodny, N. ( 1926). Pflanzenforsch. 4, 1-162. Clark, F. E., and Resnicky, J. W. (1956). Rappt., 6th Intern. Congr. Soil. Sci., Paris C , pp. 545-548. Coleman, C. S . ( 1960). J. Gen. Microbiol. 22, 423-426. Colmer, A. R. (1961). 3. Bacteriol. 83, 761-765. Colmer, A. R., and Hinkle, M. E. (1947). Science 106, 253-256. Colmer, A. R., Temple, K. L., and Hinkle, M. E. (1950). J . Bacteriol. 59, 317-328. Corrick, J. D., and Sutton, J. A. (1961). U. S . Bur. Mines, Rept. Invest. 5718, 8 pp. Cowen, R. C . (1960). “Frontiers of the Sea. The Story of Oceanography,” pp. 263-268. Doubleday, Garden City, New York. Cushman, J. A. ( 1940). “Foraminifera, Their Classification and Economic Use,” 3rd ed. Harvard Univ. Press, Cambridge, Massachusetts. Cuthbert, M. E. (1962). Econ. Geol. 57, 38-41. Davidson, C. F. (1962a). Econ. Geol. 57, 265-274. Davidson, C . F. (196213). Econ. Geol. 57, 1134-1137. Deaugard, L. (1929). Compt. Rend. Acad. Sci. 188, 1616-1618. Dessau, G., Jensen, M. L., and Nakai, N. (1962). Econ. GeoZ. 57, 410-438.

200

MELVIN 1’. SILVERMAN AND HENRY L. EHRLICH

Dietz, R. S. (1955). J. Calif. Mines Geol. 51, 209-220. Domhrowski, H. J. (1960). Zentr. Bakteriol. Parasitenk. Abt. I . Orig. 178, 83-90. Dombrowski, H. J. (1963). Zentr. Bakteriol. Parasitenk. Abt. I . Orig. 188, 248-250. Dondero, N. C. (1961). Aduan. Appl. Microhiol. 3, 77-107. Duff, R. B., and Webley, D. M. (1959). Chem. d7 2nd. (London) pp. 13761377. Duff, R. B., Webley, D. M., and Scott, R. 0. (1963). Soil Sci. 95, 105-114. Ehrlich, H. L. ( 1961). Bacteriol. Proc. p. 54. Ehrlich, H. L. (1962). In “Biogeochemistry of Sulfur Isotopes” ( M . L. Jensen, ed. ), pp. 153-168. National Science Foundation Symposium, Yale Univ., New Haven, Connecticut. Ehrlich, H. L. (1963a). Appl. Microbiol. 11, 15-19. Ehrlich, H. L. (1963b). J. Bacteriol. 86, 350-352. Ehrlich, H. L. ( 1 9 6 3 ~ ) . Econ. Geol. 58, 991-994. Ehrlich, H. L. (1963d). In “Proceedings of the 1963 Rudolfs Research Conference.” Wiley, New York (in press). Ellis, D. (1919). “Iron Bacteria,” Methuen, London. Emery, K. O., and Rittenberg, S. C. (1952). Bull. Am. Assoc. Petrol. Geologists 36, 735-806. Feely, H. W., and Kulp, J. L. (1957). Bull. Am. Assoc. Petrol. Geologists 41, 1802-1853. Ckrey, C. L., and Barber, S. A. (1952). Soil Sci. SOC. Am. Proc. 16, 173-175. Garlick, W. G. (1961). In “The Geology of the Northern Rhodesian Copperbelt” (F. Mendelsohn, ed.), pp. 146-165. Macdonald, London. Carrels, R. M., and Naeser, C. R. (1958). Geochim. Cosmochim. Acta 15, 113-130. Gillette, N. J. (1961). N . Y. State Conserv., April-May, p. 19. Gleen, H. (1950). Nature 166, 871-872. Clock, W. S. (1923). Am. J. Sci. 6, 377-408. Goldberg, E. D., and Arrhenius, G . 0. S. (1958). Geochim. Cosmochim. Acta 13, 153-212. Gruner, J. W. (1922). Econ. Geol. 17, 407-460. Guittoneau, G. (1927). Compt. Rend. Acad. Sci. 184, 45-46. Halvorson, H. 0. (1931). Soil Sci. 32, 141-185. Harder, E. C. (1919). U.S. Geol. SUTU.,Profess. Paper 113, 89 pp. Harrison, A. G., and Thode, H. G. (1958). Trurw. Faraday Soc. 54, 84-92. Harold, R., and Stanier, R. Y. (1955). Bacteriol. Reu. 19, 49-64. Hawkes, H. E. (1957). U . S. Ceol. Suru. Bull. 1000-F, pp. 225-355. Hilz, H., Kittler, M., and Knape, G. (1959). Biochem. Z. 332, 151-166. Hochster, R. M., and Quastel, J. H. (1952). Arch. Biochem. Biophys. 36, 132-146. Hodge, W. W. (1937). 2nd. Eng. Chem. 29, 1048-1055. Ingols, R. S., and Wilroy, R. D. (1962). J. Am. Water Works Assoc. 54, 203-207.

FORMATION AND DEGRADATION OF MINERALS

201

Ito, I., Wakazono, Y., Kondo, M., Yagi, S., and Katsuki, H. (1960). J. Mining Met. Inst. Japan 76, 524-529. Ivanov, M. V. (1957a). Mikrobiologiya 26, 338-345. Ivanov, M . V. (1957b). Mikrobiologiya 26, 544-550. Ivanov, M. V. (1960). Mikrobiologiya 29, 109-113. Ivanov, M. V. (1961a). Mikrobiologiya 30, 242-247. Ivanov, M. V. (1961b). Mikrohiologiya 30, 500-502. Ivanov, M. V., and Kostruva, M. F. ( 1961). Mikrobiologiya 30, 130-134. Ivanov, M. V., and Ryzhova, V. N. (1961). Mikrobiologiya 30, 280-285. Ivanov, V. I. (1962). Mikrohiologiya 31, 795-799. Ivanov, V. I., and Lyalikova, N. N. (1962). Mikrohiologiya 31, 468-469. Ivanov, V. I,. Nagirnyak, F. I., and Stepanov, B. A. (1961). Mikrobiologiya 30, 688-692. Jensen, M. L., ed. ( 1962). “Biogeochemistry of Sulfur Isotopes.” National Science Foundation Symposium, Yale Univ., New Haven, Connecticut. Jones, G. E., and Starkey, R. L. (1957). Appl. Microbiol. 5, 111-118. Jones, G. E., and Starkey, R. L. (1961). J. Bacteriol. 82, 788-789. Jones, G. E., and Starkey, H. L. (1962). In “Biogeochemistry of Sulfur Isotopes” ( M . L. Jensen, ed.), pp. 61-77. National Science Foundation Symposium, Yale Univ., New Haven, Connecticut. Jones, G. E., Starkey, R. L., Feely, H. W., and K~ilp,J. L. (1956). Science 123, 1124-1125. Kaplan, I. R., and Rittenberg, S. C. (1962a). In “Biogeochemistry of Sulfur Isotopes” (M. L. Jensen, ed. ), pp. 80-93. National Science Foundation Symposium, Yale Univ., New Haven, Connecticut. Kaplan, I. R., and Rittenberg, S. C. (1962b). Nature 194, 1098-1099. Kaplan, I. R., Emery, K. O., and Rittenberg, S. C. (1963). Geochim. Cosmochim. Actu 27, 297-331. Karavaiko, G. I ( 1959). Mikrobiologiya 28, 846-850. Karavaiko, C. I. ( 1961). Mikrobiologiya 30, 286-288. Karavaiko, G. I. ( 1962). Mikrohiologiya 31, 328-331. Kendrick, W. B. (1962). Can. J. hlicrohiol. 8, 639-647. Kindle, E. M. (1932). Am. J. Sci. 24, 496-504. Kinsel, N. A. (1960). J. Bacteriol. 80, 628-632. Kramarenko, L. E. ( 1962). Mikrohiologiya 31, 694-701. Kucera, S., and Wolfe, R. S. (1957). J. Bacteriol. 74, 344-349. Kuznetsov, V. V., and Verzhbitskaya, L. V. (1961). Mikrobiologiya 30, 511-514. Kuznetsova, V. A., and Pantskhava, E. S. (1962). Mikrohiologiya 31, 129134. Lackey, J. B. (1938). Publ. Health R e p . 53, 1499-1507. Latimer, W. M., and J , H. Hildebrand. (1940). “Reference Book of Inorganic Chemistry,” rev. ed. Macmillan, New York. Lazaroff, N. (1963). J. Bacteriol. 85, 78-83. Leathen, W. W. ( 1952). Commonwealth of Pennsylzjanio Dept. Health Industrial Fellowship No. 326B-6, 54 pp. Leathen, W. W., and Braley, S. A. ( 1954). Bacteriol. Proc. p. 44.

202

MELVIN P. SILVERMAN AND HENRY L. EHRLICH

Leathen, W. W., McIntyre, L. D., and Braley, S. A. (1951). Science 114, 280-281. Leathen, W. W., Braley, S. A., and McIntyre, L. D. (1953a). Appl. Microbid. 1, 61-64. Leathen, W. W., Braley, S. A., and McIntyre, L. D. (1953b). Appl. Microbiol. 1, 65-68. Leathen, W. W., Kinsel, N. A., and Braley, S. A. (1956). J. Bacteriol. 72, 700-704. Leeper, G. W., and Swaby, R. J. (1940). Soil Sci. 49, 163-169. Lees, H. (1960). Ann. Reo. Microbiol. 14, 83-98. Lewin, J. C. (1954). J. Gen. Physiol. 37, 589-599. Lewin, J. C. (1955). Plant Physiol. 30, 129-1.34. Lewin, J. C. (1957). Can. J. Microbiol. 3, 427-433. Lieske, R. (1911). Jahrb. Wiss. Botan. 49, 91-127. Lieske, R. (1919). Zentr. Bakteriol. Parasitenk. Abt. I1 49, 413-425. Ljunggren, P. (1960). Econ. GeoZ. 55, 531-538. Love, L. G. (1962). Econ. Geol. 57, 350-366. Lundgren, D. G., Anderson, K., and Remsen, C. C. (1962). Bacteriol. Proc. p. 25. Lyalikova, N. N. ( 1958). Mikrobiologiya 27, 546-549. Lyalikova, N. N. (1960a). Mikrobiologiya 29, 382-387. Lyalikova, N. N. (1960b). Mikrobiologiya 29, 773-779. Lyalikova, N. N. (1961). Mikrobiologiya 30, 135-139. Mager, J. (1960). Biochim. Biophys. Acta 41, 553-555. Malouf, E. E., and Prater, J. D. (1961). J. Metals 13, 353-356. Mann, P. J. G., and Quastel, J. H. (1946). Nature 158, 154-156. Marchlewitz, B., and Schwartz, W. (1961). Z. Allgem. Mikrobiol. 1, 100114. Marchlewitz, B., Hasche, D., and Schwartz, W. (1961). Z. Allgem. Mikrobiol. 1, 179-191. Mason, B. ( 1952). “Principles of Geochemistry.” Wiley, New York. Mero, J. L. (1962). Econ. Geol. 57, 747-767. Meyer, H. (1963). Zentr. Bakteriol. Parasitenk, Abt. I . Orig. 188, 245-247. Miller, L. P. (1950). Contrib. Boyce Thompson Inst. 16, 85-89. Miller, R. P., Napier, E., and Wells, R. A. (1962). Trans. Inst. Mining Met. 72, 217-234. Molisch, H. (1911). Zentr. Bakteriol. Parasitenk. Abt. I1 29, 241-243. Moore, H. B. (1958). “Marine Ecology,” p. 335. Wiley, New York. Moulton, E. Q., ed. (1957). Ohio State Uniu. Studies. Eng. Expt. Sta. Bull. No. 166, 158 pp. Mulder, E. G., and van Veen, W. L. (1963). Anionie oan Leeuwenhoek, J . Microbiol. Serol. 29, 121-153. Myskow, W. (1960). Acta Microbiol. Polon. 9, 367-393. Nadson, G. A. (1927a). Compt. Rend. Acad. Sci. 184, 896-898. Nadson, G. A. (1927b). Compt. Rend. Acad. Sci. 184, 1015-1017. Nadson, G.A. (1928). Arch. HydrobioZ. 19, 154-164. Nakai, N., and Jensen, M. L. (1960). J. Earth Sci., Nagoya Uniu. 8, 181196.

FORMATION AND DEGRADATION OF MINERALS

203

Newburgh, R. W. (1954). 1. Bacteriol. 68, 93-97. Nickerson, W. J., and Falcone, G. (1963). J. Bacteriol. 85, 763-771. Nikitia, D. I. (1959). Izvest. Akad. Nauk. Ser. Biol. 1, 118-122. Paine, S. G., Lingood, F. V., Schimmer, F., and Thrupp, T. C. (1933). Phil. Trans. Roy. Soc. London Ser. B. 222, 97-127. Parker, C. D. (1947). Nature 159, 439. Peck, H. D., Jr. (1962). Bacteriol. Rev. 26, 67-94. Perkins, E. C., and Novielli, F. (1958). Mining Congr. J . 44, 72-73. Perkins, E. C., and Novielli, F. (1962). U . S. Bur. Mines Rept. Invest. 6102, 11 PP. Pettijohn, F. J. (1949). “Sedimentary Rocks.” Harper, New York. Postgate, J. R. (1951). J. Gen. Microbiol. 5, 725-738. Postgate, J. R. (1956). J. Gen. Microbiol. 14, 547-572. Postgate, J. R. ( 1959). Ann. Rev. Microbiol. 13, 505-520. Postgate, J. R. (1960). I n “Progress in Industrial Microbiology” (D. J. D. Hockenhull, ed.), 2, 47-69. Wiley (Interscience), New York. Postgate, J. R., and Campbell, L. L. (1963). J. Bacteriol. 86, 274-279. Praeve, P. (1957). Arch. Mikrobiol. 27, 33-62. Pringsheim, E. G. (1946). Phil. Trans. Roy. SOC. London Ser. B 232, 311342. Pringsheim, E. G. (1949a). B i d . Rev. 24, 200-245. Pringsheim, E. G. (194913). Phil. Trans. Roy. SOC. London Ser. B 233, 453482. Quastel, J. H., and Scholefield, P. G. (1953). Soil Sci. 75, 279-285. Razzell, W. E., and Trussell, P. C. (1963a). J. B a c t e h l . 85, 595-603. Razzell, W. E., and Trussell, P. C. (1963b). Appl. Microbiol. 11, 105-110. Reiser, R., and Tasch, P. (1960). Trans. Kansas Acad. Sci. 63, 31-34. Remsen, C. C., and Lundgren, D. G. (1963). Bacteriol. Proc. p. 33. Rippel, A. ( 3924). Zentr. Bakteriol. Parasitenk. Abt. I1 62, 290-295. Roberts, J. L. (1947). Soil Sci. 63, 135-140. Roberts, W. M. B. (1963). Econ. Geol. 58, 52-61. Robinson, W. 0. (1929). Soil Sci. 27, 335-350. Rogoff, M. H., Wender, I., and Anderson, R. B. (1962). U.S. Bur. Mines Inform. Circ. 8075, 85 pp. Rosenthal, G. ( 1956). Heidelberger Beitr. Mineral. Petrog. 5, 146-164. Rudakov, K. J. (1929). Zentr. Bakterwl. Parasitenk. Abt. 1I 79, 229-245. Ryzhova, V. N., and Ivanov, M. V. (1961). Mikrobiologiya 30, 1075-1079. Sales, R. H. (1962). Econ. Geol. 57, 721-734. Sartory, A,, and Meyer, J. ( 1947). Compt. Rend. Acud. Sci. 225, 541-542. Sato, M. (1960). Econ. Geol. 55, 1202-1231. Schaeffer, W. I., Holbert, P. E., and Umbreit, W. W. (1963). J. Bacteriol. 85, 137-140. Schaeffer, W. I., and Umbreit, W. W. (1963). J. Bacteriol. 85, 492-493. Schatz, A. ( 1962). Naturwissenschaften 22, 518-519. Schorler, B. ( 1904). Zentr. Bakteriol. Parasitenk. Abt. I1 12, 681-695. Setchell, W. A. (1926). Proc. Am. Phil. SOC.66, 136-140. Shturm, L. D. (1948). Mikrobiologiya 17, 415-418. Silverman, M. P., and Lundgren, D. G. (1959a). J. Bacteriol. 77, 642-647.

204

MELVIN P. SILVERMAN AND HENRY L. EHRLICH

Silverman, M. P., and Lundgren, D. G. (1959b). J. Bacteriol. 78, 326-331. Silverman, M. P., and Rogoff, M. H. (1961). Nature 191, 1221-1222. Silverman, M. P., Rogoff, M. H., and Wender, I. (1961). Appl. Microhiol. 6, 491-496. Silverman, M. P., Rogoff, M. H., and Wender, I. (1963). Fuel (Lonclon) 42, 113-124. Skerman, V. B. D. (1959). “A Guide to the Identification of the Genera of Bacteria,” Williams & Wilkins, Baltimore, Maryland. Skerman, V. B. D., Dementjeva, G., and Carey, B. J. (1957). J. BacteTiol. 73, 504-512. Sokolova, G. A. (1960). Mikrobiologiya 29, 883-893. Sokolova, G. A. ( 1961 ) . Mikrobiologiya 30, 503-510. Sokolova, G . A. (1962). Mikrobiologiya 31, 324-327. Sokolova, G. A,, and Karavaiko, G. I. (1962). Mikrobiologiya 31, 984-989. Sorokin, Yu. I. (1962). Mikrobiologiya 31, 402-410. Starkey, R. L. (1934). J. Bacteriol. 28, 387-400. Starkey, R. L. (1945a). J. Am. Water Works Assoc. 37, 963-984. Starkey, R. L. (194513). Science 102, 532-533. Starkey, R. L. (1956). Ind. Eng. Chem. 48, 1429-1437. Starkey, R. L., and Halvorson, H. 0. (1927). Soil Sci. 24, 381-402. Starkey, R. L., Jones, G . E., and Frederick, L. R. (1956). 1. Gen. Microhid. 15, 329-334. Stauffer, V. R., and Thiel, G . A. (1949). Minn. Geol. Suru. Summ. Rept. N o . 3, 6 pp. Stokes, H. N. (1901). Bull. U . S. Geol. Surv. 186, 50 pp. Sugden, W. (1963). J. Inst. Petrol. 49, 65-69. Sullivan, J. D. (1930a). U . S . Bur. Mines, Tech. Paper 473. Sullivan, J. D. (1930b). U . S. BUT.Mines, Tech. Paper 487. Sullivan, 1. D. (1931). U . S. Bur. Mines, Tech. Paper 486. Sutton, J: A., and Corrick, J. D. (1961a). U . S. BUT. Mines, Inform. Circ. 8003.

Sutton, J. A., and Corrick, J. D. (1961b). U . S. Bur. Mines, Rept. 1nz;cst. 5839, 16 pp. Sutton, J. A., and Corrick, J. D. (1963). Mining Engr. 15, 37-40. Sutton, J. A,, and Corrick, J. D. (1964). U . S . Bur. Mines, Rept. Invest. 6423, in press. Tasch, P. (1960). Trans. Kunsas Acud. Sci. 63, 24-30. Taylor, J. H., and Whelm, P. F. ( 1943). Trans. Inst. Mining Met. 52, 36-96. Taylor, W. H. (1950). “Plants of Bikini and Other Northern Marshall Islands,” Univ. Mich. Press, Ann Arbor, Michigan. Temple, K. L., and Colmer, A. R. (1951). J. Bacteriol. 62, 605-611. Temple, K. L., and Delchamps, E. W. (1953). Appl. Microhiol. 1, 255-258. Temple, K. L., and Koehler, W. A. (1954). W e s t Va. Univ. Bull. Eng. Expt. Sta. Res. Bull. No. 25, 35 pp. Teodorovich, C. I. ( 1961). “Authigenic Minerals in Sedimentary Rocks” (transl. from Russian). Consultants Bureau, New York. Thiel, G. A. (1925). Econ. Geol. 20, 300-311.

FORMATION AND DEGRADATION OF MINERALS

205

Thimann, K. V. (1955). “The Life of Bacteria,” pp. 600-603. Macmillan, New York. Thode, H. C., Kleerekoper, H., and McElcheran, D. (1951). Research ( L o n d o n ) 4, 581-582. Thode, H. C . , Wanless, R. K., and Wallouch, R. (1954). Geochim. COSmochim. Acta 5, 286-298. Thode, H. C . , Dunford, H. B., and Shima, M. (1962). Econ. Geol. 57, 565-578. Thomas, J. W., Appleman, M. D., and Tucker, F. L. ( 1963). Bacteriol. Proc. p. 124. Tiller, A. K., and Booth, G . H. (1962). Trans. Faraday SOC. 58, 110-115. Timonin, M. I. (1950). Trans. 4th. Intem. Congr. Soil Sci., Amsterdam 3, 97-99. Trask, P. D. (1950). “Applied Sedimentation.” Wiley, New York. Tsubota, G. (1959). Soil Plant Food ( T o k y o ) 5, 10-15. Tucker, F. L., Walper, J. F., Appleman, M. D., and Donohue, J. (1962). I. BacterioE. 83, 1313-1314. Turner, A. W. (1949). Nature 164, 76-77. Turner, A. W. (1954). Australian J. Biol. Sci. 7, 452-478. Turner, A. W., and Legge, J. W. (1954). Australian I. Biol. Sci. 7 , 479-495. Unz, R. F., and Lundgren, D. C. (1961). Soil Sci. 92, 302-313. Van Hise, C . R., and Leith, C. K. (1911). U . S. Geol. Surv. Monograph 52, 641 pp. Van Niel, C. B. (1954). Ann. Rev. Microbial. 8, 105-132. Vavra, J. P., and Frederick, L. R. (1952). Soil Sci. Soc. A m . Proc. 16, 141-144. Vernon, L. P., Mangum, J. H., Beck, J. V., and Shafia, F. M. (1960). Arch. Biochem. Biophys. 88, 227-231. Vinogradov, A. P., and Boichenko, E. A. (1942). Compt. Rend. Acad. Sci. U . S. S. R. 37, 135-138. Vishniac, W., and Santer, M. (1957). Bacteriol. Rev. 21, 195-213. Vishniac, W., and Trudinger, P. A. (1962). Bacteriol. Rev. 26, 168-175. Volkova, 0. Yu., and Toshinskaya, A. D. (1961). Mikrohiologiya 30, 693698. Wainwright, T. (1961). Biochem. 1. 80, 27P-28P. Waksman, S. A. (1932). “Principles of Soil Microbiology.” Williams & Wilkins, Baltimore, Maryland. Webley, D. M., Henderson, M. E. K., and Taylor, I. F. (1963). J. Soil Sci. 14, 102-112. Weed, R. C. (1956). Mining Engr. 8, 721-723. Wilson, L. G., Asaki, T., and Bandurski, R. S. (1961). J. Biol. Chem. 236, 1822-1829. Winogradsky, S. (1888). Botan. Ztg. 45, 262-270. Winogradsky, S. (1922). Zentr. Bakteriol. Parasitenk. Aht. I I 57, 1-21. Wolfe, R. S. (1963). In “Proceedings of the 1963 Rudolfs Research Conference.” Wiley, New York (in press). Woolfolk, C. A. (1962). J. Bacteriol. 84, 659-668. Woolfolk, C. A., and Whiteley, H. R. (1962). 1. Bacteriol. 84, 647-658.

206

MELVIN P. SILVERMAN AND HENRY L. EHRLICH

Zalokar, M. (1953). Arch. Biochem. Biophys. 44, 330-337. Zappfe, C. ( 1931 ). Econ. Geol. 26, 799-832. Zarubina, Z. M., Lyalikova, N. N., and Shmuk, Ye. I. (1959). ZZU. Akad. Nauk S S S R , Otd. Tekhn. Nauk, Met. i. Toplivo No. 1, 117-119. Zavarzin, G . A. (1961). Mikrohiologiya 30, 393-395. Zavarzin, G. A. (1962). Mikrohiologiya 31, 586-588. Zimmerley, S . R., Wilson, D. G., and Prater, J. D. (1958). U. S. Patent No, 2,829,964. ZoBell, C. E. ( 1946). “Marine Microbiology.” Chronica Botanica, Waltham, Massachusetts.

Enzymes and Their Applications’ IRWIN W. SUER Depurtment of Biology, Massachusetts Institute of Technology, Cumbridge, Massachusetts 1. Introduction ........................................... 11. Molecular Structure of Enzymes .......................... 111. Enzyme Applications .................................... A. Industry .......................................... B. Medical Applications ................................ IV. Microbial Enzymes ..................................... V. Conclusion ............................................ References ............................................

1.

207 208 214 214 218 224 225 228

Introduction

A familiarity with the chemical reactions brought about by enzymes goes back into antiquity and antidates by thousands of years any knowledge that these biocatalysts were in fact proteins. The first application of enzymology might well have been in the fermentation of grape juice to wine brought about by the enzymes of fermentation contained within the wild yeast which contaminated the grapes. Since a living organism was required for fermentation, the production of alcohol was not clearly recognized as enzymic until Buechner separated zymase from yeast and demonstrated fermentation in vitro. The first application of an extracellular enzyme probably occurred accidentally as a result of carrying milk in flask made from a goat stomach tied off at both ends. The rennin produced by the gastric glands rapidly converted the milk to curds and whey. Microbial action further changed the curds to cheese, This enzymic coagulation of milk was further developed by the Greeks. In Homer’s “Illiad the clotting of milk in cheese making is effected by ficin by the simple expedient of stirring the milk with a branch from a fig tree (Gaughran, 1962). The application of enzymes to the manufacture of leather resulted from the very early observation that animal feces applied to 1 Presented at a symposium on “Applications of Fundamental Biology to the Needs of Man” held at Rutgers University, New Brunswick, New Jersey.

207

208

IRWIN W. SIZER

the flesh side of hide resulted in removal of noncollagenous material and the softening of the leather. Hundreds of years passed before the active ingredients in the excreta were identified as proteases, hence making it possible to substitute pancreatic or mold enzymes for the bating of hides (Gustavson, 1956). From these early beginnings in fermentation, cheese making, and the tanning of leather the application of enzymes has spread to the textile (desizing) and paper (starch modification) industries as well as to nutrition and foods and also to pharmaceuticals and medicine. It is fair to say that the applications of enzymes are growing at a very rapid pace, and it is estimated that by 1965 the market for enzymes sold in the United States will have reached the figure of 60 million dollars. This figure can be considered evidence that enzymes are being employed in many different ways for the welfare of mankind. On the other hand, this utilization of enzymes is not very impressive if one considers the fact that almost all chemical reactions which take place in living organisms are catalyzed by enzymes, and yet our knowledge of enzymology enables us to control only a very few of these. Similarly, a consideration of the several thousand different enzymes produced by animals, plants, and microorganisms shows that only a very small percentage of enzymes has been made available for application to industry, nutrition and foods, and the field of medicine. The failure in the past of applied enzymology to develop rapidly stems in part from a lack of understanding of the fundamental properties of enzymes, and from a tendency to use enzymes like chemical reagents without due regard for their unique properties as biochemical catalysts consisting of protein, which often has attached to it a coenzyme or trace metal. The relatively recent developments on the chemical structure of enzymes and their kinetics should make possible a more basic approach to enzyme systems and their applications ( Sizer, 1962).

II. Molecular Structure of Enzymes Although the size and general shape of enzyme molecules has been known for some time as a result of physicochemical studies, it is only recently that the detailed anatomy of any protein molecule has been revealed by high resolution X-ray crystallography. Myoglobin, in the laboratory of Kendrew et al. (1958), was the very

ENZYMES AND THEIR APPLICATIONS

209

first protein whose three-dimensional architecture was described in detail (Fig. 1). Hemoglobin appears as though it were made of four myoglobin molecules clumped together, and we can imagine that heme enzymes such as catalase, peroxidase, and the cytochromes would look quite similar, with heme groups oriented in a comparable fashion with reference to the polypeptide chain.

FIG. 1. The arrangement of the polypeptide chain of myoglobin as determined by means of X-ray crystaliography. The gray disk like structure is the heme prosthetic group of the molecule (Kendrew et al., 1958).

While physicists were preoccupied with the description of the polypeptide chain of proteins as an a-helix, chemists were busy with the determination of the specific sequence of the twenty different amino acids along this chain. Using the techniques developed especially by Sanger (see Ryle et al., 1955), the amino acid sequence of the first enzyme, ribonuclease, has been determined by Hirs et al. ( 1956) and by Anfinsen ( 1961) (Fig. 2 ) . Although the three-dimensional configuration has not yet been determined by crystallography, the specific coiling of the peptide chain of ribonuclease is partially fixed by the coupling of loops of the chain by disulfide bonds. If the physical structure is disrupted by severing these disulfide links the coiling becomes randomized, and a denatured, inactive enzyme is produced. Hence the gross architecture of an enzyme is of critical importance in determining its catalytic activity. Of equal importance is the amino acid sequence along the polypeptide chain. For example, Ingram (1957) in his studies of

210

IRWIN W. SIZER

sickle-cell hemoglobin has shown that the substitution of valine in place of glutamic acid in the peptide chain of normal hemoglobin constitutes a lethal change in a man who is homozygous for the sickling gene. Preliminary studies already indicate that a similar situation holds true for enzymes and that single groups in the

FIG.2. Amino acid sequence in bovine pancreatic ribonuclease (Anfinsen, 1961).

molecule might critically determine either the specificity or the activity of an enzyme (Coombs and Yoshimori, 1962). Chemical and enzymic attacks on various groupings in the enzyme molecule are yielding information concerning the role of various parts of the molecule with reference to binding of substrate to enzyme and the specificity and catalytic efficiency of the active site. In the case of ribonuclease, much of the N-terminal peptide can be removed with little effect, but, when all of the tail piece is split off, activity is destroyed. Surprisingly enough, activity is restored when the tail piece is reattached nonspecifically by ionic

ENZYMES AND THEIR APPLICATIONS

211

bonds rather than by a covalent peptide linkage (Anfinsen, 1961). The enzyme is far more sensitive to attach at the C-terminal end of the chain, and, while the successive removal of valine, serine, and arginine is innocuous, the removal of the tetrapeptide which includes aspartic acid destroys enzyme activity. Independent chemical evidence indicates the critical importance of the histidine which is one residue removed from this aspartic acid group. Chemical reagents specific for individual amino acids are needed in order to study the significance of each residue in determining the properties of individual enzymes. Our armamentarium is by no means empty, and inhibitors such as diisopropyl fluorophosphate (for serine residues ) and certain antimetabolites are rapidly contributing to our knowledge of functional groups of enzymes. Many enzymes, especially those of respiration and intermediary metabolism, contain a coenzyme or trace metal (Fe, Cu, Zn, Mo, Co, etc.) located at the active site. These non-amino acid prosthetic groups may be bound firmly or quite loosely to the protein apoenzyme (Fig. 3 ) ; hence some may be removed by simple dialysis,

Subst rote

+

Apornryma (protein)

Coenzyme or m e t a l

Product8

+ -

free Enzyme Enzymr-Subrtratr complex

FIG.3. Schematic diagram illustrating the pathway of many enzyme-catalyzed reactions. The step involving coenzyme or metal is not always required (Sizer, 1957).

while others can only be split off the enzyme by vigorous chemical treatment (Sizer, 1957). Coenzymes and trace metals are not only concerned with the formation of the enzyme-substrate complex, but also with the intimate mechanism of catalysis. For example,

212

IRWIN W. SIZER

vitamin Bo in the form of pyridoxal phosphate or pyridoxamine phosphate attached to transaminases is involved in the acceptance and donation of amino groups from amino acids to keto acids in transamination reactions (Fig. 4 ) . Similarly in metalloenzymes, the

10 0 c 0

El u)

n Q

0.5

350

400

450

Wavelength ( m p )

FIG.4. Spectra of the pyridoxal and pyridoxamine forms of the glutamicaspartic transaminase. The pyridoxal transaminase was made b y the addition of ketoglutarate (0.0005 M ) to the pyridoxamine form. For the p H 4.8 spectrum, 0.15 M acetate buffer was used, and for p H 8.5, 0.1 M pyrophosphate buffer was employed (Jenkins and Sizer, 1960).

zinc atom in carboxypeptidase, bound to the sulfhydryl group of cysteine and the amide group of the N-terminal asparagine, is directly involved in the enzymic splitting of peptide bonds (Coombs and Yoshimori, 1962). The metal incorporated into the active site is also concerned with enzyme specificity, since, when the zinc is replaced by cobalt or other metal, the relative specificity of carboxypeptidase toward peptide and ester linkages may be completely reversed. Major progress in the understanding of the mechanism of enzyme action has come from studies of the kinetics of enzyme systems. Studies of rate as a function of time, enzyme concentration, substrate concentration, pH, temperature, oxidation-reduction potential, and as affected by inhibitors have been especially fruitful in providing information on the way in which enzymes bring about catalysis ( Sizer, 1962). An understanding of the critical factors which control enzyme-catalyzed reactions has made possible the application of enzymes to industry, foods, agriculture, and medicine. Of special

ENZYMES AND THEIR APPLICATIONS

213

interest has been the use of inhibitors to elucidate the nature of the active site (Koshland, 1960) (Fig. 5 ) , as well as to control enzyme systems in their applications to human welfare. Competitive inhibitors such as the antivitamins and antimetabolites have assumed

FIG.5 . A schematic active site. The cross-hatched area indicates the bond to be broken by the enzyme. The black lines represent the polypeptide chain, while the R’s represent the amino acid residues protruding from the backbone (Koshland, 1960).

paramount importance in the prevention and cure of disease such as bacterial infection, cancer, and certain types of mental disease. Even more important is the hypothesis that new chemicals and pharmaceuticals fashioned to be specific for the active site of particular enzymes may become the “magic bullets” of the future (Wilson, 1955) (Fig. 6 ) in the field of chemotherapy. Anionic site

Esteratic site

t

t

CH3 FIG.6. Schematic presentation of interaction between the active groups of acetylcholinesterase and its substrate (Wilson, 1955).

214

IRWIN W. SIZER

111.

Enzyme Applications

With few exceptions (see Section 111, A, 6 on oxidases), enzymes which have found industrial applications are hydrolytic. This is primarily because they are much simpler to deal with and historically have been easier to prepare. Now that excellent and inexpensive methods are available and basic properties of most enzymes are understood, one is justied in forecasting that uses will be found in the near future for respiratory, biosynthetic, and metabolic enzymes. Although many industrial applications of nonhydrolytic enzymes will probably be found, it is likely that their major role will be in the service of medicine. A. INDUSTRY 1. Proteases

Doubtless the first industrial application of enzymes was in the manufacture of cheese in which the initial step is the coagulation of milk by rennin followed by a separation of the curds and whey. Waugh (1958) (Fig. 7 ) has recently shown that a stable micelle

FIG. 7. A scheme for the a,-c-casein complex. The a,-caseins to K-casein ratio here is 4. If the ratio is greater than 4, excess a,-caseins precipitate, leaving a stable micelle suspension; micelle size decreases with decrease in the ratio (Waugh, 1958).

of milk is made of four a,,-caseins plus one r-casein stabilized by calcium and phosphate ions. In a secondary reaction, p-casein combines with this complex. When the proteolytic enzyme rennin is added to milk the rennin first hydrolyzes K-casein, thereby disrupting the colloidal system and causing the formation of a clot. The

ENZYMES AND THEIR APPLICATIONS

215

clotting of milk by rennin is involved in the making of junket desserts as well as in the production of cheese. The tanning of leather also dates from antiquity, for it was common practice to clean hides free of flesh and noncollagenous material by smearing them with dung. I t was a long time before the active ingredients were identified as proteolytic enzymes from the pancreas. For economic reasons, microbial proteases are used today for the bating of hides instead of a pancreatic powder. Enzymic treatment of hides is an indispensable step in the preparation of pliable and attractive leather of fine grain (Gustavson, 1956). The application of proteases to the food industry is rapidly becoming one of their major uses. Pure proteins are quite tasteless while some of the amino acids and certain peptides produced by protein hydrolysis are highly flavorful. Hence, treatment with proteases of many foods rich in proteins may be expected to increase their flavor. Even more important is the softening, tenderizing, increased digestibility ( and higher nutritional value), increased solubility, and shortened cooking time which result from partial digestion of many foods with proteolytic enzymes. For economic reasons, plant proteases, especially papain, ficin, and bromelin, have been used the most. Now that Bacillus subtilis and Aspergillus enzymes have been cleared for food use, microbial enzymes should become competitive with those from plants. The largest food use of enzymes is for the prevention of the cloudiness in beer which develops when beer is chilled. This “chillproofing” involves the digestion of the small amount of beer proteins by the addition of a protease. Some 4 million dollars worth of enzymes are used for this purpose each year. The most rapidly growing food application is the use of proteases (especially papain) as a meat tenderizer. A tough steak treated by spreading a papain powder over it hour before cooking becomes “tenderized and more succulent as a result of this treatment. For this application it is an advantage to use an enzyme with a high inactivation temperature so that proteolysis occurs during the early stages of cooking. Because all housewives are not skilled in enzyme kinetics, this meat tenderization technique in the home has met with variable success. Much more exciting is the possibility of tenderizing meat “on the hoof.” In this procedure papain is injected intravenously into steers 8 minutes before slaughter. Since the enzyme is distributed to every capillary of the

216

IRWIN

W.

SIZER

carcass, conditions are ideal for enzymic tenderization of steaks and roasts. From a theoretical viewpoint this is an ideal way to tenderize meat, and it is gratifying that this procedure has met with such dramatic initial success. A relatively new application of proteases is to the treatment of stock feeds. For example, enzymic attack on barley can improve its nutritional value in poultry feed. Similarly the enzymic digestion of whole fish powder can increase the nutritional value of animal feeds. An alternative is to feed the enzyme to the stock along with the untreated fodder. To successfully apply this technique, one must employ an enzyme which hydrolyzes the feed before it itself is destroyed in the digestive tract of the livestock. An example of this procedure is in hog raising where young shoats can be weaned at a much earlier age if they are fed pepsin. Still relatively new is the application of proteases to the baking industry by the hydrolysis of wheat gluten by proteases leading to greatly improved dough. Similarly, in the field of “quick-cook cereals a preliminary treatment of the cereal with a protease may shorten the cooking period and render the cereal more digestible by infants. 2. Carbohydrate-Splitting Enzymes One of the largest uses of enzymes is in the desizing of cloth by the hydrolysis of starch by amylases from Aspergillus. In this way starch and dextrins can be readily removed from cloth. Modified starches can also be prepared by treatment with amylase, and many of these find application in the paper industry. More extensive amylase or amyloglucosidase action converts the tasteless corn starch to sweet glucose syrups, which are widely used in candy manufacture. Candy containing sucrose can be made even sweeter by its hydrolysis by invertase to glucose and fructose. Fruit juices, wines, and vinegar often are cloudy and viscous due to the dispersion of pectin in the suspension. After hydrolysis of the pectin by pectinase the fruit extract becomes very fluid, crystal clear, and amenable to fiItration, should this be necessary. 3. Lipases

In theory the hydrolysis of fats and phospholipids by lipases could improve or modify their flavor and consistency. Only preliminary work has been done in this field, however.

ENZYMES AND THEIR APPLICATIONS

217

4. Nuclemes Nucleic acids are fibrous molecules of molecular weight in the millions. These tasteless molecules can be readily converted by ribonuclease or desoxyribonuclease to highly flavorful nucleotides. These in turn, by enzyme action, could be hydrolyzed to pentose phosphates, nucleosides, purines, pyrimidines, and inorganic phosphate. Many of these products of hydrolysis are flavorful compounds, but few seem to be used in foods as yet. 5. Other Hydrolytic Enzymes

Many enzymes have potential application to the food industry, especially those which might destroy toxic or undesirable components. For example, penicillinase might be added to milk from penicillin-treated cows so that bacteria employed in cheese making would not be destroyed by the penicillin. Similarly, an enzyme which metabolized cholesterol or converted a saturated fat to an unsaturated one would easily earn a place for itself in this age of worry about cardiovascular problems. Destruction of antigens in foods by enzymes would be a great boon to people suffering from certain kinds of allergy. Examples of this type of potential application of enzymes exist in large number. 6. Oxidases

Although the industrial applications of oxidative enzymes is at present quite limited, their use should increase greatly now that their purification and general properties have become so well understood. The most promising of this group is glucose oxidase produced from mold mycelium. This enzyme utilizes atmospheric oxygen for the oxidation of glucose to gluconic acid and hydrogen peroxide. Most glucose oxidase preparations also contain catalase, which acts upon this peroxide. In the dehydration of eggs the offcolor and unpleasant flavor developed is due to the "browning reaction," the reaction of glucose with egg albumin at elevated temperatures. This combination can be prevented by removal of all the glucose by the addition of the glucose oxidase system. Other applications in which the removal of glucose from foods is desirable are under consideration. Since this enzyme utilizes oxygen even at extremely low pressure, it can also be employed to create an essentially anaerobic environment. Because of its ability to scavenge

218

IRWIN W. SIZER

oxygen the glucose oxidase system (with added glucose when necessary) can prove useful in many systems in which foods develop off-flavor and color due to oxidation. As examples of such applications its use in whole milk solids, ice cream mixes, soft drinks containing fruit juices, and even chewing gum may be mentioned. Unstable pharmaceuticals such as vitamins and hormones can also be protected by this enzyme. A somewhat unexpected application of glucose oxidase is in the diagnosis of diabetes by analysis for glucose in the urine. A pH indicator shows how much gluconic acid is produced and makes possible a "do-it-yourself" kit for diabetes diagnosis available in rest rooms throughout the nation. Catalase may find an important application in the sterilization or pasteurization of food (e.g., milk) by hydrogen peroxide. After sterilization is complete, residual peroxide is readily removed by the addition of catalase. Although little is known about the way in which flavor can be protected or actually improved by the action of oxidases on foods, this subject is being investigated in several laboratories with encouraging results. B. MEDICALAPPLICATIONS One of the most exciting and significant applications of enzymes is to the field of medicine and pharmaceuticals. Already pharmaceuticals account for half the total enzyme industry and this application has high growth potential in the future. 1. Digestive Aids

Perhaps the oldest medical application of enzymes is their use as digestive aids. Since most enzymes are destroyed in the stomach it is not surprising that historically pepsin has been used more than any other enzyme for the hydrolysis of proteins in the digestive system. By the use of enteric coatings, however, it is possible to protect enzymes from the action of gastric juice so that they can catalyze the digestion of food after reaching the intestine. Crude extracts of the pancreas are especially valuable aids to digestion since they contain a galaxy of enzymes which digest proteins, fats, carbohydrates, and nucleic acids. By utilizing bacterial sources it is possible to prepare a combination of protease, carbohydrase, and lipase which is active at the pH of the stomach instead of the intestine. Recently, plant enzymes have also been introduced into this field and preparations of papain, bromelin, and ficin are available.

ENZYMES AND THEIR APPLICATIONS

219

The latter has the advantage that it can function as an effective vermifuge against roundworm infection in the intestine (Dixon and Webb, 1958).

2. Blood Coagluution For many years, thrombin (and now autoprothrombin C ) has been available for checking bleeding in a variety of sanguinary situations. Even more important, however, is the opposite problem of the dissolution of clots which have spontaneously formed in the blood system. To deal with this situation the pharmaceutical industry has made available, for intravenous injection, profibrinolysin prepared from human plasma. This enzyme is activated to fibrinolysin by streptokinase, a protease from streptococcus. It is already apparent that fibrinolysin can be useful in the dissolving of blood clots in many medical problems of the cardiovascular system ( Sherry and Alkjaersig, 1957). 3. Debridement of Burns and Ulcers

A severe second or third degree burn becomes covered with a tough, leathery eschar which remains for months. It must be removed surgically before skin grafting can be accomplished. Since surgical removal is traumatic, many clinical investigators have turned to proteases for loosening or even dissolution of the eschar. Similarly, many ulcers are coated with pus or necrotic and dead tissue which must be removed before healing can take place. Many different proteases have been tried on burns and ulcers with variable success. Among these are pepsin, trypsin, chymotrypsin, bacterial protease, collagenase, streptokinase, bromelin, papain, penguinain, fibrinolysin, and ficin. Although none of these is ideal, it seems likely that some will find a place in the armamentarium available to the clinician in the treatment of certain types of burns and ulcers (Spier et al., 1956) (Fig. 8 ) . 4. Anti-Inftammatory Agents

Inflammation in various parts of the body can often be relieved by anti-inflammatory agents which are usually proteolytic enzymes. Inflammation may be caused by infection, accumulation of toxins, edema, injuries and bruises, and many other causes. It was observed some time ago that the intravenous, intramuscular, or intraperitoneal injection of certain nontoxic proteases could often alleviate certain

220

IRWIN W. SIZER

FIG. 8. An ulcer of the abdominal wall treated by enzymic debridement (Spier et al., 1956). ( a ) Onset of treatment. ( b ) Twenty days after onset.

ENZYMES AND THEIR APPLICATIONS

221

types of inflammation. More recently, it has been found that some enzymes are eqiially effective if dissolved in the mouth or swallowed. At the same time, certain undesirable side effects associated with injection of enzyme could be avoided. Anti-inflammatory enzymes now available include preparations of papain, bromelin, bacterial proteases, trypsin, and chymotrypsin. Rather surprisingly, a crude microbial a-amylase has also been recommended for inclusion in this group. The actual mechanism by which antiinflammatory enzymes produce their action is generally not known. Among other effects it is claimed that (1) they may reduce a hematoina and thereby decrease inflammation and edema, ( 2 ) they may liquify mucopolysaccharides and nucleic acids, thus relieving respiratory disorders and areas of infection, ( 3 ) in the case of wound areas encased in fibrin walls, the fibrin may be hydrolyzed and the injured tissues may become accessible to perfusion by blood or other fluids, (4)by attack on bacterial or viral membranes these microorganisms may become susceptible to antibiotics. Similarly, by increased tissue permeability produced by enzymes (e.g., hyaluronidase ) certain drugs may be rendered more effective. Despite our lack of understanding of the mechanism of action of antiinflammatory enzymes, the pragmatic approach seems to have demonstrated their usefulness in a number of medical situations (Anonymous, 1962) (Fig. 9 ) .

FIG.9. ( a ) Administration of a protease tablet for the relief of black eye. ( b ) In 3 days the eye shows marked improvement. Without the use of enzyme this would normally have taken 2 weeks or more.

222

IRWIN W. SIZER

5. Prostheses Enzymically cleaned and purified tissue products are beginning to find medical uses. Bone and cartilage treated this way are being investigated by several clinical workers. Animal tissues and their components cannot in general be used in humans because of foreign body reaction. Since collagen, of all proteins, is almost completely free of such allergic properties, considerable success has been achieved in medical applications of purified beef tendon collagen freed of other proteins by treatment with ficin or other protease. Collagen sponges and membranes have been used to prevent adhesions, to support tissues, and in other ways as an adjunct to surgery. Of special significance is the use of tanned beef arteries freed of noncollagenous proteins by ficin treatment ( Bothwell and Simmons, 1962) (Fig. 10) as heterographs for the replacement of arteries in dogs and humans, These transplanted collagenous arteries are completely functional for years and gradually are invaded by host connective tissue over a period of months. Initial results of surgeons indicate that these enzyme-treated heterographs may be quite useful for the repair of certain blood vessels. 6. Diagnostics

Assay of enzymes in blood or tissues is becoming a very useful tool in the diagnosis of disease, the efficacy of medication, and the course of recovery. It appears that the pattern of a considerable number of enzymes may be modified in a diseased organ, and this change in pattern will usually be reflected in a change in the enzyme composition of the blood. For those enzymes which exist in several forms (isoenzymes) in different tissues not only the total amount but also the relative concentrations of the individual isoenzymes may change in a diseased organ or in the blood. As examples of enzymes which are useful in clinical diagnosis one might mention lactic dehydrogenase and glutamic-aspartic transaminase for myocardial infarction, alkaline phosphatase for jaundice, ceruloplasmin for Wilson’s disease, alcohol dehydrogenase for cirrhosis of the liver, a-amylase and lipase for malfunction of the pancreas, leucine aminopeptidase for liver function, isocitric dehydrogenase for brain disease, etc. Although this field is new, enzyme assay as a diagnostic aid is rapidly becoming an important tool for the clinician ( Ruyssen, 1961 ) . The identification and quantitative measurement of many bio-

ENZYMES AND THEIR APPLICATIONS

223

FIG. 10. ( a ) Bovine carotid after enzyme treatment and tanning. ( b ) Heterograph temporarily implanted in a patient ( Bothwell and Simmons, 1962).

224

IRWIN W. SIZER

chemicals is best done using highly specific enzymes in the assay system. In clinical chemistry these tests are routinely applied to blood and urine. Two great advantages in the use of enzymes for assay of organic chemicals are: ( 1 ) mild conditions of pH and temperature which can be used and ( 2 ) the high substrate specificity of most enzymes. Typical of such assay is the determination of glucose in diabetic urine using glucose oxidase referred to previously. Perhaps the oldest application of an enzyme to medical assay is the use of urease in the measurement of urea in blood and urine. Since most organic compounds in the body can be attacked by enzymes, the potential use of enzymes in the assay of such compounds is very high.

IV. Microbial Enzymes The increased use of enzymes for a wide variety of applications makes the economic production of enzymes of standard quality and purity a matter of great concern. Properties of individual enzymes may vary somewhat depending on the species of origin but there are no characteristic differences which can be related to their derivation from animals, higher plants, or microorganisms. Hence, economics may dictate the source of origin. An excellent example of this is seen in the bating of hides by proteolytic enzymes. For years crude trypsin (desiccated, powdered pancreas) was the enzyme of choice until competition for pancreas by manufacturers of insulin made the cost of trypsin prohibitive. In a relatively short time fungal enzymes were produced by fermentation which proved highly satisfactory to the leather industry. As animal products become relatively more costly, there will be a continual attempt to replace animal enzymes with comparable ones from other sources. While certain plant enzymes (e.g., papain) will continue to have important applications, the replacement of animal enzymes will be primarily by microbial enzymes. With the replacement of flasks and trays by large fermentation vessels or continuous flow techniques, it has been possible to grow bacteria and fungi in large quantities under relatively economic conditions. Much of the knowledge learned from the microbial production of antibiotics is directly applicable to the production of enzymes. Careful control of such factors as temperature, pH, salt concentration, nutrients, vitamins, aeration, and time of harvest has resulted in greatly

ENZYMES AND THEIR APPLICATIONS

225

increased yields. Search for strains of microorganisms or isolation of mutants after irradiation has led to the establishment of special cultures which produce many times more enzyme than the wildtype organism. Basic studies on inducible enzymes have made possible the increased yield of certain enzymes by adding to the fermentation the substrate or substrate analog which acts as an inducer. In a similar way our understanding of repression of microbial enzyme production caused by the end product liberated by that enzyme is making possible the increased yield of the particular enzyme by keeping to a minimum the concentration of that particular end product in the fermentation. In the case of alkaline phosphatase production by Escherichia coli, the inorganic phosphate liberated by phosphatase produced “feedback inhibition” of the synthesis of this enzyme. Reduction of inorganic phosphate in growth medium is only partly successful in increasing yield since the organism requires phosphate for growth. This dilemma was finally solved by the discovery of a mutant which is not susceptible to phosphate repression of phosphatase synthesis. This mutant is now used to commercially produce high yields of phosphatase of high purity. It is safe to predict that knowledge of fermentation gained from production of antibiotics plus recent understanding of microbial genetics and protein biosynthesis will make microorganisms the major source of enzymes. Now that a protein-synthesizing system of bacterial ribosomes has been separated from cells and shown to function in oitro, it may be possible to use this system for “cell-free’’ synthesis of enzymes. In theory this system supplied with twenty amino acids plus adenosine triphosphate ( ATP ) could synthesize an enzyme for which the corresponding messenger ribonucleic acid (RNA) was added to furnish the proper “blueprint.” It may be some time, however, before an enzyme such as human pepsin is produced commercially from a cell-free ribosomal system from E. coli even though this is theoretically possible.

V. Conclusion The recent growth in the industrial and medical applications of enzymes has resulted from greatly improved methods for their production, especially from plant and microbial sources. In no small degree it has also evolved as a result of our deeper understanding

226

IRWIN W. SIZER

of the chemical and physical properties of enzymes and of the kinetics of the total enzyme-substrate system, with special reference to the role of activators and inhibitors. This fundamental knowledge of enzyme systems will inevitably lead to major growth in the industrial and medical applications of enzymes in the near future.

REFERENCES Anfinsen, C. B. ( 1961). “The Molecular Basis of Evolution.” Wiley, New York. Anonymous. ( 1962). Editorial. Chem. Week pp. 49-50. Bothwell, J. W., and Simmons, G. W. (1962). Federation Proc. 21, 169a. Coombs, T. L., and Yoshimori, 0. (1962). Federation Proc. 21, 234f. Dixon, M., and Webb, E. C. (1958). “Enzymes.” Academic Press, New York. Gaughran, E. R. L. (1962). Preparation, characteristics and uses of ficin. Unpublished. Gustavson, K. H. ( 1956). “The Chemistry of Tanning Processes.” Academic Press, New York. Hirs, C. H. W., Stein, W. H., and Moore, S. (1956). J. Biol. Chem. 221, 151-169. Ingram, V. M. (1957). Nature 180, 326-328. Jenkins, W. T., and Sizer, I. W. (1960). J . Biol. Chem. 235, 620-624. Kendrew, J. C., Bodo, G., Dintzis, H. M., Parrish, R. G., Wyckoff, H., and Phillips, D. C. (1958). Nature 181, 662-668. Koshland, D. E., Jr. (1960). Aduan. Enzymol. 22, 119-125. Ruyssen, R. (1961). Proc. 2nd Intern. Symp. on Enzymes Clin. Chem. Ghent, 1961. I. Pure and Appl. Chem. 3, 383-384. Ryle, A. P., Sanger, F., Smith, L. F., and Kitai, R. (1955). Biochem. J. 60, 541-556. Sherry, S., and Alkjaersig, N. (1957). Ann. N . Y. Acad. Sci. 68, 52-66. Sizer, I. W. (1957). Science 125, 54-59. Sizer, I. W. (1962). Proc. Symp. in Appl. Math. 14, 189-203. Spier, I. R., Rees, T., and Cliffton, E. E. (1956). Am. J , Surg. 92, 496-506. Waugh, D. F. (1958). Discussions Farudny Soc. 25, 186-192. Wilson, I. B. (1955). Discussions Faraday Soc. 20, 119-125.

A Discussion of the Training of Applied Microbiologists B. W. KOFTAND W. W. UMBREIT Depurtment of Bacteriology, Nelson Biological Laboratories, Rutgers University, New Brunswick, New Jersey I. 11. 111. IV. V.

Introduction ........................................... Undergraduate Training ................................. Bachelor Degree with Orientation toward Graduate School . . . . Graduate School Training ................................ Other Prablems ........................................

227 231 235 235 238

I. Introduction We wish to discuss a problem of concern to applied microbiologists which is not strictly scientific in nature, but which, if properly understood, could constitute a real advance in applied microbiology. This problem is the training required for a modem, successful, applied microbiologist. Most of the essays written for Advances are written by professional applied microbiologists. There has been no real discussion of the training and abilities necessary for success in this field. We therefore take an editorial prerogative and have prepared a discussion of this subject since we are engaged in training such individuals, In the course of our experience in this area, we have developed certain opinions which we would like to point out; we will elaborate on some of the more important ones. While in one sense Advances is not the place for such discussion, in another sense it is, if for no other reasons than that scientific journals are not willing to provide space for such discussion and that trade media are often too narrow and too superficial for adequate discussion. The eighteenth century essay is still a suitable medium for conveying information, but its present function is the exposition of ideas without the somewhat rigid restrictions of format presumably necessary in contemporary journals. With this explanation we shall return to the subject. We have discussed the matter of the proper training of applied microbiologists with quite a few people, and we are of the opinion that it will be exceedingly difficult to obtain general agreement on 227

288

B. W. KOFT AND W. W. UMBREIT

the subject. It is therefore necessary to set up certain guidelines so that one can stay within the area of pertinent discussion. The first of these guidelines is that we are talking about the training of the average applied microbiologist, not the genius or the man of oustanding ability. It seems reasonable that the kind of training necessary should be directed more to the average person than to the genius. For the genius, nothing is difficult; the trick is to be able to do things with a minimum of natural talent. It seems to us, also, that it is simply not likely that applied microbiology can attract the genius and that we shall therefore have to do with men of adequate but not unusual ability. There seems to be several reasons for this. First, there are excellent opportunities for outstanding men which provide freedom to do research on any subject desired, in stimulating environments, at excellent salaries, with opportunities to travel and enjoy some of the amenities of life. So broad are these opportunities and so uncertain our judgments that we often see men of less than first rank successfully managing to stay for a time, a t least, within this charmed circle. For the average applied microbiology laboratory to compete with this situation is not very likely. Fortunately, the vast majority of men are capable of a great deal of achievement, and it is with them that one must, in fact, work. Next, there is the problem of the great variety of laboratories in applied microbiology which have different requirements and different demands. Each laboratory wishes to find a man trained to fill its special needs and, of course, full of enthusiasm to undertake the routine work which the present personnel is tired of doing. Yet he must be preceptive enough to solve the problems that the laboratory has been struggling with for a long period of time. While we have perhaps exaggerated the situation, this is frequently what appears to happen. The concern with the present problems shows up in those few surveys made to determine what the employer feels the training of applied microbiologists should be. I t appears that he wants them trained either specifically in a technique used in the laboratory in question or so broadly as to be meaningless. We have had men tell us that the most important training we could give a B.A. or B.S. microbiologist is to teach him how to use the library. We have had others who insist that every B.A. or B.S. should have physical chemistry, statistics, biochemistry, and/or immunology, when, in fact, the man is to

DISCUSSION: TRAINING APPLIED MICROBIOLOGISTS

229

inoculate tubes of lactose broth for surveys of water contamination or determine the rates of decay of inadequately treated strawberries. There is, then, in our estimation a great deal of “off the CUP opinion about the training required which, if one were to follow, would lead to a complete morass. We have, thus far, been somewhat critical of the applied laboratories by choosing some not too extreme examples of inadequate approach. But the colleges and universities do not present a very much happier picture. Often the microbiology courses are oriented so far away from the applied fields that not only are men inadequately trained, they are actually discouraged from contemplating a career in the applied areas. Sometimes the applied areas are treated in so antiquated a manner as to be useless. This is not always the fault of the universities; sometimes the necessary information, equipment, or know-how is not freely available. There are some other difficulties. If one examines the matter closely, it is surprising to find that much of what is done in a teaching laboratory depends not as much as it should on the ability of the teacher or his knowledge, but on the physical and environmental conditions in the institution-on schedules, on media cooks, on whether or not there is water piped to the desks, on how many classes must share a laboratory, etc. Similarly, what is done in the college research laboratory is dependent on the space, time, and the choice of facilities available. This curious limitation on what one does in fact, in contrast to what one could or should do, may not be as important in the future, but it is actually a limiting parameter at present. “But we have always done it this way’’ is as hard, if not harder, to overcome in the college laboratory as in the applied laboratory. Persons who have been away from the undergraduate or graduate programs of the modern university for some time tend to think in terms of their own experiences and neglect two factors. One is that the pace has stepped up a bit; there is more to learn now than there was a decade ago. The other is that they have forgotten how little they knew when they started. Once one has learned something, before too long he makes the assumption that he has “always” known this and soon thereafter that “everybody” knows it. But any training program must start with the knowledge possessed by its trainees, and it is sometimes impossible to teach molecular biology to nonreaders. As such, the university can be expected to provide

230

B. W. KOFT AND W. W. UMBREIT

men trained to a given level but the extra information, which comes from further experience and which, in fact, is probably acquired only in this fashion, it cannot provide. There is, we think, a good deal of justifiable discontent on both sides of the picture, and much of the disagreement on means comes about because the picture we possess of the “ e n d is unrealistic. Perhaps we can reach a better understanding, which is, in fact, the purpose of this essay, if we recognize two things about any training program. First, no institution can really train a person for a specific position or organizational niche. This can be approached very closely in preparing new programs “in shop” as it were and can be specifically done in technical institutes devoted to a particular specific program. This kind of “technician” training is not what we are talking about. The vast majority of applied microbiologists are not educated in this fashion. We expect that, without doubt, there will be an enormous expansion in the need for such specific training in the future as our operations become more complex, more interdependent, and more technical. But the majority of applied microbiologists at all levels are not trained as technicians; they obtain their education at colleges and universities. Here a different system prevails. A man normally enters such an institution without a distinct and definite idea as to precisely what he wishes to be. Even if he does, he is likely to change his mind at least once before he is finished. Even if he has very firm opinions as to what he wishes to be, circumstance may dictate otherwise. In short, one can never tell what a man will end up doing or what contribution he can make. It is therefore necessary to provide him with enough knowledge to permit a variety of alternatives. It is, we assume, generally agreed that a college-trained man should have a broad general basis on which to develop. But people disagree on what constitutes a broad general basis. Is it as broad as “the ability to use the library” or as narrow as “another course in statistics”? We shall present our ideas later. But one certainly must be aware that the young man who now wishes to be a biochemical physiologist may in 10 years become a taxonomist and make his best contribution in developing a diagnostic procedure for ringworm, all of which he has not dreamed of as yet. Any program for his education must permit him to move and work in fields for which he is not specifically trained. Most of us are working or have worked in areas for which we had no specific training at the student level.

DISCUSSION : TRAINING APPLIED MICROBIOLOGISTS

231

Second, any educational program faces a very fundamental difficulty. It is educating for the future with the methods and concepts of the present or sometimes of the antiquated past. How do you provide for the yet unknown developments when all you know about them is that they will occur? The young people in our colleges now have different methodology, concepts, and tools (and we might add much better) than we had when we were at their stage of learning. But what they shall actually use in the future will still be more markedly advanced. How does one manage? We think our present famili’arity with various types of chromatography, for example, which was hardly heard of two decades ago, comes about because we “grew up” with its development. In the same fashion the present generation will grow up with (and in fact may discover and develop) the new approaches and concepts which will constitute their world. What is needed then is a training program which permits such “growing up”; the problem is “how do you do it?” We feel that there are several levels of accomplishment which bear some discussion, since the problems of each are somewhat different. We shall therefore divide the problems into three large areas: that of undergraduate education where the B.A. or the B.S. is thought to be the terminal degree; that of undergraduate training as preparatory for graduate school; and finally, the graduate school training per se. We have omitted any consideration of postdoctoral work, but devote some comment to continued training.

It. Undergraduate Training In modern applied microbiology a man with training to the B.A. or B.S. level may occupy either of two positions. In larger organizations he is a technician doing laboratory work under the direction of someone with more experience (or more status), but in a smaller organization he may be “the microbiologist” with an enormous range of responsibility. As we have said, we regard it as unlikely that he will be the smartest man in the class. If he were, there would be plenty of opportunity to go on to graduate school; sheer poverty is no longer a barrier to the man with outstanding ability and intelligence. We recognize that there may be some debate on this proposition but we feel it valid as a general rule. As such, then, the man involved has not been outstandingly successful in his college work, nor has he attracted the professors’ attention because of any other outstanding characteristic. As a young man, it is quite

232

B. W. KOFT AND W. W. UMBREIT

all right for him to be employed as a laboratory worker, but in the long run it is a blind alley, and he should at this point begin preparations to get out of it. There are two routes out. First, he may obtain more technical training, sometimes at technical institutes, but more normally by returning full-time or part-time to graduate schools, Here there are some difficulties. Graduate schools are designed for a different kind of person, and are scheduled to accommodate students and professors engaged in full-time graduate work. They are devoted to research work generally of a fundamental nature and offer no lures of comfort, security, or well-being. Rather, they offer challenge and harsh competition, and support of students in them is not based on need but on excellence. Further, graduate schools are highly selective, and the chances are not always good that a man with anything but an outstanding record will be able to enter or that once in he will be able to compete. The second route out is to enter into areas where experience and ability to deal with people becomes gradually more important than technical know-how. These areas are administration, production, sales, etc., and in these areas further technical training, aside from that provided by the institution involved and at the bench, is usually not a necessary prerequisite. Assuming then that we have a young man of the characteristics described above and to whom the bachelor’s degree is the terminal point in his education, what should he have as his undergraduate program if he is to be successful in the future as an applied microbiologist? What follows is, of course, our own opinion, not necessarily our own program. First, it should be pointed out that as a freshman in college such a young man does not have an exactly free choice of what he should take. It is one of the theories of our educational system that all students should have a core of similar studies; hence, there are certain “general education” requirements usually applicable to essentially all students. These are the following: English composition Foreign language Humanities Social studies

1 2 2 2

year years years years

A student normally takes five courses a year and, thus in the 4 years has twenty 1-year course equivalents of which seven are used

DISCUSSION: TRAINING APPLIED MICROBIOLOGISTS

233

in the above requirements. For training in microbiology the following are regarded as universally required: General chemistry Biology College math Physics Organic chemistry

1 year 1 year 1 year 1 year 1 year

using another five 1-year course equivalents. Therefore, there are available eight 1-year course units. For the person to whom the bachelor’s degree is to be the final one, we regard the following as a suitable program, assuming that some minor variations may be inserted depending on individual interest: % year % year % year % year % year This leaves five %-year course units available. For strictly microbiological training, we feel that the following is reasonable: Analytical chemistry General physiology Genetics Mycology Parasitology

General microbiology Microbiology related to disease “Applied microbiology,” which comprises a consolidation of food, dairy, water, sanitary, and soil bacteriology, rather than separate courses in each Introductory microbial genetics and/or Tissue culture and introductory virology

% year % year

% year % year

% year

The remainder, the three %-year course units still uncommitted, could suitably be spent by a young man, whose bachelor’s degree is to be the terminal point in his technical education, in acquiring some elementary knowledge of business administration, basic engineering, economics, and statistics. But under normal circumstances he will not do this, In part, this is due to his own inclination; he rather likes microbiology and he wants to take more of it, although he has little idea that he will become an applied microbiologist; in fact, he has no real notion of what he would be faced with should he become one. Further, he has no way of knowing what he should take nor is he inclined to listen to any advice on the matter, assuming he was given any in the first place. The usual

234

B. W. KOFT AND W. W. UMBREIT

advice he will get is to “take more fundamentals,” which, depending on whom the advice comes from, may range from courses in physical chemistry and biochemistry to research problems. This advice normally comes from people concerned with graduate work. We feel, and perhaps we differ from most of our colleagues, that a great deal of what is taught in modern general chemistry today is what used to be taught in physical chemistry, and that the latter is now so far removed from microbiological application that it is not especially useful. Biochemistry is so much a part of microbiological or other biological physiology (and even modern courses in cytology have a great deal of biochemistry) that the student has acquired a great deal of this information without taking a course in biochemistry. We are not at all enthusiastic about a research problem course. Such undergraduate research courses have their place, but they are most inefficient as a teaching device, and they are not applicable to the situation facing the student when he graduates . Another reason for a man not normally obtaining what he should is that he does not really know what area of endeavor he wishes to enter until he reaches, and sometimes after, his junior year. In most institutions there is no major available in applied microbiology, although in some cases its equivalent is hidden in terms of a specified applied area, such as “food science” or “dairy science,” which by its very name indicates a restriction in the program in microbiology as such. Most students must choose a “major” at the start of their junior year, and “applied microbiology” is not among the choices available. Before his choice of a major field he explores various areas of knowledge and tries to determine what he excels in. It is rare indeed to find a man starting college committed to applied microbiology, or even one who has seroiusly heard of it, and there will therefore be no direct line to a single objective in his program, but a certain amount of wandering. This is as it should be, because applied microbiology needs men of demonstrated talent and flair for this field and not those trained in a narrow specialty. Persons should be attracted to the field of applied microbiology because it fits with their interests and talents rather than their having chosen this kind of work purely by chance or in ignorance of its nature and opportunities. Incidently, the program we have outlined will provide the necessary information and courses to

DISCUSSION : TRAINING APPLIED MICROBIOLOGISTS

235

qualify as a “Registered Microbiologist” in the National Registry of Microbiology.

111. Bachelor Degree with Orientation toward Graduate School

For the young man going on to graduate school, we believe the program outlined above should be modified in only three particulars. First, in addition to math through analytical geometry, he should have a year of calculus. Second, in his senior year he should be given a course in research problems. The latter, while an inefficient teaching device, gives him something of the attitude of the graduate school he will soon enter and a chance to see whether or not he is temperamentally suited to microbiological research. Our experience is that while we can make a fairly good guess, we are sometimes surprised at the lack of research ability in people doing well in course work and the occasional presence of rare talent in those doing only a little better than average work otherwise. Third, we feel that training at the bachelor’s level should be broad and not too concentrated in microbiology as such. However, while the student always wishes to take more and more microbiology, we feel that he should be limited to no more than three semesters of formal course work in microbiology as an undergraduate, so that more of the information will come at the graduate level where it is possible to penetrate the subject to greater depths.

IV. Graduate School Training There are many variations in the progression of events leading

up to completion of a doctoral degree, but one may define three main program categories. An integrated doctoral program describes the situation in which a master’s degree is awarded to the doctoral candidate as a definite step in an uninterrupted drive for the top degree. In the direct doctoral program the master’s degree is either completely ignored or awarded as a consolation prize to those deemed not qualified for the higher degree. An interrupted program describes the situation in which an individual either moves from one school to another after the M.S. degree or has left school for 3 or more years after his M.S. and then returns for Ph.D. work in the same department. In any event, in the interrupted program it is

236

B. W. KOFT AND W. W. UMBREIT

most likely that the thesis work will change completely or the emphasis and approach will change drastically. We will use the integrated program as our model, for we tend to favor this program for reasons to be outlined later and it is not too difficult to relate the special conditions of the others to this model. First, let us state unequivocally that the doctorate is a research degree. In order to qualify for such a degree the individual should demonstrate that he is a person able to recognize that theories bearing investigation do not come from the mere accumulation of facts but rather from the enlightened use of knowledge. The doctoral training program, although it includes the master‘s program or its equivalent, only begins once the student has proved his potential for fulfillment of the degree requirements. In the integrated program the qualifications of the graduate student can be thoroughly evaluated at the master’s plateau and it can be ascertained whether he is indeed potential doctoral material. The qualifications which must be measured are: (1)comprehension of fundamental information; ( 2 ) facility for independent investigation; ( 3 ) capacity to equate data obtained in the laboratory with that found in the literature; and (4) ability to formulate ideas and theories and to speculate on future courses of investigation. In the direct and interrupted doctoral programs, special qualifying examinations are necessary to measure these qualifications. In general, the measurement of the individual qualifications by such an exam does not adequately evaluate the candidates’ abilities in categories ( 3 ) and (4) above. Unless there are published papers prior to doctoral thesis writing, there is relatively little to evaluate the potential in these two qualifications. The person clearing the hurdle at the master’s level, who is to go on for a doctorate, must have shown good promise of developing all the attributes required of a Ph.D. Acquisition of the M.S. degree is really only a parenthetical interruption of the program. Therefore we will accept that most courses dealing with basic fundamentals and breadth of program have been accomplished and that approximately 50% of the course work has been completed. The remaining program beyond the master’s should be divided with about 3035% of the time spent on formal course work and 65-70% of the time spent on research. The academic courses now should be particularly chosen to bring the student up-to-date in the related

DISCUSSION: TRAINING APPLIFD MICROBIOLOGISTS

237

fields of biochemistry, physiology, pathogenesis, and genetics and to allow a reasonable depth in a particular applied field. In our age of rapidly expanding and changing concepts of science, the Ph.D. must be prepared to think in frames of reference which today may not seem directly related to applied microbiology, for tomorrow the relationship may become intimate. The impact of genetics on the applied field within the last decade is only one example of this. It is generally easier to learn to think in terms of a related field and to “grow up” with new advances when the “language” is learned at an earlier age. This is not meant to rule out “the expert self-taught,” but the complexities of science today dictates a reasonable amount of formal introduction to subject matter. Also the human frailty of resistance to “drinking at a new fountain” is not totally absent among members of the scientific community. A training steeped only in a very limited applied area would rob the individual of what little versatility he can get in this age of specialization. The situations in research today dictate the development in breadth over specialization in a particular field. As the microbiology course work of a master’s program will vary greatly with the bachelor offerings, the academic course work for a Ph.D. may vary with the training at the two lower levels. In some instances it might be necessary to add some M.S. deficiencies to the Ph.D. program, but it is hoped that this is very limited. At any rate we would expect the M.S. to have completed (at either the B.S. or M.S. level) seven of the following one-semester courses: general bacteriology, pathogenic bacteriology, applied bacteriology, tissue culture, public health bacteriology, soil microbiology, mycology, bacterial physiology, parasitology, diagnostic microbiology, and basic research methods. In addition, two semesters of general biochemistry would be a must and one semester of either physical chemistry or biophysics would be desirable. To the above basic program we should add as essential basic courses at the Ph.D. level one semester each of microbial genetics and fermentations. We would recommend a course in biological engineering of one semester, but this is seldom offered. The remaining course work should consist of five or six elective courses which should be tailored to fit the needs and interests of each individual. These electives should be chosen with some distribution in the following three areas:

238

B. W. KOFT AND W. W. UMBREIT

C. A. Biochemistry 1. Metabolism 2. Microbial biochemistry 3. Nucleic acids 4. Enzymology 5 . Immunochemistry (or Immunology)

Other miscellaneous areas 1. Antibiotics 2. Advanced applied microbiology 3. Pathogenesis 4. Advanced soil microbiology 5. Advanced mycology 6. Protozoology

B. Virology 1. Plant 2. Animal 3. Bacteria

V. Other Problems As we see it, there are some points of difficulty in the relationships between the applied microbiology laboratory and the college or university training centers. We mention these in an effort to clarify the problems without in the least proposing a solution. While we might grant that at several levels of training many laboratories of applied microbiology are engaged in a somewhat stable and predictable operation, we must also recognize that many others are engaged in highIy competitive research operations or even in assay operations in which the rapidity and economics of the situation may rapidly change. As such, one real problem is that of technical obsolescence, not only in equipment but in know-how and in the ability of personnel to be aware of and to respond rapidly to technical advances in the field. None of the training programs we have discussed even approach a solution to this problem. We think that applied microbiologists should begin to consider what can be done in this area. There is perhaps a more delicate subject which should also be mentioned for it is in the response from the student as a potential employee that certain deficiencies in the applied microbiology approach becomes evident. The research director of an applied laboratory and, even more, the personnel directors with whom the prospective employee has contact seem to operate on the general assumption that young men of ability are attracted by facilities, salary scales, achievement awards, and security provisions, and that a “happy family” atmosphere, no matter how artificial, is all that is necessary. We submit that the kind of person attracted by these matters is not the one most applied laboratories really want.

DISCUSSION: TRAINING APPLIED MICROBIOLOGISTS

239

There is one more problem to which we would like to give our attention. At present there is an increasing tendency to stereotype graduate requirements and programs resulting in a decreasing amount of over-all program flexibility. If the prospective graduate student does not present an undergraduate record showing definite promise of genius, with straight A’s of course, as well as completion of 3% years of chemistry, 1 year of calculus, 1 year of physics, and 3 years of biology, he has a slim chance of being accepted. (We must beware of this tendency to require the preparation for a science to begin with ninth grade science.) This is due in part to the great tendency of many graduate departments to accept only those thoroughly trained individuals who, before even starting graduate school, they feel sure will attain the doctorate degree. The staff has fewer distractions from its research this way. This type of emphasis on the doctorate program has also resulted in a general lack of appreciation of the master’s degree. There is a definitely positive need and place for the M.S. in the applied microbiology laboratory. The M.S. offers a training in depth that cannot be achieved in the bachelor’s program and a sincere interest in his chosen field that is difficult to assess accurately in the individual with lesser training. The void between the B.S. or B.A. level of training and the doctorate level is great and the difference in work performance is extreme. There is an important level of competence between these two extremes needed in the applied laboratories which should be represented by the person with a master’s degree. A carefully administered flexible master’s program would allow: ( 1 ) those of definite ability who have discovered the field of applied microbiology at a late stage of their schooling to develop the necessary background; ( 2 ) those having completed the background work to get training in depth not possible at an undergraduate level; and ( 3 ) a chance for those nongeniuses looking toward the doctorate to prove under proper circumstance their potential for this degree. It appears to us that the training of applied microbiologists is too important a problem to be left solely to chance to the “off the cuff” opinions of either the applied laboratories or the training centers, and we hope that this essay may stimulate some thinking and discussion of this matter.

This Page Intentionally Left Blank

AUTHOR INDEX Numbers in italics show the page on which the complete reference is listed. BaddiIey, J., 45, 63 Baer, F. E.,158, 198 Baker, N.,35, 62 Baker, S. J., 36, 66 Baldwin, I. L., 79, 89 Baldwin, R., 52, 53,67 Balotta, R., 84, 90 Ban, Y., 54, 63 Bandurski, R. S., 166, 205 Barber, S. A., 164, 191, 195,200 Barghoom, E. S., 187, 198 Barker, H.A., 27, 28, 49, 50, 51, 53, 57, 62, 68 Barlow, G. H., 36, 62 Bames, E. M.,109,112, 115, 118 Bamett, H.L.,49,50,65 Barnett, S. M., 99, 115 Barth, E. F., 135, 150 Bassett, E.,78, 88 Bassett, E. W., 45,62, 68 Bateman, A. M.,156, 174, 184, 186, 190, 191, 197, 198 Baumgartner, A. W., 197, 198 Bavendamm, W., 162, 169, 198 Bayly, R. J., 35, 62 Beck, J. V., 27, 28, 62, 163, 167, 177, 178, 179, 180, 182, 194, 196, 198, 199, 205 Beerstecher, E., 154, 198 Beijerinck, M.W.,153, 190, 191, 198 Benedict, R. A,, 104, 115 Bennett, E. L.,59, 68 Bentley, R.,28, 45,57, 62 Berky, J., 45, 46, 66 Berner, R. A., 164, 175,198 Bernhauer, K.,79, 88 Bernlohr, R. W., 45, 62 Bhat, J. V., 57, 62 B Bhuyan, B. K.,45, 62 Baars, J. K.,191, 195,198 Birch, A. J., 37,45,62,63,64,68 Baas Becking, L. G. M., 162, 163, Birkinshaw, J. H.,83, 88 168, 176, 196, 197, 198 Bider, B. A., 31, 45,64 Baba, S.,55, 62 Blackwood, A. C., 45,52,53,63,65

A Abraham, E. P., 45, 68 Achenbach, H.,45,64 Adams, F., 165, 198 AdIer, H.H.,161, 198 Aiba, S., 55, 61 Ainsworth, G. C., 109, 114 Akabori, S., 50, 68 Al-Wakil, S. J., 54, 64 Alexander, G.J., 45,54, 61,67 Alexander, M., 154, 158, 160, 164, 165, 166, 167, 169, 193, 196, 198 Alkjaersig, N.,219, 226 Allison, D.,36, 37, 67 Alpen, E. L., 60, 61 Anderson, A. A., 100, 114 Anderson, D. G.,49, 62 Anderson, H.V., 88, 89 Anderson, K., 179, 202 Anderson, R., 165, 178, 182,199 Anderson, R. B., 154, 203 Anderson, R. F.,81,83,88, 89 Anfinsen, C.B., 209,210, 211,226 Anonymous, 221, 226 Anthony, D.S., 57, 63,64 Appleman, M. D.,168, 205 Amold, M.,81, 83, 88 Amstein, H. R. V., 28, 45, 46, 57, 62, 64 Arrhenius, G. 0. S . , 197,200 Asaski, T.,166, 205 Ash, S. H.,186, 198 Ashirov, K. B., 162, 198 Ashmead, D.,177, 185, 198 Audley, B. G.,49, 59,62 AudsIey, A., 177, 185, 198 Ayres, J. C.,103, 115

241

242

AUTHOR INDEX

Blakeley, E. R., 53, 63 Blanche, G. E., 45, 62, 63 Blau, M., 52, 63, 64 Blaylock, B. A,, 196, 198 Bloch, K., 51, 64 Blumson, N. L., 45, 63 Bodo, G., 208, 209, 226 Bogan, R. H., 136, 150, 151 Bohner, C. W., 100, 115 Bohonos, N., 98, 117 Boichenko, E. A., 167, 205 Bolton, E. T., 52, 63 Bomstein, R. A., 57, 63 Bonde, R., 103, 115 Booth, G. H., 176, 180, 197, 198, 205 Bothwell, J. W., 222, 223, 226 Boswell, G. A., 54, 63 Bourns, A. N., 73, 90 Brack, A., 78, 88 Braley, S. A., 163, 177, 178, 179, 180, 182, 186, 192, 194, 199, 201, 202 Branian, H. D., 97, 116 Bray, R., 49, 50, 63 Breed, R. S., 162, 164, 165, 166, 193, 199

Brenner, W., 167, 199 Brian, P. W., 98, 115 Brin, M., 57, 63 Bringmann, G., 147, 150 Broberg, P. L., 49, 66 Brodie, H. J., 55, 62 Brody, H. D., 103, 115 Bromfield, S. M., 159, 164, 166, 191, 192, 195, 199 Broquist, H. P., 95, 96, 115 Brown, R., 106, 116 Brunner, M. P., 88, 89 Bryner, L. C., 163, 165,167, 177, 178, 179, 182, 199 Bu'Lock, J. D., 45, 63, 78, 88 Bunch, R. L., 135, 150 Bungay, H. R., 45, 67 Burroughs, J. D., 100, 115 Buser, W., 191, 199 Buss, C. D., 82, 83, 89 Butkevitch, V. S., 191, 199 Butlin, K. R., 162, 165, 166, 171, 187, 199

Butte, J. C., 45, 63, 65 Buyske, D. A,, 45, 47, 65 Byme, A. F., 108, 115 Bywater, A., 122, 123, 151

C Calvin, M., 33, 53, 59, 66, 68 Cameron, D. W., 45, 62 Cameron, E. J., 100, 115 Campbell, L. L., Jr., 164, 166, 199, 203 Candy, D. J., 45, 63 Canellakis, E. S., 50, 51, 68 Carey, B. J., 165, 204 Carey, B. W., 111, 115 Carlson, C. W., 95, 115 Carroll, V. J., 104, 115, 116 Carson, S. F., 27, 57, 63, 64 Castro, T., 35, 50, 51, 53, 59, 65 Catch, J. R., 28, 34, 46, 50, 52, 63,64 Cayeux, L., 155, 162, 199 Chaiet, L., 49, 63, 64 Chalk, K. J. I., 57, 63 Chambers, H. H., 197, 199 Chen, S. L., 52, 63 Cheney, E. S., 174, 199 Cheney, L. C., 76, 89 Chichester, C. O., 82, 83, 89 Cholodny, N., 192, 199 Chomey, W., 33, 52, 53, 63, 67 Christensen, J. J., 95, 115 Chughtai, I. D., 81, 88 Ciegler, A., 81, 82, 83, 88 Clark, F. E., 159, 191, 199 Clegg, F. G., 97, 115 Clem, L. W., 108, 115 Cliffton, E. E., 219, 220, 226 Clubb, M. E., 45, 62 Coats, M. E., 97, 115 Cochrane, V. W., 71, 88 Coffey, G. L., 32, 45, 66 Coghill, R. D., 69, 90 Coleman, C. S., 166, 199 Colmer, A. R., 163, 177, 179, 192, 194, 196, 199, 204 Connstein, W., 72, 88 Conrad, J. P., 165, 198 Cook, C. D., 110, 116

AUTHOR INDEX

Coombs, T. L., 210, 212, 226 Cooper, P. D., 45, 67 Corcoran, J. W., 45, 49, 50, 63, 65, 67, 68 Corrick, J. D., 177, 178, 182, 185, 199, 204 Corven, R. C., 197, 199 Corvie, D. B., 52, 63 Cox, R. S., 103, 104, 115 Crabb, W. E., 113, 117 Craig, J. T., 30, 37, 52, 53, 63, 67 Crawhill, J. C., 46, 62 Crespi, H. L., 33, 63 Crowell, E. A,, 57, 64 Cruess, V. W., 70, 88 Cruickshank, R., 106, 108, 115 Currie, J. N., 77, 88 Curtis, P. J., 98, 115 Cushman, J. A,, 162, 169, 199 Cuthbert, M. E., 175, 199

D Daborn, G. R., 177, 185, 198 Dale, J. F., 52, 53, 67 Dal Pozzo, A., 86, 88 Dam, R., 86, 88 Dansi, A., 86, 88 Dauben, W. G., 54, 63 David, S., 45, 63 Davidson, C. F., 174, 199 Davies, M. K., 97, 115 Davis, D. B., 163, 167, 177, 178, 182, 199 Davis, J. B., 57, 64 Davisson, J. W., 85, 87, 89 Dean, F. M., 76, 89 Deaugard, L., 191, 199 DeFerrari, G. A., 77, 90 Delchamps, E. W., 182, 204 DeLong, C. W., 45, 68 Dementjeva, G., 165, 204 Denny, C. B., 113, 115 Dessau, G., 173, 199 DeVay, J. E., 57, 68 Dierks, H. A., 186, 198 Dietz, R. S., 191, 200 Dintzis, H. M., 208, 209, 226 Dixon, M., 219, 226

243

Djerassi, C., 37, 45, 62, 64 Doerschuk, A. P., 31, 45, 64, 66 Dombrowski, H . J., 155, 200 Donald, G. M. S., 73, 89 Dondero, N. C., 160, 200 Donohue, J., 168, 205 Donovick, R., 47, 64 Dorfman, R. I., 55, 65 Dorrell, W., 79, 89 Dowler, W. N., 100, 116 Downing, A. L., 121, 122, 138, 144, 145, 146, 150 Drescher, D., 45, 64 Duff, R. B., 159, 167, 200 Dulaney, E. L., 45, 64, 65 Duodoroff, M., 53, 68 Dunford, H. B., 155, 187, 205 Dunn, M. S., 50, 51, 67 Dutcher, J. D., 37, 45, 62, 64

E Eade, R. A., 76, 89 Eble, 86, 89 Ehrensvard, G., 51, 64 Ehrlich, H. L., 157, 159, 162, 163, 164, 178, 180, 182, 183, 191, 195, 200 Ellis, D., 192, 197, 200 Ellner, P. D., 60, 64 Elvehem, C. A., 93, 116 Emerson, R. L., 98, 117 Emery, K. O., 175, 200, 201 Engel, B. G., 82, 83, 89 English, R. J., 45, 62 Eoff, J. R., 74, 89 Erb, W., 50, 64 Ettinger, M. B., 135, 150 Evans, C. C., 28, 64 Evenson, A., 93, 116 F Fair, G. M., 119, 121, 135, 150 Falcone, G., 167, 203 Faulds, W. F., 45, 65 Feeley, H. W., 161, 173, 200, 201 Feigelman, H., 50, 51, 64 Felegy, E. W., 186, 198 Fenendegen, L., 51, 67

244

AUTHOR INDEX

Fenn, W.O.,126, 150 Ferguson, J. H., 85, 87,89 Fernando, R., 93, 115 Ferretti, L. D., 57, 66 Finn, R. K., 70, 89 Firman, M. C., 74, 89 Fish, C.A,, 54, 67 Fitton, P.,45, 62 Foley, G. E., 110, 116 Fonken, G. J., 54, 63 Forbes, J. J., 97, 115 Foster, J. W., 27, 57, 63, 64, 74, 89 Francis, F. J., 103,115 Frank, H. A,, 164, 166, 199 Frantz, I. D., 50,51,64 Fraser, D.,50, 51, 68 Frederick, L. R., 164, 174, 191, 195, 204, 205 Freeman, G. G., 73,89 Fuhs, G. W., 136, 150 Fusari, S. A., 32,45,66

G Gaden, E. L., 70, 89 Gallo, G. G., 77, 90 Gander, J. E., 53, 64 Garey, C.L., 164,191,195,200 Garlick, W.G., 174, 200 Garrels, R. M.,169, 187, 200 Gastel, R.,57, 64 Gatenbeck, S., 45, 64 Gaudy, A. F., 136, 150 Gaughran, E. R. L., 207, 226 Gerard, R. W., 126, 150 Gerhardt, P.,79, 89 Ghose, T. K., 132, 151 Gibbons, A. P., 35, 62 Gibbs, M.,57, 64 Gillette, N. J., 191,200 Gilvarg, C.,51, 64 Glazko, A. J., 32,45,66 Gleen, H., 177, 200 Glock, W.S., 155, 162,200 Glover, J., 49, 64 Godzesky, C.,45, 46,66 Gold, A. M., 45,54,61, 67 Gold, W., 106, 115 Goldberg, E.D., 197, 200

Goldberg, H. S., 96, 100, 101, 102, 104, 106, 108, 109, 110, 111, 112, 114, 115, 116 Goodman, J. J., 31,45,64, 77,89 Goodman, R. N., 98, 99, 100, 103, 104, 110, 115, 116 Goodwin, T. W.,49,59,62, 64 Gordon, H. A., 97, 116 Gordon, M.,45,46, 52,64, 66 Gordon, W . S., 97, 117 Grant, P. T., 45,46, 62 Greathouse, G.A., 53, 66 Greco, A. M., 84, 90 Greenberg, R. A., 100, 113, 116 Grenner, R.,35,50,51,52,53,59,65 Griesebach, H., 45, 64 Griesebach, U. C., 45, 64 Gross, G., 79, 88 Growich, J. A., 31, 45,64 Gruetter, A., 191, 199 Gruner, J. W., 191, 200 Guittoneau, G., 166, 200 Gundersen, K., 136, 150 Gustavson, K. H., 208,215, 226 Gut, M.,55, 65 Gutcho, S., 35,50,51,52,53,59,65 Guyman, J. F., 57, 64 Gwynn, G. W., 32, 45,66

H Hajny, G. J., 73, 89 Hale, C.W., 45, 68 Hall, E. R., 164, 166, 199 Halliday, W . J., 45, 64 Halvorson, H. O., 158, 168, 200, 204 Hamilton, J. M., 99, 116 Hanahan, D.J., 54, 64 Hanson, F. R., 88, 89 Hansson, E.,52, 64 Harder, E. C., 159, 161, 190, 191, 200 Harold, R., 200 Harris, J. F., 73,89 Harris, M.,53, 66 Harrison, A. G., 172,200 Hart, E.B., 93,116 Hasche, D.,178,180,183,202 Hassid, W.Z., 53,68

245

AUTHOR INDEX

Hau, H. H., 82, 88 Hawkes, H. E., 185,200 Hayano, M., 55,62, 65 Hazen, E. L., 106,116 Hemming, H. G., 98,115 Henderson, M. E. K., 160, 167,205 Hendlin, D., 64 Herbert, D., 121, 122, 150 Herbert, M., 30, 45, 65 Hesseltine, C. W., 81, 83, 89 Hildebrand, J. H., 186, 201 Hill, D. C., 97, 116 Hilz, H., 166, 200 Hines, L. R., 103,116 Hinkle, M. E., 177, 192, 199 Hirs, C. H., 209, 226 Hirsch, A,, 100, 116 Hirsch, P., 73, 81, 89 Hitchens, A. P., 162, 164, 165, 166, 193, 199 Hobbs, G., 108, 116 Hochstein, F. A., 45,67 Hochster, R. M., 164, 195,200 Hock, A., 51, 67 Hockenhull, D. J. D., 30, 45, 46, 47, 49, 65, 67 Hodge, E. B., 52, 53,67 Hodge, W. W., 186,200 Hodgkiss, W., 108, 116 Hoeksema, H., 86, 89 Hofheinz, W., 45, 64 Hofmann, K., 57, 65 Holbert, P. E., 174, 203 Holloway, P. N., 45, 62 Holzapfel, C. W., 37, 45, 64 Homer, W. H., 45, 65 Horton, A. A., 49,64 Howell, J. F., 37, 45, 65 Huang, H. T., 85, 87, 89 Hummel, O., 51, 67 Hunter, G. D., 30,45, 47,65 Huston, K. M., 186, 198

I Ikeda, K., 49,50,67 Imhoff, K., 121, 150 Inamine, E., 45, 68 Ingols, R. S., 197, 199, 200

Ingraham, J. L., 57, 64 Ingram, J. M., 45, 65 Ingram, M., 104, 116 Ingram, V. M., 209,226 Ito, I., 177, 201 Ivanov, M. V., 171,172,201,203 Ivanov, V. I., 177, 178, 181, 201

J Jackson, M., 45, 65 Jacquet, J., Jr. 93, 115 Jameson, A. K., 163, 164, 177, 178, 179, 199 JQrai, M., 77, 89 Jarvis, F. G., 86, 87, 89 Jefferson, W. E., 27,57, 63, 64 Jenkins, W. T., 212,226 Jensen, E. R., 31, 45, 64 Jensen, H. L., 136, 150 Jensen, M. L., 172, 173, 174, 187, 199, 201, 202 Johnson, M. J., 37, 45, 46, 57, 62, 63, 65, 66, 67, 69, 70, 71, 80, 86, 87, 89, 90 Johnston, M. R., 104,116 JoliEe, N., 110, 116 Jones, G. E., 161, 172, 173, 174, 201, 204 Jones, W. T. G., 49,64 Jones, S. M., 109, 116 Judkins, H. F., 70, 89 Jukes, H. G., 97,116 Jukes, T. H., 93, 96, 97,116

K Kamen, M. D., 27,62,64 Kaneda, T., 45, 63,65 Kanegas, L. A., 45,46,65 Kaplan, I. R., 172, 173, 174, 175,201 Kar, S. K., 97, 115 Karavaiko, G. I., 173, 186, 201 Karnemaat, J. N., 88, 89 Karow, E. O., 30, 45, 47, 65 Katsuki, H., 177, 201 Katz, E., 45, 65, 67 Katz, J. J., 33, 63 Keener, H. L., 70, 89 Keil, J. G., 45, 62

246

AUTHOR INDEX

Kelly, C. G., 45, 47, 65 Kendrew, J. C., 208, 209, 226 Kendrick, W. B., 185, 201 Kennedy, D. O., 186, 198 Kindle, E. M., 164, 191, 192, 201 Kinsel, N. A., 163, 177, 179, 180, 194, 201, 202 Kirk, M., 59, 68 Kitai, R., 209, 226 Kittler, M., 166, 200 Kleerekoper, H., 172, 205 Knape, G., 166, 200 Knothe, H., 111, 114, 116 Knowles, G., 121, 122, 138, 144, 146, 150 Koch, G., 104, 116 Koch, R. B., 108, 115 Kodicek, E., 57, 63 Koehler, W. A., 186, 204 Kohler, A. R., 95, 96, 115 Kollir, J., 77, 89 Kondo, M.,177, 201 Koshland, D. E., Jr. 213, 226 Kostruva, M. F., 171, 201 Kozinski, A. W., 33, 65 Kramarenko, L. E., 175, 178, 183, 185, 197, 201 Krause, R. F., 49, 50, 65 Kucera, S., 163, 194, 201 Kuehn, R., 147, 150 Kulczycki, L. L., 110, 116 Kulp, J. L., 161, 173, 200, 201 Kuroki, S., 104, 117 Kutzner, H. J., 45, 65 Kuwahara, S., 111, 114, 116 Kuznetova, V. A., 171, 201 Kuznetsov, V. V., 197, 201

L Labaw, L. B., 37, 45, 65 Lackey, J. B., 183, 201 Lager, 86, 89 Lanning, B., 110, 115 Latimer, W. M., 186, 201 Laufer, L., 35, 50, 51, 52, 53, 59, 65 Lazaroff, N., 177, 180, 201 Leaders, F., 97, 116

Leathen, W. W., 163, 177, 178, 179, 180,182, 186, 192, 194,201,202 Leddicotte, G. W., 52, 65 Leeper, G. W., 190, 191, 202 Lees, H., 142, 150, 170, 202 Legge, J. W., 162, 205 Lein, J,, 76, 89 Leith, C. K., 190, 205 Lev, M., 97, 115 Lewin, J. C., 167, 202 Lieske, R., 161, 163, 191, 194, 195, 202 Lilly, V. G., 49, 50, 65 Lingood, F. V., 163, 203 Liu, T. Y., 57, 65 Lis, H., 45, 68 Liston, J., 108, 116 Ljunggren, P., 170, 202 Long, J. P., 116 Long, M. V., 27, 57,64 Lorenz, 186 Lotspeich, F. J., 49, 50, 65 Loughlin, E. H., 111, 116 Love, L. G., 174, 202 Luckey, T. D., 93,97,98,116 Ludecke, K., 72, 88 Lukton, A., 82, 89 Lunan, K. D., 49, 65 Lundgren, D. G., 177, 179, 180, 194, 196, 202, 203, 204, 205 Lutz, A., 109, 116 Lyalikova, N. N., 177, 183, 186, 196, 201, 202, 206

M Maass, E. A., 37, 45, 65 McClure, L. E., 50, 51, 67 McConnell, K. P., 52, 65 McCormick, J. R. D., 31, 45, 64, 66, 77, 89 McCoy, E., 93, 116 McElcheran, D., 172, 205 MacDonald, J. C., 45, 65 McIntyre, L. D., 178, 179, 182, 192, 194, 202 MacKinney, G., 82, 83, 89 McKinney, R. E., 125, 150 McMahan, 102, 116

247

AUTHOR INDEX

McNutt, W. S., 49, 65 McVay, L. V., 110, 117 Mager, J., 166, 202 Majumdar, S. K., 45, 65 Maley, G. F., 49, 65, 66 Malouf, E. E., 168, 178, 180, 184, 202 Mangum, J. H., 196, 205 Mann, K. M., 88, 89 Mann, P. J . G., 191, 195, 202 Marchlewitz, B., 177, 178, 179, 180, 183, 202 Margalith, P., 84, 87, 89 Marmur, J., 33, 66 Marr, A. G., 50, 66 Marshall, R. O., 60, 67 Martin, E., 45, 46, 66 Martin, S. M., 80, 89, 90 Marvo, B., 50, 68 Mason, B., 154, 156, 202 Mattick, A. T. R., 100, 116 Maurer, W., 50, 51, 64, 67 Mautner, H . G., 52, 65 Massy-Westropp, R. A,, 45, 62 Matrishin, M., 31, 45, 64, 77, 89 Mechsner, Kl., 147, 150, 151 Meiklejohn, J., 142, 150 Meloni, M. L., 45, 67 Mercer, S. J., 176, 180,198 Mero, J. L., 197, 202 Meyer, H., 155, 202 Meyer, J., 191, 194, 195, 203 Michener, H. D., 100, 114 Middleton, F. M., 135, 150 Miller, G. A., 45, 68 Miller, I. A., 49, 50, 67 Miller, I. M., 49, 50, 67 Miller, J., 37, 45, 67 Miller, L. P., 163, 165, 168, 175, 176, 202 Miller, P., 45, 46, 66 Miller, P. A., 31, 45, 64, 66 Miller, P. S., 186, 198 Miller, R. P., 177, 185, 202 Miller, W. J., 64 Mills, G. S., 51, 68 Minieri, P. P., 74, 89 Minor, F. W., 53, 66 Myskow, W., 202

Mitsui, H., 50, 68 Mohberg, J., 45, 46, 62, 66 Moldave, K., 59, 66 Molisch, H., 192, 194, 195, 202 Mollin, D. L., 36, 66 Moore, D., 162, 163, 168, 175, 198 Moore, H. B., 162, 202 Moore, P. R., 93, 116 Moore, S., 209, 226 Monod, J., 128, 135, 151 Morgan, E. N., 83, 88 Morgan, G. B., 60, 66 Morrison, A. B., 86, 88 Mosbach, E. H., 57, 66 Moses, V., 53, 66 Moubasher, R. A., 76, 89 Moulton, E. Q., 186, 202 Moyer, A. J., 85, 89 Mulder, E. G., 163, 191, 192, 202 Miintz, A., 142, 151 Murray, A., 28, 66 Murray, E. G. D., 162, 164, 165, 166, 193, 199 Musgrave, 0. C., 45, 62 Myskow, W., 165, 202

N Nadson, G. A., 162,163,169,202 Naeser, C. R., 169, 187, 200 Nagimyak, F. L., 178, 201 Nakai, N., 172, 173,199, 202 Nakayama, T., 82, 83, 89 Napier, E., 177, 185, 202 Nara, T., 45, 66 Nason, A,, 142, 151, 196, 198 Nathorst-Westfelt, L., 45, 46, 66 Neish, A. C., 45, 52, 63 Nelson, G. E. N., 81, 82, 88 Neuberg, C., 72, 90 Newburgh, R. W., 174, 203 Newton, G. G. F., 45,68 Nichols, R. L., 187, 198 Nickerson, W. J.. 167, 203 Nikitia, D. I., 165, 203 Nobili, F., 45, 67 Nomura, M., 104, 117 Nomura, N., 50, 68 Norgard, D. W., 49, 62

248

AUTHOR INDEX

Norris, L. C., 86, 88 Novelli, G. D.,45, 62 Novielli, F., 164, 191, 193, 195, 197, 203 Numerof, P., 45,46, 52, 64,66

0 Ober, R. E., 32, 45, 66 O’Brien, E., 45, 66 O’Conell, P. W., 88, 89 Olson, R. E., 57, 63 Oxford, A. E., 77, 90

P Pagani, H., 84, 87, 89 Paine, S. G.,163, 203 Painter, H. A., 121, 122, 123, 124, 138, 144, 146,150,151 Pan, S. C., 45, 46,52, 64 Pantskhava, E. S., 171, 201 Parker, C. D., 163, 203 Parks, G. S., 196, 197, 198 Parks, J. T.,97, 115 Parrish, R. G.,208, 209,226 Passorn, P. N. A., 59, 64 Pasteur, L., 71, 90 Peck, H. D., Jr. 185, 166, 170, 203 Peck, R. L., 30,45, 48,65 Perkins, E. C., 164, 191, 193, 195, 197, 203 Perkinson, J. D., 28, 51, 68 Perlman, D., 45, 62, 106, 116 Perret, C. J., 36, 37, 45, 66 Peterson, D. H., 55, 62,65 Pettijohn, F. J., 159, 203 Petty, M. A., 31, 45, 64, 70, 74, 77, 90 Peyser, P., 50, 51, 59, 66 Phares, E. F., 57, 63 Phelps, A. S., 31, 45, 64 Phillips, D. C., 208, 209, 226 Pillai, S . C., 123, 151 Pittenger, R. C., 45, 67 Pittinger, C. B., 116 Plaut, G.W. E., 49, 50, 65, 66 Porges, N., 121, 151 Porter, J. W., 49, 62, 68 Postgate, J. R., 186, 167, 170, 171, 199, 203

Praeve, P., 183, 194, 195,203 Pramer, D., 100, 116 Prater, J. D., 168, 178, 180, 184, 185, 202, 206 Pride, E., 45, 62 Pringsheim, E. G., 159, 160, 161, 163, 169, 192, 194, 203 Prockop, D. J., 45, 65 Putter, I., 45, 64, 65 Putnam, E. W., 53, 68

Q Quastel, J. H., 162, 164, 191, 195, 200, 202, 203 Quilter, A. K. J., 49, 67

R Ramakrishnan, C. V., 80, 90 Rauch, J., 79, 88 Razzell, W. E., 165, 177, 178, 179, 180, 182, 183,185, 194,203 Read, B. E., 110, 115 Rees, T., 219, 220, 226 Reinfurth, E., 72, 90 Reis, L., 51, 64 Reiser, R., 155, 203 Remsen, C. C., 179,202,203 Renoux, G., 109, 116 Resnicky, J. W., 159, 191, 199 Rhodes, A., 99, 116 Richards, J. H., 54, 57 Richards, J. W., 70, 90 Rickards, R. W., 37,45, 62,64 Rinehart, K. L., 30, 66 Rippel, A,, 168, 203 Rittenberg, S. C., 172, 173, 174, 175, 200, 201 Robert, J. L., 164, 191,195,203 Roberts, R. W., 45, 67 Roberts, W. M. B., 175,203 Robertson, A., 76, 89 Robinson, W. O.,191, 203 Rogoff, M. H., 154, 178, 182, 186, 192, 196, 203, 204 Roholt, 0. A., 45, 68 Rolland, G., 77, 90 Rosella, J. J., 186, 198 Rosen, A. A.,135, 150

AUTHOR INDEX

Rosenblum, C., 30, 45, 47, 49, 50, 55, 63, 65, 66, 67 Rosenblum, C. H., 34, 35, 66 Rosenthal, G., 175, 203 Rotta, L., 86, 88 Rowlands, S., 37, 45, 67 Rowley, D., 37, 45, 67 Ruben, S., 27, 28, 62, 63, 64 Rudakov, K. J., 165, 203 Ruyssen, R., 222, 226 Ryan, A. J., 45, 62, 63 Ryle, A. P., 209, 226 Ryzhova, V. N., 171,172,201,203

S Sales, R. H., 174, 203 Saluste, E., 51, 64 Sanderson, N. D., 36, 62 Sands, M. K., 52, 63 Sanger, F., 209, 226 Santer, M., 165, 166, 170, 174,205 Santer, U. V., 45, 67 Sartory, A., 191,194,195,203 Sastry, C. A., 123, 151 Sato, M., 183, 203 Saunders, A. P., 45, 46, 67 Sawmiller, L. F., 76, 89 Sawyer, C. N., 136, 150, 151 Sazanova, I. V., 162, 198 Schaeffer, W. I., 174, 203 Schafer, H. M., 65 Schatz, A,, 159, 203 Schatz, B. A,, 46, 66 Schepartz, S. A., 37,67 Schieler, L., 50, 51, 67 Schildkraut, C. L., 33, 66 Schimmer, F., 163, 203 Schloesing, T., 142, 151 Schliissel, H., 51, 67 Scholefield, P. G., 162, 203 Schorler, B., 194, 203 Schubert, A., 55, 65 Schwachman, H., 110, 116 Schwartz, A. M., 53,66 Schwartz, S., 49, 50, 67 Schwartz, W., 177, 178, 179, 180, 183, 202

249

Schweiger, L. B., 79, 85, 90 Schwenk, E., 45, 54, 61,67 Scott, R. O., 167,200 Scully, N. J., 33, 52, 53 Seaman, A,, 108, 109, 116, 117 Sebek, 0. K., 45, 46, 55, 62,65, 67 Segal, S., 52, 67 Segel, I. H., 46, 67 Seidel, P. C., 37, 45, 64 Senkus, M., 37, 63 Sensi, P., 77, 84, 90 Sephton, H. H., 77, 90 Setchell, W. A., 162, 203 Shafer, H. M., 45, 65 Shafia, F. M., 196,205 Shah, P. P., 49, 64 Shemin, D., 49, 50,63, 67 Sheppard, W. A., 73, 90 Sherry, S., 219, 226 Shewan, J. M., 108, 116 Shima, M., 155, 187,205 Shipley, R. A., 35, 62 Shirk, H. G., 53, 66 Shmuk, Ye. I., 177, 186, 206 Shturm, L. D., 166, 203 Shu, P. A., 80, 90 Siebert, R., 55, 65 Silliker, J. H., 100, 113, 116 Silverman, M. P., 178, 179, 180, 182, 186, 192,194, 196,203,204 Simmons, G. W., 222,223,226 Simpson, J. R., 136, 151 Singh, K., 70, 90 Sivak, A., 45, 67 Sizer, I. W., 208,211,212,226 Skerman, V. B. D., 160, 162, 164, 165, 193, 194,199,204 Skinner, F. A., 142, 151 Skon, J., 52, 53, 67 Slaytor, M., 45, 62 Smalley, H. M., 45, 63 Smith, C. G., 75, 86, 89, 90 Smith, E. L., 36, 37, 45, 46, 47, 49, 50, 67 Smith, G . N., 60,67 Smith, H., 45, 62 Smith, H. W., 113, 117 Smith, L. F., 209,226

250

AUTHOR INDEX

Smith, L. W., 103, 117 Smith, R. L., 45,86,67,90 Smythe, M. P., 50, 51, 64 Smeath, P. H. A., 109,114 Snell, J. F., 30, 45, 67, 68 Snell, R. L., 79, 85, 90 Snoke, J. E., 45, 68 Sokol, H., 74, 89 Sokolova, G. A., 171, 172, 173,204 Sorokin, Yu. I., 175, 204 Spier, I. R., 219, 220,226 Sprecher, E., 83, 90 Sprunt, D. H., 110, 117 Stanier, R. Y.,200 Stanley, A. R., 52, 53, 67 Stare, F. J., 57, 63 Stark, W. M., 86, 90 Starkey, R. L., 159, 161, 164, 165, 166, 167, 168, 170, 172, 173, 174, 193, 194, 195, 201,204 Stauffer, V. R., 191, 204 Stavely, H. E., 52, 53, 67 Stein, W. H., 209, 226 Stepanov, B. A., 178, 201 Stevens, C. M., 45, 68 Stevens, D. F., 54, 67 Stjernholm, R., 51, 64 Stokes, H. N., 181, 204 Stone, R. W., 45, 46, 66 Stonier, T., 60, 68 Stoudt, T. H., 67, 70, 90 Stowens, D., 110, 117 Strange, L., 59, 68 Subrahmanyan, P. V. R., 123,151 Sugden, W., 175, 204 Suhadolnik, R. J., 45, 68 Sullivan, J. D., 182, 184,204 Sutton, J. A., 177, 178, 182, 185, 199, 204 Suzuki, I., 57, 68 Swaby, R. J., 190, 191, 202 Szumaski, S. A., 31,45, 64 Sylvester, J. C., 45, 46, 67, 69, 90

T Tabachnick, M., 50, 51, 68 Taber, W. A., 60,68 Takida, T., 85, 90

Tamiya, H., 50, 68 Tanebaum, S., 78, 88 Tanenbaum, S. W., 45,62,68 Taniguchi, Sh., 142, 151 Tarr, H. L. A,, 102, 104,117 Tarver, H., 50, 51, 68 Tasch, P., 155, 203, 204 Taubman, S. B., 45,65,68 Tarvara, K., 85, 90 Taylor, I. F., 160, 167, 205 Taylor, J. H., 97, 117, 185, 204 Taylor, W. R., 162, 204 Temple, K. L., 163, 177, 179, 186, 192, 194, 196,199,204 Teodorovich, G. I., 169, 186, 191, 204 Thayer, J. D., 37, 45,65 Thelin, H., 46, 66 Thiel, G. A,, 190, 191, 204 Thimann, K. V., 160,205 Thode, H. G., 155, 172, 173, 200, 205 Thomas, J. W., 168, 205 Thomas, R., 37, 45, 64,68 Thomas, S. L., 28, 68 Thomson, P. J., 45, 62 Thomson, P. L., 45, 68 Thrupp, T. C., 163,203 Tiller, A. K., 197, 198, 205 Timonin, M. I., 164,205 Tindall, J. B., 37, 63 Tome, J., 45, 46, 66 Tomiyama, T., 104, 117 Tomlinson, T. G., 128, 151 Toohey, J. I., 49, 68 Topper, Y. J., 52, 67 Toshinskaya, A. D., 171,205 Trask, P. D., 167,205 Trown, P. W., 45, 68 Trudinger, P. A., 165, 205 Trussell, P. C., 165, 177, 178, 180, 182, 183, 185, 194, 203 Tsubota, G., 165, 205 Tucker, F. L., 168,205 Turbitt, P. A., 109, 117 Turner, A. W., 162,205 Turner, H. S., 28, 68, Tuve, T., 52, 68

182, 190,

187,

179,

AUTHOR INDEX

U Udenfriend, S., 45, 65 Umbreit, W. W., 174, 203 Underkoffer, L. A., 73, 79, 90 Unz, R. F., 177, 179, 205

v Van der Westhuizen, G. C. A,, 77, 90 Van Hise, C. R., 190, 205 Van Niel, C. B., 153, 170, 205 Van Veen, W. L., 163, 191, 192,202 Vavra, J. P., 164, 191, 195, 205 Vernon, L. P., 196, 205 Verzhbitskaya, L. V., 197, 201 Viney, M., 122, 123, 124, 151 Vining, L. C., 60, 68 Vinogradov, A. P., 167, 205 Virgona, A., 45, 64, 66 Vishniac, W., 165, 166, 170, 174, 205 Vohra, P., 45, 68 Vogel, H. J., 45, 67 Volkova, 0. Yu., 171, 205 Von, Beust, F., 132, 151

W Wada, T., 50, 59, 68 Wagner, B., 147, 150 Wagner, M., 97, 116 Wagner, R. L., 45, 67 Wainwright, T., 166, 205 Wakazono, Y., 177, 201 Waksman, S. A., 160, 205 Walker, N., 142, 151 Walker, T. K., 81, 88 Wallouch, R., 173, 205 Walper, J. F., 198, 205 Walter, F. G., 71, 90 Wanless, R. K., 173, 205 Warburg, O., 126, 151 Watanbe, R., 52, 53, 67 Watso, C. J., 49, 50, 67 Waugh, D. F., 214, 226 Webb, E. C., 219, 226 Webley, D. M., 159, 160, 167, 200, 205 Weed, R. C., 185, 205 Wehmer, C., 79, 90

251

Weigel, H., 35, 62 Weinberger, P., 60, 68 Weiser, H. H., 102, 117 Weissbach, H., 65 Wells, R. A., 177, 185, 202 Wender, I., 154, 178, 182, 186, 192, 203, 204 Werkman, C. H., 57, 68 Werner, A. S., 50, 51, 64 West, C. A., 49, 65 Westley, J., 37, 45, 64 Whalley, W. B., 45, 62 Wheatland, A. B., 121, 122, 150 Wheaton, I. E., 100, 115 Whelan, P. F., 185, 204 Whiffin, A. J., 98, 117 White, J., 69, 71, 90 Whiteley, H. R., 163, 164, 165, 166, 167, 168, 205 Williams, D. I., 99, 117 Williams, D. L., 28, 66 Williams, H. H., 52, 68 Williams, W. L., 97, 117 Wilroy, R. D., 197, 200 Wilson, D. G., 163, 167, 177, 178, 182, 185, 199, 206 Wilson, I. B., 213, 226 Wilson, J. B., 50, 66 Wilson, L. G., 166, 205 Winnick, R. E., 45, 68 Winnick, T., 45, 68 Winogradsky, S., 153, 191, 193, 205 Winstead, J. A., 45, 68 Wix, P., 109, 117 Wolf, F. J., 45, 64, 65 Wolfe, R. S., 163, 193, 194,201,206 Wolochow, H., 53, 68 Wong, P. S., 82, 83, 89 Wood, J. L., 28, 51, 68 Woodbine, M., 108, 109, 166, 117 Woodbury, D. T., 30, 45, 47, 49, 63, 65, 66 Woodruff, H. B., 45, 65 Woolfolk, C. A., 162, 163, 164, 165, 166, 167, 168, 205 Wostmann, B., 97, 116 Wrenshall, C. L., 115

252

AUTHOR INDEX

Wrenshall, L., 101, 117 Wuhrmann, K., 128, 127, 129, 130, 131, 132, 134, 141, 147, 148, 150, 151, Wursch, J., 82, 83, 89 Wyckoff, H., 208, 209, 226

Y Yagi, S., 177, 201 Yamamoto, A., 55, 61 Yoshimori, O., 210, 212, 226 Young, F. E., 45, 68 Young, R. W., 77, 89

Z Zalokar, M., 187, 206 Zanini, C., 88, 88 Zappfe, C., 160, 161, 191, 197, 206 Zaprometov, M. N., 57, 68 Zarubina, Z. M.,177, 186,206 Zaumeyer, W. J., 99, 117 Zavarzin, G. A., 184, 190, 191, 192, 206 Zimmerley, S. R., 185, 206 Zimmerman, M., 82, 83, 89 Ziveig, G., 57, 68 %Bell, C. E.,187, 189,175,206

SUBJECT INDEX A Absorption of isotopes, 60 Acetic acid-CI4, 56 Acetyl-p-nitrophenylserinol, 75 Actinomycin-C14, 38 Activated sludge process, 121, 125 Adenine-U-C14, 58 Adenosine-5’-P04-C14, 58 Adsorption of minerals to cells, 160 Aerobiology, 8 Aerosol vaccination, 8 Aggregates of microorganisms, 125 Agricultural productivity, 12 Air contamination, 8 Air-sterilization filters, 60 Alcohols, radioactive, 55 Algae, 11, 20 Alkaline process for production of glycerol, 74 Alternariol-Cl4, 38 Amino acids, radioactive, 50 Amphotericin B, 106, 108 Amphotericin B, tissue culture, 107 Amy1 alcohol, 0 4 , 56 Amylases, 216 Animal feed supplements, 92 Antibiograms, 105, 108 Antibiotic growth stimulation, modes of action, 97 Antibiotic level in feeds, 94 Antibiotic residues from plant disease control, 100 Antibiotic sensitivity patterns, 108 Antibiotic treatment of fruits and vegetables, 103 Antibiotics, 9, 91 Antibiotics as adjuncts in microbiological procedures, 108 Antibiotics, feed residues, 96 Antibiotics, growth-promoting agent in, 111 Antibiotics in animal nutrition, 93 Antibiotics in canned foods, 100 Antibiotics in food preservation, 100 Antibiotics in milk preservation, 101

Antibiotics in plant disease, 103 Antibiotics in plant disease control, 98 Antibiotics, meats and poultry, 102 Antibiotics, non-medical uses of, 91 Antibiotics permitted for food preservation, 105 Antibiotics, radioactive, 37 Antibiotics, vegetable products, 104 Antifungal agents in phytopathology, 99 Antifungals, 108 Antihalogenating activity, 77 Anti-inflammatory agents, 219 Applied microbiologists, 227 Aspergillic acid-Cl4, 38 Authigenic deposits, 157

B Bacitracin, 38, 93, 113 Bacterial contamination in tissue cultures, 106 Bacterial oxidation of sulfide minerals, 182 Bacterial regeneration of spent leach solutions, 185 Bacterial soft rot in vegetables, 103 Bacterial spoilage, meat, 102 Barbiturates, in fermentations, 84 Barbituric acid, streptomycin, 87 Bating of hides, 208 Bdellovibrio, 12 Beet molasses, 79 Benzoxazolethiol, 76 Benzylpenicillin, 36, 37, 42 Bioengineering, 21, 24 Biogenic sulfur deposits, 171 Biological control of insects, 15 Biological fuel cells, 11 Biological mineral transformation, 157 Biological waste purification, 120 Biosphere, 154 Biosynthetic capacities of bacteria and molds, 8 Bisulfite ion in fermentation, 73 Blackstrap, 79

253

254

SUBJECT INDEX

Blood coagulation, 219 BOD, 122, 129 Bone, 222 Botulism, 100 Broad-spectrum antibiotics in milk preservation, 101 Bromelin, 215 Browning reactions, 217 Butyric acid, C14, 56

C Canning preservation of foods and antibiotics, 100 Carbonates, microbial aspects, 190 Carbon labeled penicillins, 37 Carbon labeled sterols, 55 Carotenes, 48, 50, 81, 82 Cartilage, 222 Casein, 214 Catalase, 218 Cell aggregates, 125 Cellulose-C14, 53 Cellulose into protein, 19 Cephalosporin-C14, 38 Characterization of microorganisms, 106 Cheese products, antibiotics in, 102 Chemical composition of sewage, 123 Chemosynthetic autotrophs, 158 “Chill proofing”, 215 Chloramphenicol, 75 Chloride ions, 74 Chlorides, secondary effect in fermentation, 77 Chloromycetin, 108 Chlortetracycline, 44, 74, 75, 93, 95, 96, 101, 102, 105, 110 Citral, 83 Citric acid, 56, 76, 77 Citrinin, 39 Citromycetin, 39 Civilization, 6 Cloudiness in beer, 215 COD, 122 Collagen, 222 Composts, 12 Continuous fermentations, waste treatments, 121 Continuous fermentors, 138

Control of infectious disease, 7 Crop protection, 14, 192 Crystal structure and susceptibility to oxidation, 182 Curvularin-C14, 39 Cyanides, 79 Cyanocobalamin, 35 Cycloheximide, 98 Cystine-95, 51, 52 Cytidine-5’-P04-C14, 58 D Dairy products, preservation by antibiotics, 101 Debridement of bums and ulcers, 219 Decomposition of sulfur, 170 Degradation of metal sulfides, 177 Degradation of minerals, 153 Degradation of sulfate deposits, 173, 187 Dehalo analogs, 74 Dehydration of eggs, 217 Demethyl-7-chlortetracycline-C14,44 Denitrscation, 141, 145 Deoxyribonucleic acid-Cl4, 59 Deoxyuridine-2-Cl4, 58 Desizing of cloth, 216 Detergents in sewage, 136 Deterioration, 17 Determination of the purity of labeled compounds, 33 Deuterium, 32 Dextran-C14, 52, 53 Dextran-Cl4 sulfate, 36 Diagnostic enzymes, 222 Diauxic growth, 235 Diffusion of metabolites into aggregates, 126 Digestion of metal complexes, 158 Digestive acids, 218 Dihydroxyacetone-1,3-C14, 52 Distillers solubles, novobiocin, 86 Documentation, 21 Domestic sewage, 124

E Eburicoic acid-Cl4, 54 Echinulin-C14, 39

SUBJECT INDEX

255

Ecological conditions for denitrifica- Glucose oxidase, 217 tion, 147 Glutathione-96, 52 Ecology, 8 Glycerol, 53, 72, 73, 74 “Ecology of science”, 6 Glycogen-C14, 52 Electric energy by microbiological Graduate Schools, 235 means, 11 Gramicidin-C14, 40 Ensilage, 17 Growing cultures in preparing labeled Enteric flora, antibiotic affect, 97 compounds, 30 Environment, 6 Griseofulvin, 40, 76, 97, 98 Environmental sanitation, 120 Griseofulvin-2-ethoxy derivative, 40 Enzymes, 207, 208 Guanine, 58 Epigenetic mineral deposits, 156 Guanosine-5’-P04, 98 Ergot alkaloids, 8, 60 H Ergosterol-(214, 59 Erythromycin, 30, 39, 40, 86, 93, 109 Halogens in fermentation, 74 Hazards of antibiotics in feed, 95 Erythrose-C14-phosphate, 53 Exploitation of biological resources, 9 Human biosphere, 3 Human ecology, 2, 4 “Exponential feeding”, 71 Hunger, 7 Hydrocarbons as microbial nutrients, F 20 Fermentation process, 69 Fermentation processes, secondary fac- Hydrogen peroxide milk preservative, 102 tors, 70 Fermentative upgrading of natural Hydrogen sulfide formation, 171 Hydrosphere, 154 resources, 18 Hydrothermal fluids, 155 Fermented sawdust, 19 5-Hydroxy-4-N-ethylmethyltetraFerric ions, 80 eyeIine-C14, 44 Ferro cyanide, inhibition of isocitric a-Hydroxysulfonic acid, 73 acid dehydrogenase, 80 Hypersensitivity, 110 Ficin, 215 Fish, 104 I Flocculation of bacteria, 125 Igneous rock, 155 Fodder, 17 Immunity, 8 Food preservation, 92 Immunization programs, 7 Food production, 2, 15, 16, 24 Inactivated antibiotics and growth re~-Fucose-C14,52 sponse, 97 Fulvic acid, 76 Industrial microbiology in developing Fumaric acid, (214, 56 countries, 25 Industrial wastes, 124 G Infectious disease, 4 Galactose, 52, 53 Inoculation of legume seed, 13 Gangue rocks, 156 Inorganic factors in fermentation Gentisyl alcohol, 78 processes, 71 Geomicrobial activity, 154 Insects, 4, 15 Gibberellins, C14, 57 International association of microbioGliotoxin, 40 logical societies, 1, 23 “Global Impacts of Microbiology”, 1 Ionones, 81, 82 Glucose-Cl4, 35, 52, 53 Iron, 78

256

SUBJECT INDEX

Iron corrosion, 197 Iron deposits, 187 Iron oxides, 191 Iron-oxidizing thiobacilli, 177 Isochlortetracycline, 112 Isotopes, 27 Isotopes of sulfur, 172

K Kerosene, 82 Kojic acid, C14, 56 1 Labeled antibiotics, 37 Labeled compounds, 27 Labeled toxin, 60 Laboratories in applied microbiology, 228 Lactic acid, 0 4 , 58 Lactobacillic acid, 0 4 , 56 Lactose, 87 Lanosterol-Cl4, 54 Leaching of ores, 184 Leaching of phosphorus in soil, 13 Legal protection of microbiological processes, 10 Lipases, 216 Lithosphere, 154 Livestock, 16 Low-level feeding, antibiotic, 94

M

Magma, 155 Malaria, 4 Malic acid, C14, 57 Malnutrition, 7 Manganese, 78 Manganese deposits, 187 Manganese oxide, 191 Mannan-Cld, 52 MannitoLC14, 53 Mannose-Cl4, 52, 53 Manufacture of leather, 207 Marine environments, 11 Marine farming, 11 Marine foods, 11 Mass developments at low substrate, 132 Meats, antibiotics, 102

Meat tenderizer, 215 Mechanism of enzyme action, 212 Mechanisms of metal sulfide oxidation, 180 Metabolic C1402, 30 Metabolic products and mineral transformations, 159 Metal sulfide formation, 174 Metamorphic rock, 155 Methionine-!W, 51, 52 Methods of addition of precursor to microbial systems, 29 Methyl heptenone, 83 Methymycin-Cl4, 40 Microbes as natural resources, 6 Microbial aerosols, 8 Microbial classification, 108 Microbial enzymes, 224 Microbial enzymes in foods, 215 Microbial foods, 19, 20 Microbial formation of minerals, 153 Microbial genetics, 22 Microbial halogenation, 77 Microbial interactions with inorganic substances, 161, 192 Microbial mineral interactions, 157 Microbial processes, primary factors, 70 radioactive compounds, 27 Microbial prospecting for ore deposits, 185 Microbial protein hydrolyzates, (214, 51 Microbial transformations of minerals, 154 Microbiological leaching of mine workings, 13 Microbiological syntheses of pharmacologically active substances, 8 Microbiological waste treatment, 120 Microbiology in biological research, 22 in water pollution control, 119 Microfermentation scale, 31 “Microorganisms decade”, 23 Mineral deposits, 156 Mineralization of organic impurities, 141 Mineral prospecting, 197

257

SUBJECT INDEX

Mode of action of antibiotic growth stimulation, 97, 98 Molasses, 79 ferrocyanide, 80 Molecular biology, 22 Molecular structure of enzymes, 208 Mycelianamide-C14, 40 Mycophenolic acid-Cz4, 41 Myo-Inositol, 53

N National registry of microbiology, 235 Neomycin, 41, 104 Nisin, 100, 102, 105, 113 in cheeses, 102 Nitrification phase in sewage, 142 Nitrifying organisms, 142 Nitrogen deficits in sewage disposal, 142 Nitrogen fixation, 13, 14 p-Nitrophenylserinol, 75 Nonmedical uses of antibiotics, 91 Novobiocin, 14,86, 109 Nucleases, 217 Nucleic acids, radioactive, 55, 58 Nystatin, 41, 97, 105, 106, 108

0 Oleandomycin, 93, 113 Ore-forming processes and microbial action, 184 Organic acids, radioactive, 55 Oxalic acid, 57, 77 Oxaloacetic acid-CI4, 57 Oxides of iron, manganese; microbial aspects, 191 Oxygen supply, 71, 126 Oxytetracycline, 93, 101, 102, 103, 104, 105, 110

P Papain, 215 Paromomycin, 32, 41 Pasteurization of food by hydrogen peroxide, 218 Patulin, 78 labeled, 41 Pectinase, 216

Penicillic acid-Cl4, 41 Penicillin, 36, 86, 93, 100, 112 Penicillin G, 48 Penicillin G-S35, 37 Penicillin with C13, 37 Penicillinase, 217 Pepsin, 218 pH as a secondary factor in fermentation processes, 71 Phytopathogenic bacteria, 12 Phytotoxicity, antibiotics, 99 Plant design, 25 Plant disease control, antibiotics, 99 Plant pathogens, 15 Plant protection, 15 Plant roots, 12 Plasma volume expanders, 8 Polymyxin, 104 Population crisis, 2 Population density concept, 3 Poultry, antibiotics, 102 Preservation of foods, 17 Primary factors, microbial processes, 70 Problems of developing areas, 24 Prodigiosin-C14, 43 Productivity of the seas, 11 n-Propanol, C14, 57 Prophylactic feeding, antibiotic, 95 Proteases, 214, 222 treatment of stock feeds with, 216 Protection of animal resources, 16 Protein, 20 Protozal diseases, 7 Puberulic acid, (214, 57 Public health aspects of antibiotics, 96, 109 Purines, radioactive, 55 Pyocyanine-C14, 43 Pyridoxamine-C14, 48

R Radioactive labeled compounds, 27 Radioactive precursors, 30 “Recombined milk‘‘, 17 Recovery of labeled materials, 33 Redox reactions, 158 “Refractory” materials, 133

258

SUBJECr INDEX

“Registered microbiologist”, 235 Removal of nitrogen from wastes, 141 Rennin, 214 Repression of chlorination, 76 Residue of antibiotics, 96 Residue levels of antibiotics in milk, 101 Resistant compounds, 137, 139 Resistant strains, to antibiotics, 9 Resting cells, 32 Retrieval of information, 21 Rhizosphere, 14 Riboflavin, 48, 50 Ribonucleic acid-Cx4, 59 Rifomycin, 84 Rock, 155 Root microflora, 13

S Screening of microorganisms, 9 Sea, 11 Sea food, 104 Secondary pollution, 141 Sedimentary rock, 155 Selective isolation of specific microorganisms, 100 Selenomethionine-Se76, 52 Sewage as a growth medium, 122 Shikimic acid, C14, 57 Siderochromes, 9 Slowly decomposable substances in wastes, 133 Sludge concentrations, 138 Soft rot, 103 Soil microbiology, 12 Solubilization of minerals, 13 Sources of food, 3 Spiramycin, 43 Spoilage, 17 Squalene-Cl4, 54 Stability of labeled compounds, 34 Starvation, 3 Steroids, transformation of, 55 Sterols, radioactive, 55 Stipitatic acid, C14, 57 Stream pollution, 185 Streptomycin, 30, 47, 93, 98, 103, 104, 108, 112

Streptomycin -C14, 43, 44 Substrate removal from dilute media, 129 Subtilin, 100, 113 Sucrose-CI4, 52, 53 Sulfate deposits, 186 Sulfites in fermentation, 72 Sulfur, 170 Sulfur mineral deposits, 169 Sulfate-reducing bacteria, 172, 187 Survival, 4 Symbiotic nitrogen fixation, 13 Syngenetic deposits, 156 Synnematin-S35,43 Synthesis of fat, 19 Synthetic antibiotics, 9

T Tanning of leather, 215 Tenderizing, 215 Tetracycline, 74, 75, 101, 102, 110 Therapeutic feeding antibiotic, 95 Thermophilic spoilage bacteria, 100 Thio organics, 26 Thymidine, 35 Thymidine-2-Cl4, 58 Thymidine-CH33, 58 Thymine, 35 Tissue culture, 106 Tissue products, 222 Tissue residues, antibiotic, 95 Toxin, growth rate and, 97 labeled, 60 Training of applied microbiologists, 222 Training programs, 230 Trans-B-carotene, 83 Transport of substrates to microorganisms in purification systems, 125 Trehalose-C14, 52 Trickling filters, 121 Turbulence, 128 Tylosin, 93, 100, 113

U Uridine-2-Cl4, 58 Utilization of human resources, 20 of resistant compounds, 138

SUBJECT INDEX

V Vaccines, 7 Valinomycin-(214, 45 Vitamin B,,, 35, 36, 47, 49 Vitamins, 8 radioactive, 47

W Washed cells, 32 Waste materials, 18 Water microbiology, 21

Water pollution control, 119 Water resources, 10 Whey, 18

Y Yeast for food or fodder, 69 Z Zero-order substrate, 129 Zinc, 78, 80 ZymosteroLC14, 54

259

This Page Intentionally Left Blank